HIV-1 Gag Gene - Understanding Current Trends in the Development of Promising ARV Treatments

Georges Teto^1*, Pierre Zoelong Emini², Beatrice Dambaya¹, Alex Durand Nka¹, Ezechiel Ngoufack Jagni Semengue¹, Desiré Takou¹, Collins Chenwi¹, Aude Christelle Ka’e¹, Grace Angong Beloumou¹, Constant Anatole Pieme³, Joseph Fokam^{1,3,4,5, 6, 7} and Alexis Ndjolo^1,4

¹Chantal Biya International Reference Centre for Research on HIV/AIDS Prevention and Management, Yaoundé´, Cameroon

²Faculty of sciences, University of Yaoundé I, Yaoundé, Cameroon

³National Advanced School of Engineering, University of Maroua, Maroua, Cameroon

⁴Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, Yaoundé, Cameroon

⁵Faculty of Health Sciences, University of Buea, Buea, Cameroon

⁶National HIV Drug Resistance Working Group, Yaoundé, Cameroon

⁷National AIDS Control Committee, Ministry of Public Health, Yaoundé, Cameroon

^*Corresponding author: Georges Teto, Chantal Biya International Reference Centre for Research on HIV/AIDS Prevention and Management, Yaoundé´, Cameroon.

Received: 25 May 2026; Accepted: 05 June 2026; Published: 17 June 2026

1. The Gag Gene in HIV/AIDS Infection

HIV infection is a chronic infection due to Human Immunodeficiency Virus. Since its discovery between 1981 and 1983 [1], HIV/AIDS infection remains incurable and widespread throughout the world. In 2024, according to estimates by the Joint United Nations Program on HIV/AIDS (UNAIDS), 40.8 million people were living with HIV worldwide, of whom 31.6 million (77%) had access to antiretroviral therapy, 1.3 million people were newly infected and 630,000

people had died of HIV-related opportunistic infections [2]. This infection remains a major public health concern. To overcome this infection more effectively, it is vital to take a look back on its pathogen. HIV-1 is the most widespread as compare to HIV-2, and is responsible for the pandemic [3]. HIV-1 is an enveloped retrovirus with a genome made up of two identical RNA molecules [4], each of which encodes for the virus' nine genes: structural genes (gag, pol, env), regulatory genes (tat, rev) and accessory genes (vif, vpr, vpu, nef). The HIV-1 gag gene encodes the structural proteins of the viral particle and the information required for the assembly of retroviruses. During the viral replication cycle (Figure1), viral RNA first undergoes reverse transcription, using the enzyme reverse transcriptase, resulting in pro-viral DNA. Thanks to the action of viral enzyme integrase, the pro-viral DNA is integrated into the host cell genome and then transcribed to form messenger RNA, which is translated to produce viral proteins. The part of the messenger RNA representing the gag gene codes directly for the Gag polyprotein and, by shifting the reading frame from -1 to the end of the gag gene, the Gag-Pol polyprotein is produced. Under the enzymatic action of the viral protease, these polyprotein precursors are cleaved into mature viral proteins (Figure2). The Gag polyprotein (Pr55Gag) plays a key role in genomic RNA packaging, specifically selecting whole (unspliced) strands of viral genomic RNA from a large number of other RNAs. This process takes place through specific interactions between Pr55Gag and the 5' region of the genomic RNA [5,6]. The Pr55 multimers Gag, in association with HIV-1 genomic RNA molecules, form a ribonucleoprotein complex inside cells [7,8] that travels to the plasma membrane for viral particle assembly [5]. Gag can bind, fuse and reorganize pre-existing lipid domains in the plasma membrane [9,10]. The association of the Gag polyprotein with the plasma membrane depends on the lipid composition of the membrane; studies suggest that electrostatic interactions with acidic lipids result in different lipid concentrations at the membrane surface, and therefore specific hydrophobic interactions; this creates an affinity between Gag and membrane lipids [10]. The HIV-1 Pr55Gag precursor is composed of several structural domains: the matrix (MA), the capsid (CA), the nucleocapsid (NC), the P6 protein and two spacer peptides (P2 and P1). Each of these structural domains and spacer peptides plays a role in the assembly and maturation of the viral particle (Figure3).

The MA domain, formed by P17 proteins, allows interaction with the plasma membrane, thanks to a highly basic zone it contains [11,12,13]. It has been established that the targeting and binding of Gag proteins to the plasma membrane is made possible by interactions between the basic region of the MA domain and acidic phospholipids such as phosphatidylinositol (4,5)- bisphosphate (PI (4,5) P2) in the plasma membrane. Studies have shown that binding between the MA domain and RNA leads to a specific interaction between the Gag polyprotein and the plasma membrane; specifically, oligomerisation of this polyprotein on the plasma membrane[14,15]. The CA domain (P24) is essential for Gag multimerisation during assembly and capsid formation during maturation [16]. During the final stages of viral particle maturation, the CA domain, derived from Gag cleavage by protease, forms a core containing the viral genetic material (RNA) and other proteins (reverse transcriptase, Integrase) essential for replication of the new viral particles [17].

Containing several basic residues, the NC domain (P7) is highly important for specific interaction with genomic RNA. It is also involved in reverse transcription, thanks to its ability to facilitate the incorporation of transfer RNA, the tRNALys3, which then serves as a primer [18]. This domain behaves like nucleic acid chaperone proteins [19]. The P6 domain contains binding motifs involved in interaction with the host cell, and budding of the viral particle for release from the host cell [20,21]; it makes use of the endosomal sorting complex required for transport (ESCRT) system to perform this activity, which is its primary one [22,23]. However, it has multiple implications in the viral replication cycle: it plays a role in viral replication through a series of post-translational modifications such as sumoylation, ubiquitination and phosphorylation; it mediates the incorporation of Vpr accessory proteins into virions, thereby promoting Vpr-induced replication. The P6 domain of the Gag gene would therefore be a promising therapeutic target [22]. The two spacer peptides (P2 and P1) are involved in the maturation kinetics of the Pr55 Gag precursor [5].

Figure 1: HIV life cycle and site of inhibitors of viral replication [24].

Figure 2: The scheme of Gag and Gag-Pol processing by HIV-1 PR [25].

Abbreviations: MA – matrix, CA – capsid, SP1 – spacer peptide 1, NC – nucleocapsid, SP2 – spacer peptide 2, TFP –

transframe peptide, PR – protease, RT – reverse transcriptase, IN – integrase. Boxes representing individual domains are not drawn to scale.

Figure 3: Gag proteins and their main functions.

2. HIV/AIDS and Antiretroviral Treatment

Research studies carried out since the beginning of the pandemic have led to significant advances in the management of the disease, resulting in the gradual disappearance of the term AIDS. These studies have enabled us to learn more and more about the virus and its pathophysiology, opening the way to various therapeutic approaches. As a result of this research, antiretroviral treatments (ART) have proved highly effective in the fight against HIV and in improving patients' quality life. Starting with the first discovery of an anti-HIV molecule (Zidovudine) in 1987, several other ARV molecules have been developed, including nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), integrase inhibitors (INs), entry inhibitors [26] , and newly developed ones, namely capsid inhibitors, nucleoside reverse transcriptase translocation inhibitor, maturation inhibitors, broadly neutralising antibodies and in some extend, the pharmacokinetic enhancers.

Triple therapies are combinations of three antiretroviral molecules that inhibit the multiplication of the virus by at least two mechanisms of action [27]. Generally, this involves a combination of two NRTIs with one PI or one NNRTI or one IN. The ARV treatments currently in use help to reduce the viral load to undetectable levels. Thanks to these treatments, progression to AIDS is on the way out, and HIV infection is no longer perceived as a disaster or an inevitability. However, ART does not eradicate or cure HIV infection. The factors explaining this are mainly the existence of multidimensional viral reservoirs where the virus remains in a latent state and escapes the action of ART and the immune system [28], the loss of ART efficacy due to viral resistance, limited access to ART particularly in Low and Middle Income Countries (LMICs), among others. [29,30]. In addition, these antiretroviral are also responsible of numerous adverse effects [30, 18, 31]. There was a need to find out the above mentioned new therapeutics that would provide an option for people living with HIV who have limited treatment options due to

comorbid conditions, side effects, or drug–drug interactions.

Ideally new drugs should exhibit robust HIV-1 antiviral activity against a broad range of subtypes without cross-resistance to currently used therapies, have few drug interactions, and an excellent side effect profile. In addition, simplified dosing regimens, especially having long-acting formulations and being used in two-drug combinations. All

these characteristics are the reason why more attention should be paid to the development of new drugs research. In this context, new classes of ARV drugs have been particularly targeted these recent years, like the capsid inhibitors, maturation inhibitors and broadly neutralizing antibodies among others.

2.1 Interactions between HIV-1 Gag and reverse transcriptase, Protease, Integrase, and entry inhibitors

Reverse transcriptase (RT) is a viral enzyme that acts during the HIV-1 replication cycle by means of DNA polymerase activity, which allows reverse transcription of single- stranded RNA into double- stranded proviral DNA, and endonuclease activity, which results in hydrolysis of the RNA strand contained in the RNA/DNA hybrid molecule. Reverse transcriptase inhibitors are molecules that bind competitively (NRTI) or non-competitively (NNRTI) to this enzyme [32]. Studies have shown that treatment with Efavirenz, a non-nucleoside reverse transcriptase inhibitor, results in significantly lower virus yields due to increased Gag cleavage by the protease [33]. RT instability has been

correlated with impaired Gag cleavage by the protease, suggesting that RT stability is critical for protease activation [34]. Mutations in the RT gene can therefore have a significant impact on protease activation; certain mutations in the RT gene can considerably reduce virus production rates or the release of inadequately processed virions due to enhance or reduce Gag cleavage [35]. It should also be noted that several studies have shown that mutations in the HIV-1 gag gene are associated with treatment failure (ARV combinations) including reverse transcriptase inhibitors. mutations in the Gag cleavage sites can increase viral infectivity and resistance to NRTIs [36]

2.2 Interactions between HIV-1 Gag and Protease inhibitors

Protease inhibitors (PIs) block the action of the viral protease. The gag and polgenes are flanking sequences, and when the viral mRNA is translated, they are translated into Gag polyproteins and then, thanks to a change in the -1 reading frame, into Gag-Pol polyproteins. Viral protease is an enzyme that catalyzes the hydrolysis of these polyprotein precursors into mature viral proteins (Figure4). Gag and Gag-Pol are significantly associated with the final stages of the viral replication cycle, in particular, assembly, genome packaging, budding, release and maturation of viral particles [32]. Viral protease therefore plays a major role in the maturation and acquisition of infectivity of viral particles. PIs are antiretroviral molecules whose main function is to prevent the cleavage of these polyprotein precursors and consequently the maturation of viral particles. However, treatment of HIV infection with PIs is often confronted with cases of HIV-1 resistance to these drugs. Several studies have reported that this resistance is due to mutations that may be located not only in the protease gene, but also in other HIV-1 genes, in this case, the gag gene [37, 38, 39]. Studies of HIV-1, namely subtype B, showed that mutations on Gag or Gag cleavage sites can induce resistance to antiretroviral drugs and treatment failure, independent of mutations in other segments of the viral genome, and directly contribute to resistance to PIs [36,40,41,42,43,44]. However, the mechanisms by which these mutations in the Gag gene lead to failure or resistance to ARV treatments are not yet well understood. Studies by teto and collaborators in 2022, Abdullahi and collaborators in 2023, found a correlation between some protease mutations and the Gag mutations, for instance the correlation between the mutation V82A, M46I, L10F and the mutations L449F, P453L, D480E in gag gene [38,45,46], of course, the mechanism explaining the above mentioned correlation has not yet been explained, future studies should focus on such mechanism.

Figure 4: Cleavage site of HIV protease in the Gag and Pol polyproteins

2.3 Interactions between HIV-1 Gag and Integrase inhibitors

Integrase inhibitors block viral integrase action, an enzyme essential for HIV replication in target cells. Following their identification as innovative medicines, remarkable progress has been made in the development and approval of several compounds belonging to this class of ARVs, including dolutegravir, raltegravir, elvitégravir, cabotegravir and bictegravir. The latter, also known as integrase strand transfer inhibitors (INSTI), not only effectively block HIV-1 replication, but also have a higher genetic barrier to resistance than reverse transcriptase inhibitors [47]. In recent years, treatments including dolutegravir have demonstrated much greater efficacy and are

recommended by the WHO as the treatment of first choice, especially in countries with limited resources. However, there have been cases of antiretroviral resistance in patients receiving treatment protocols including integrase inhibitors such as dolutegravir [47; 48]. There is strong evidence that viral integrase may be involved in assembly and maturation, in addition to its primary role in pro-viral DNA integration [49, 50]. Indeed, mutational analyses have shown that mutations in the integrase gene prevent the correct formation of the virion, leading to the condensation of ribonucleoproteins outside the capsid and thus an empty capsid; this alters the infectivity of viruses that have undergone such mutations [51, 49]. It has been shown that patients receiving integrase strand transfer inhibitors have been reported to experience virologic failure in the absence of resistance mutations in integrase gene. These mutations are selected in the viral nucleocapsid of the Gag rather than integrase. It has been shown that these nucleocapsid mutations accelerate the kinetics of viral DNA integration, suggesting that they limit the window of time available for integrase inhibitors to block viral DNA integration. Moreover, the selected nucleocapsid and integrase mutations act in concert to reduce the susceptibility of the virus to integrase inhibitors. This underscores the importance of genotypic analysis outside IN in patients experiencing failure on integrase inhibitors-containing drug regimens [52, 53, 54, 55, 56, 57].

2.4 Interactions between HIV-1 Gag and entry inhibitors

Entry inhibitors are molecules that prevent the virus from entering the high cell. These entry inhibitors can be grouped into three groups according to their target: those that prevent binding between the glycoprotein gp120 and the CD4 receptor, those that target binding between gp120 and coreceptors, and fusion inhibitors [32]. It has been suggested that incorporation of the envelope into forming viral particles (budding) is facilitated by the interaction between glycoprotein GP41 and the MA domain of polyprotein Gag. A good understanding of the mechanisms of interaction between Gag, GP41 and the plasma membrane would help in the development of new molecules to prevent the

assembly of viral particles [58].

3. Gag Based Inhibitors and Novel Developed Antiretroviral Therapies

3.1 Capsid Inhibition: Lenacapavir

The HIV-1 capsid is a crucial structural component of the virus, forming a conical protein shell that encases the viral RNA and essential enzymes for replication. Primarily composed of the capsid protein, this complex structure plays a pivotal role in various stages of the viral life cycle, including assembly, uncoating, and nuclear import of the viral

genome [59]. Lenacapavir (figure 5), represents the first-in-class HIV-1 capsid inhibitor, a novel mechanism that targets multiple stages of the viral lifecycle (figure 6) [60,61]. By binding to a conserved pocket at the interface of capsid protein (p24) monomers, it allosterically disrupts the delicate processes of viral core assembly, a late-stage event and

disassembly, an early-stage event [32, 62]. This dual mechanism confers picomolar potency and activity against all major HIV-1 subtypes, with no cross-resistance to existing antiretroviral classes [63]. The pivotal evidence for Lenacapavir in treatment comes from the CAPELLA trial, a Phase 2/3 study in heavily treatment-experienced individuals with multidrug-resistant virus [32]. After a functional monotherapy period, participants were switched to an optimized regimen plus subcutaneous Lenacapavir every 26 weeks. At week 52, 83% of participants achieved virologic suppression (HIV-1 RNA <50 copies/mL) [64]. These high rates of suppression were durable, with 82% of those remaining on study maintaining suppression at week 104, accompanied by a robust mean increase in CD4 count of 122 cells/μL [65]. The unique pharmacokinetic profile of Lenacapavir, characterized by a very long half-life, enables its semi-annual subcutaneous dosing and its use as a long acting drug. Studies have also confirmed that an oral dose of 300 mg once weekly can be used effectively as a bridging therapy if a subcutaneous injection is missed [66]. Furthermore, a new intramuscular formulation has shown potential for once- yearly administration [67]. Resistance to Lenacapavir is primarily associated with mutations in the Gag protein at or near the capsid binding site, such as M66I [64,68]. While highly effective, the relatively low genetic barrier to resistance for lenacapavir is a key clinical consideration. A low genetic barrier implies that a small number of viral mutations, sometimes just a single nucleotide change, can confer a significant reduction in susceptibility to the drug.

For lenacapavir, several key mutations (e.g., M66I, Q67H, K70N) have been identified that can reduce its activity [68]. The emergence of resistance during functional monotherapy in trials underscores the critical need for it to be paired with other active agents, especially given its long pharmacokinetic tail, which can create extended periods of low-level drug exposure if adherence is suboptimal or if a follow-up dose is missed. This long tail is a double-edged sword: while it provides forgiveness for slightly delayed doses, a significantly late or missed dose can result in a prolonged period of sub- therapeutic drug concentrations, an ideal scenario for the selection and amplification of pre-existing resistant variants. Recent analyses from clinical trials have further characterized these resistance pathways, emphasizing that the risk of resistance emergence is highest when lenacapavir is not combined with at least one other fully active agent in the regimen [69]. In vitro studies have shown no cross-resistance between Lenacapavir and other antiretroviral classes, including entry inhibitors, underscoring its value in salvage therapy [70]. Beyond treatment, Lenacapavir has demonstrated transformative potential in HIV prevention (PrEP). [71,72]. A study evaluated subcutaneous twice-yearly lenacapavir for HIV prevention in cisgender women yielded impressive results, with no participants in the lenacapavir arm acquiring HIV infection. This trial established that HIV incidence with lenacapavir was significantly lower than both background HIV incidence and the incidence observed with daily oral

emtricitabine/tenofovir disoproxil fumarate (F/TDF) [73]. The PURPOSE 1 trial showed that twice-yearly lenacapavir achieved 100% efficacy in preventing HIV among young women in sub-Saharan Africa. It outperformed both emtricitabine/tenofovir alafenamide (F/TAF) and F/TDF, with no infections observed over 52 weeks [74]. This represents a significant advance in HIV prevention for at risk young women. Additionally, a report detailed the first case of lenacapavir administration during pregnancy to prevent mother-to-child HIV transmission (PMTCT) in a woman with multi-drug resistance HIV. Despite her resistance and high viral load, the newborn was HIV-negative, and lenacapavir was cleared from the infant, suggesting its potential as a safe treatment for pregnant women with adherence issues [75, 76].

Figure 5: Lenacapavir’s chemical structure

Figure 6: The mechanism of action of capsid inhibitor, lenacapavir at the multiple stages of the HIV life’s cycle [77].

3.2 Lenacapavir and Broadly Neutralizing Antibodies

Broadly neutralizing antibodies (bNAbs) are a form of immunotherapy for HIV [78]. They are selected for clinical development based on their potency and breadth, with combinations chosen to target distinct, non-overlapping epitopes on the viral envelope protein. This multi-target strategy creates a higher barrier to resistance, forcing the virus

to mutate at several sites to escape neutralization. The HIV envelope is highly variable, and the virus can rapidly mutate to escape the pressure of a single antibody. However, by combining two or three bNAbs that target conserved regions—for example, the CD4 binding site, the V3 loop, and the MPER region—the virus would need to accumulate

multiple simultaneous mutations to evade neutralization, a far less probable event. This strategy mimics the principles of combination ART. Combinations of two or three bNAbs have demonstrated the ability to maintain virologic suppression in individuals who pause ART [79,80,81], though success is highly dependent on baseline viral sensitivity [82]. Indeed, overcoming the challenges of pre-existing viral resistance and subsequent viral escape remains a primary hurdle for bNAb therapy, requiring the development of more potent and broader antibody combinations or engineered antibodies to increase the barrier to resistance [83]. Their role in cure strategies when combined with latency- reversing agents (LRAs) has shown modest results in trials like TITAN (LRA lefitolimod) and ROADMAP (LRA romidepsin), where no significant additional benefit was observed [84,85]. For prevention, the AMP trials provided proof-of-concept that bNAbs can prevent acquisition of sensitive HIV strains [86]. The future lies in improved combinations,

including long acting drugs like lenacapavir [87].

3.3 Maturation Inhibitors: Zabofiravir

Maturation inhibitors target the final step of Gag polyprotein cleavage. Zabofiravir (figure 7) (GSK3640254) also known as Fipravirimat is a second-generation agent with potent activity across diverse HIV-1 subtypes and baseline Gag polymorphisms [88]. A Phase IIa monotherapy study confirmed its dose-dependent antiviral activity [89], and it has shown no clinically significant pharmacokinetic interaction when co-administered with dolutegravir [90]. Resistance to this class typically involves mutations in Gag at the cleavage site between capsid (p24) and spacer peptide 1 (SP1). A key advantage of this mechanism is its novel resistance profile, which is not expected to show cross-resistance with existing antiretroviral classes, making it a potentially important component of future combination regimens for both treatment-naïve and experienced individuals. For heavily treatment-experienced patients who have accumulated extensive resistance mutations to protease inhibitors, NRTIs, NNRTIs, and even integrase inhibitors, the availability of a new class with a completely distinct resistance pathway is of paramount clinical value. It offers a new tool to construct a viable, suppressive regimen where options were previously exhausted.

Figure 7: zabofiravir’s chemical structure

3.4 Matrix inhibitors

The MA domain anchors Gag into the plasma membrane through electrostatic and hydrophobic interactions, but binding is mediated predominantly by dynamic electrostatic interactions. The MA domain is myristoylated on its N terminus and forms ionic interactions with acidic polar heads of phosphatidylinositol-(4,5)-bisphosphate (PI (4,5) P2) through its highly basic region (conserved stretch of lysine and arginine residues). The MA domain is also capable of binding RNA just like NC domain. The specificity of MA binding to PI (4,5) P2 might be attributed to MA-RNA binding because this binding prevents Gag from interacting with other lipid interfaces until it reaches the plasma membrane. It was reported that MA presented a hexamer-of-trimers arrangement for Env incorporation by interacting with the cytoplasmic tail of the Env gp41 protein and some residual mutations hinder this trimeric formation and subsequent Env incorporation for successful virus particle assembly [91,92,93,94,95]. It was shown that both the divalent phosphate groups and the acyl chains of PI (4,5) P2 [95] were essential for strong binding to MA [96]. Zentner et al. 2013 [97] developed compound 7, inhibitor of HIV-1 assembly (Figure8) using a virtual screening method. They further reported that this compound showed potential anti-HIV-1 activity with IC50 values of 7.5–15.6 µM. It was also determined that compound 7 directly interacted with HIV-1 MA, competing with PI (4,5) P2 for MA binding and blocking the generation of new viruses.

Figure 8: chemical structure of compound 7

In addition, Alfadhli et al. 2013 [98] targeted MA-RNA binding, since RNA binding might protect MA from interacting with other cellular membranes before Gag delivery to the cell surface. They determined four compounds including compound 7 and three thiadiazolanes (TD 1-3) that compete with RNA for MA binding. Thiadiazolanes (figure 9) were found to halt HIV-1 replication but they were subject to toxicity.

Figure 9: Chemical structure of the three thiadiazolanes TD 1-3

Conclusion

In the search for even more effective and definitive solutions to HIV infection, studies are exploring all possible therapeutic targets. In this context, Gag inhibitors namely capsid inhibitors, maturation inhibitors, matrix inhibitors have been studied. One of these, the capsid inhibitors, lenacapavir has been clinically approved, representing a new and highly promising class of antiretroviral agents, offering excellent potential for reducing viral load, few adverse effects and a long half-life. This long half-life allows twice-yearly administration and greatly improve patient adherence. This ARV class is especially useful for heavily treatment-experienced patients battling multi-drug-resistant HIV. Furthermore, prevention trials showed high efficacy in high-risk groups previously underserved by existing PrEP options. This drug was also effective in preventing mother-to-child HIV transmission of HIV. Despite these advances, several limitations need consideration. The emergence of resistance mutations to lenacapavir highlights the importance of combination therapy to maintain a high genetic barrier to resistance and optimal therapeutic responses. Additionally, concerns regarding potential neurotoxicity through synaptic disruption require ongoing surveillance. Even though pharmacokinetic studies support lenacapavir’s use across diverse populations, real-world effectiveness data in resource-constrained settings remains limited. Future research should prioritize developing clinically approved maturation, matrix inhibitors and also second generation capsid inhibitors that maintain potency against emerging lenacapavir-resistant HIV variants while preserving the favourable pharmacokinetic properties that enable long-acting formulations. Working to develop newly long-acting antiretroviral therapy to use in combination with lenacapavir will also be useful to preserve its long term efficacy in the field of HIV infection treatment and prevention.

Ethics and Consent to Participate declarations

not applicable.

Clinical trial number

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Competing interest

Authors declared there is no competing interest.

Funding

No funding has been received for the present work.

Authors’ contributions

GT conceived and wrote the manuscript, PZE, BD, AN, ENJS, DT, CC, ACK and GAB reviewed the manuscript, CP, J F and AN reviewed and supervised the work. All authors read and approved the manuscript.

Acknowledgement

We acknowledge all those who participate to the manuscript by sending us useful manuscripts to be considered in the present work.

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