Sema7A, a brain immune regulator, regulates seizure activity in PTZ‐kindled epileptic rats

Abstract Aims Semaphorin7A (Sema7A) plays an important role in the immunoregulation of the brain. In our study, we aimed to investigate the expression patterns of Sema7A in epilepsy and further explore the roles of Sema7A in the regulation of seizure activity and the inflammatory response in PTZ‐kindled epileptic rats. Methods First, we measured the Sema7A expression levels in patients with temporal lobe epilepsy (TLE) and in rats of a PTZ‐kindled epilepsy rat model. Second, to explore the role of Sema7A in the regulation of seizure activity, we conducted epilepsy‐related behavioral experiments after knockdown and overexpression of Sema7A in the rat hippocampal dentate gyrus (DG). Possible Sema7A‐related brain immune regulators (eg, ERK phosphorylation, IL‐6, and TNF‐α) were also investigated. Additionally, the growth of mossy fibers was visualized by anterograde tracing using injections of biotinylated dextran amine (BDA) into the DG region. Results Sema7A expression was markedly upregulated in the brain tissues of TLE patients and rats of the epileptic model after PTZ kindling. After knockdown of Sema7A, seizure activity was suppressed based on the latency to the first epileptic seizure, number of seizures, and duration of seizures. Conversely, overexpression of Sema7A promoted seizures. Overexpression of Sema7A increased the expression levels of the inflammatory cytokines, IL‐6 and TNF‐α, ERK phosphorylation, and growth of mossy fibers in PTZ‐kindled epileptic rats. Conclusion Sema7A is upregulated in the epileptic brain and plays a potential role in the regulation of seizure activity in PTZ‐kindled epileptic rats, which may be related to neuroinflammation. Sema7A promotes the inflammatory cytokines TNF‐α and IL‐6 as well as the growth of mossy fibers through the ERK pathway, suggesting that Sema7A may promote seizures by increasing neuroinflammation and activating pathological neural circuits. Sema7A plays a critical role in epilepsy and could be a potential therapeutic target for this neurological disorder.


| BACKG ROU N D
Epilepsy, currently one of the most common chronic neurological disorders, is characterized by persistent brain susceptibility to generating epileptic seizures. Up to ~1% of people worldwide suffer from epilepsy, 1 and temporal lobe epilepsy (TLE), the most common type of focal epilepsy, has the highest incidence rate compared with other seizure types as well as a high refractory rate, leading to an elevated risk of neurological deficits. 2 TLE features a variety of molecular and anatomical changes, including inflammation, mossy fiber sprouting, neuronal death, neurogenesis, gliosis, and neuronal network remodeling. [3][4][5] Recently, numerous studies have suggested that the immune system and inflammatory processes in the central nervous system (CNS) may be important mechanisms involved in the pathophysiology of epilepsy and seizures. [6][7][8] Downstream inflammatory cytokines such as interleukin IL-1β, IL-6, tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2), as well as some inflammatory mediators including nuclear factor kappa B (NF-κB) and cyclooxygenase (COX)-2, are activated in the progression of seizures. 9 In the PTZ-kindled epileptic rat model, increased expression levels of IL-6 and TNF-α were found. [10][11][12] Epileptic seizures induce the release of IL-6 and TNF-α cytokines from glial cells, likely increasing neuronal cell loss, enhancing extraneuronal glutamate concentrations, and decreasing K + and glutamate uptake by glia. Such inflammatory responses cause hyperexcitability of neurons and recurrent seizures, eventually leading to the development of refractory epilepsy. [13][14][15][16] Furthermore, the mitogen-activated protein kinase (MAPK) signaling pathway is known to play an important role in epilepsy. The phosphorylation of extracellular regulated protein kinase (ERK) was significantly increased in the brains of TLE patients compared with that in the brains of controls, suggesting that ERK is activated in epilepsy. 17 In PTZ-kindled rats, inhibiting the TGF-β-mediated ERK signaling pathway can alleviate inflammation responses in epilepsy. 18 However, the mechanism by which inflammation is involved in the pathogenesis of epilepsy remains unclear and requires further exploration. Semaphorins (Semas), consisting of 20 family members in vertebrates, are one of the largest families of guiding clues and have a variety of important functions in the peripheral and CNSs. Five types of semaphores exist, named classes 3, 4, 5, 6, and 7. 19,20 Semaphorin7A (Sema7A) is the only membrane-associated glycosylphosphatidylinositol (GPI)-linked semaphorin and plays a role in the regulation of neuronal axon outgrowth. Importantly, Sema7A also mediates the immune response in multiple systems, such as the lungs, liver, eyes, and bones. [21][22][23][24] Emerging evidence suggests that Sema7A not only promotes inflammatory responses by inducing proinflammatory cytokines, such as TNF-α and IL-6, but also acts as a potent autocrine stimulator of monocytes, stimulating chemotaxis and inflammatory cytokine production and superoxide release. 25 Sema7A initiates inflammatory responses by inducing phosphorylation of ERK 1 and ERK 2, as indicated by research results obtained using a human monocytic cell line. 26 In the CNS, Sema7A is expressed in multiple regions, including the hippocampus, olfactory system, hypothalamic-pituitary system, mesodiencephalic dopamine system, and spinal cord. 27 Sema7A is reportedly involved in CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). 28 In neuronal cells, Sema7A also activates MAPK signaling cascades, which are derived from the β1 integrin receptor and affect processes of neuronal network formation, such as axonal and dendritic growth, branching, guidance and pruning, target recognition, and synapse formation. 29,30 These characteristics of Sema7A suggest that it functions in a variety of ways to regulate neuroinflammation and axon guidance, which may lead to epileptogenesis.
The role of Sema7A in epilepsy remains unknown. In this study, the effects of Sema7A on TLE patients and epileptic rat models were initially investigated. First, we investigated the protein expression of Sema7A in PTZ-kindled epileptic rats and in TLE patients. Additionally, we analyzed behavioral activity and hippocampal morphological changes in PTZ-kindled rats after lentivirus-mediated knockdown and overexpression of Sema7A. Furthermore, the mechanism underlying Sema7A-related inflammation in epilepsy was explored. As the control group, twelve brain tissue samples were also obtained from the Second Affiliated Hospital of Army Medical University. We obtained temporal neocortex samples from patients who underwent craniotomy due to severe traumatic brain injury.

| Human brain tissues
Patients of the control group had no history of epilepsy or any other neurological disease and had never used AEDs. Pathological diagnosis revealed that all resected brain tissues were normal. The clinical features of the control patients are listed in Table 2.

| Experimental animals
Adult male Sprague-Dawley rats (200-230 g) were obtained from the Experimental Animal Center of Chongqing Medical University.
The animals were housed in a specific-pathogen-free (SPF) animal facility at constant temperature (22-24°C) and humidity (55-65%) under an artificial 12/12-hours dark-light cycle. Food and water were available ad libitum. All animal experimental procedures and applicable regulations on animal welfare were in accordance with the guidelines of the Animal Welfare Agency of Chongqing Medical University and were approved by the Ethical Committee for Experimental Animals.

| Pentylenetetrazole (PTZ) kindling
The rats received a daily subconvulsive dose of PTZ (35 mg/kg, Sigma-Aldrich), and their behaviors were then observed for 30 minutes in a Plexiglas chamber. Seizure behavior was monitored according to Racine's standard criteria as follows: grade 0, no response; grade 1, facial myoclonus; grade 2, head nodding; grade 3, forelimb clonus; grade 4, rearing and severe forelimb clonus; grade 5, rearing, falling, and severe forelimb clonus; and grade 6, death. 33 Only rats with at least three consecutive seizures of grade 4 or 5 within 28 days were considered fully kindled and were assigned to the epilepsy group. The controls, in contrast, received an equal amount TA B L E 2 Clinical features of control patients of saline instead of PTZ. The PTZ injections were administered between 9:00 am and 12:00 am to minimize possible complicating effects on the circadian rhythms of the rats.

| Recording of local field potentials (LFPs) during PTZ-induced chronic seizures
Each rat was deeply anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg) and placed on a stereotaxic apparatus (RWD Life Science, Shenzhen, China). Then, a U-shaped frame was fixed to the skull to hold the head. 34 Before LFP recording, electrodes were implanted into the dorsal hippocampus (anterior-posterior: −3.6 mm; medial-lateral: −2.8 mm; dorsal-ventral: −3.0 mm) and attached to an LFP signal connector, which was fixed to the skull using dental acrylic cement. LFPs were recorded with a MAP data acquisition system (Plexon) after the last PTZ injection. The signals were filtered (0.1-500 Hz), preamplified (1000×), and digitized at 4 kHz. The recordings were carried out after the rats had been successfully kindled according to Racine's classification.

| Lentiviral vector production
Lentiviral vectors expressing Sema7A (Sema7A) and containing an enhanced green fluorescent protein (EGFP) sequence were produced by GeneCopoeia Inc. The same lentiviral vectors, encoding only EGFP, were used as controls (Con-Sema7A). For Sema7A knockdown, we constructed lentiviral vectors expressing Sema7A RNAi (Sema7A-RNAi), which was designed and synthesized based on the following sequence: GCCATAACCGCACTGTCATTC. The same lentiviral vectors, but coding for EGFP (Con-RNAi), were used as the control. The viruses were prepared according to a previously described method. 35 The titer of the lentiviral vectors for transfection was 2 × 10 8 TU/mL.

| Stereotaxic injections and U0126 treatment
Stereotaxic injections were performed as described previously with minor modifications. 36 Rats were deeply anesthetized by intraperi- analysis was conducted at 2 weeks after injection into the transfected brain tissues of rats. PTZ was administered for kindling starting at 14 days after lentivirus injection.

U0126 (MedChemExpress) is a specific inhibitor of MAPK kinase
(MEK-1 and MEK-2) that effectively blocks ERK1/2 phosphorylation. U0126 was dissolved in 1% dimethyl sulfoxide (DMSO) solvent, and 30 mg/kg was injected intraperitoneally once per day. 37,38 As described in Figure 9A, the intraperitoneal injections were started 3 days before the initial administration of PTZ, and U0126 was administered 1 hour before each PTZ injection, while the same volume of DMSO was administered to the vehicle group.

| Behavioral assessment
Two weeks after lentivirus injection, each group was given a daily dose of PTZ (35 mg/kg, ip,), and behavioral changes were accessed.
Rats with grade 4 or 5 seizures were selected for behavioral studies, and the latency to the first seizure, number of seizures, and total duration of seizures were separately recorded. 39,40 Two weeks after lentivirus (Sema7A) injection, U0126 or DMSO was intraperitoneally injected once per day. PTZ kindling was performed at 3 days after U0126 injection. Behavioral assessments were performed in Sema7A + U0126+PTZ and Sema7A + DMSO + PTZ groups. (n = 10 biological replicates).

| Human
One portion of each brain tissue sample was immediately stored in liquid nitrogen for Western blot analysis. Another portion of each sample was immersed in 4% paraformaldehyde for 48 hours. Then, a portion of the fixed tissue was removed for paraffin embedding and sliced at a thickness of 5 μm for immunohistochemistry analysis. The remaining tissues were frozen and sliced at 10 μm for immunofluorescence analysis. (n = 5 biological replicates).

| Rat
The rats were anesthetized with pentobarbital (

| Western blotting
Total protein from brain tissues was extracted using a total protein extraction kit (Beyotime), and the protein concentration was detected using a bicinchoninic acid (BCA) Protein Concentration Assay Kit (Beyotime) according to the manufacturer's instructions.
The samples were denatured and stored at −8°C. Protein was separated by SDS-PAGE with a 5% stacking gel and a 10% separating gel and then transferred to a PVDF membrane (Millipore). After being blocked with 5% defatted milk for 1 hour at 37°C, the membranes were incubated at 4°C overnight with the following pri- washing, the immunoreactivity on the membrane was visualized by enhanced chemiluminescence (ECL) substrates. Quantity One software (Bio-Rad) was used to analyze the data. 38 The experiments were repeated three times under the same conditions. The final intensity ratio was obtained by averaging the three experimental values.  The software programs SPSS 20.0 (IBM) and GraphPad Prism 7 (GraphPad software) were used for statistical analyses and graphing. The significance was set at P < 0.05.

| Clinical characteristics of TLE and control subjects
The average age of the sixteen TLE patients was 20.75 ± 2.541 (nine males and seven females), and the average age of the twelve controls was 24 ± 2.637 (six males and six females). Analysis of gender distribution (chi-squared = 0.1077, P = 0.7428) and age distribution

| Sema7A expression in human brain tissues
To determine the cellular localization and distribution of Sema7A in human brain tissue, we used double immunofluorescence labeling and immunohistochemistry. In human brains, Sema7A was mainly costained with a neuron-specific marker NSE but not with the astrocyte marker GFAP in the TLE and control groups, suggesting that Sema7A was mainly expressed in the cell membrane and cytoplasm of neurons but not in astrocytes in the human temporal neocortex ( Figure 1A). Quantitative analysis of immunofluorescence showed a stronger intensity in the TLE group than in the control group ( Figure 1B). Immunohistochemical analysis also showed that Sema7A was present in the cell membrane and cytoplasm ( Figure 1C), and the staining of Sema7A in the brain tissues of TLE patients was significantly stronger than in brain tissues of the control group (P < 0.05; Figure 1D). Western blot analysis was used to further detect the expression of Sema7A in the temporal neocortex of TLE patients and controls ( Figure 1E), revealing that the expression of Sema7A was increased significantly in TLE patients compared with that in control group patients (P < 0.05; Figure 1F).

| Sema7A expression in PTZ-kindled epileptic rat models
To further investigate the expression of Sema7A in epilepsy, we detected the expression level of Sema7A in PTZ-kindled rat models of epilepsy. After the rats were kindled by injection of a subconvulsive dose of PTZ (35 mg/kg) every day for 28 days, LFPs were recorded to assess epileptiform discharges in the PTZ-induced epileptic rat model (Figure 2A). In this model, we detected epileptiform discharge during an episode of behavioral seizure ( Figure 2B). As revealed by double immunofluorescence labeling analysis, Sema7A was observed in the cell membrane and cytoplasm of neurons in the DG region ( Figure 3A) and temporal cortex ( Figure 3C).
Quantitative analysis of immunofluorescence showed that the mean intensity of Sema7A in the hippocampus ( Figure 3B) and adjacent temporal cortex ( Figure 3D) of epileptic rats was increased, indicating that the expression of Sema7A in the hippocampus of epileptic rats was significantly higher than that in control group rats (P < 0.05). Furthermore, the location of Sema7A between the two groups revealed no difference. Similar results were also obtained by immunohistochemistry analysis, as Sema7A was observed in the cell membrane and cytoplasm, and enhanced staining of Sema7A was detected in the DG region of the hippocampus ( Figure 4A) and temporal cortex.

| Sema7A expression after transfection with recombinant lentivirus
The function and mechanisms of Sema7A in epilepsy were investi-

| Sema7A affects seizure susceptibility and activity
Fourteen days after the intracranial injection of lentivirus, a subconvulsant dose (35 mg/kg) of PTZ was intraperitoneally injected daily for behavioral evaluation. A significant increase in latency was observed (P < 0.05; Figure 6A), while both the total duration and the number of seizures were decreased in the Sema7A-RNAi group compared with that in the Con-RNAi group (P < 0.05; Figure 6B, 6). In addition, we also investigated whether overexpression of Sema7A could have an adverse effect on rat behavior. A significantly reduced latency was observed in the Sema7A group compared with that in the Con-Sema7A group (P < 0.05; Figure 6A), while the duration and number of seizures were increased (P < 0.05; Figure 6B

| Effect of Sema7A on mossy fiber projection of the hippocampus
Previous studies suggest that Sema7A is an axonal guidance regulator. 29 In this study, we investigated the effect of Sema7A on the growth of hippocampal mossy fibers in epileptic models. After 2 weeks of stereotaxic injection of BDA into the hippocampal DG region of rats, BDA-positive fibers were detected to assess the development of mossy fiber projections from point to point. We examined the mossy fiber projections from the DG to CA3 in rats. In the control group, the main axons of dentate granule cells (mossy fibers) grew along the inner surface of CA3 in the stratum lacunosum-moleculare (SLM) layer, while only a few grew into the distal layer ( Figure 7A, 7). However, in the Sema7A group, substantially more mossy fibers projected from the hilus to the CA3 SLM, and grew abnormally far laterally in the CA3 field, deeply penetrating the pyramidal (SP) layer of CA3 and, in some cases, extending to the stratum oriens (SO; Figure 7D).The mossy fiber staining in the CA3 region was much stronger in the Sema7A group than in the Con-Sema7A group, while that in the CA3 region was fainter in the Sema7A-RNAi group ( Figure 7B) than in the Con-RNAi group.
These results suggest that downregulation of Sema7A in the DG region inhibits mossy fiber projections, while upregulation of Sema7A promotes the projections.

| Sema7A induced changes in ERK phosphorylation and inflammatory cytokines
In PTZ-kindled epileptic rats, we assessed the phosphorylation of ERK 1/2 and the levels of the inflammatory cytokines IL-6 and TNF-α after Sema7A knockdown or overexpression. The expression levels of pERK1/tERK1, pERK2/tERK2 ( Figure 8A, 8), IL-6, and TNF-α ( Figure 8C-E) were significantly decreased in the Sema7A-RNAi group compared with those in the Con-RNAi group (P < 0.05), while their levels were significantly increased in the Sema7A group compared with those in the Con-Sema7A group (P < 0.05).

| U0126 suppresses seizure severity and reduces the inflammatory response in Sema7Aoverexpressing rats
To determine whether ERK activation is involved in the function of Sema7A in epilepsy, we detected the levels of IL-6 and TNF-α in F I G U R E 5 Expression of Sema7A and distribution of EGFP in the hippocampus after injection of LV vectors. A, Immunofluorescence image of EGFP expression in the dentate gyrus of the hippocampus at 14 d after stereotactic injection of lentivirus. DAPI staining (blue) represents the cellular nucleus (scale bar = 500 μm). B, Western blot images of Sema7A expression in the hippocampus at 14 and 42 days after injection of Sema7A and Sema7A-RNAi. C, At different time points after injection, the mean intensity of Sema7A was decreased significantly in the Sema7A-RNAi group and increased significantly in the Sema7A group. (*P < 0.05, compared with Con-RNAi group; # P < 0.05, compared with Con-Sema7A group, one-way ANOVA test, n = 5) the presence of U0126 after injecting the lentiviral vector Sema7A ( Figure 9A), and the phosphorylation levels of ERK1 and ERK2 were significantly inhibited in the U0126-treated group compared with those in the DMSO-treated group after PTZ kindling (P < 0.05; Figure 9B). Analysis of the behavioral performance showed that the latency was significantly increased (P < 0.05; Figure 9C) and that the duration and number of seizures were decreased in the U0126treated group in comparison with those in the DMSO-treated group.
We also examined the changes in the inflammatory cytokines IL-6 and TNF-α after U0126. In the Sema7A + U0126 + PTZ group, the protein levels of IL-6 and TNF-α were significantly decreased com-

| D ISCUSS I ON
The expression of Sema7A in the CNS increases as development progresses. 27 As we report, Sema7A localizes to the cytomembrane and cytoplasm of neuronal subsets in the hippocampus and temporal neocortex. Interestingly, a significant increase in the expression of Sema7A was found in the temporal neocortex of patients with intractable epilepsy and PTZ-kindled epileptic rats. Given the abnormal expression of Sema7A, we hypothesized that Sema7A might play an important role in TLE, and behavioral analysis confirmed this role.
Overexpression of Sema7A in the hippocampus increases seizure susceptibility and severity in PTZ-treated rats, whereas knockdown of Sema7A results in decreased seizure susceptibility and severity.
Therefore, our data indicate that Sema7A contributes to epileptic seizures. As this study is the first on the role of Sema7A in epilepsy, further investigations are needed to elucidate the possible mechanistic role of this protein in epilepsy.
Sema7A is considered an important effector molecule in T cellmediated inflammation. By stimulating the production of cytokines in monocytes and macrophages by binding α1β1 integrin, Sema7A plays a critical role in the effector stage of the inflammatory immune response. 26 Sema7A is a potent monocyte activator that can stimulate the production of chemotaxis and inflammatory cytokines, such as TNF-alpha and IL-6. 25 In the pathogenesis of multiple sclerosis (MS), Sema7A participates in peripheral immunity and CNS inflammation and promotes cytokines, such as IL-6 and TNF-α. 28 Increasing evidence highlights that inflammation caused by brain damage may be involved in epileptogenesis. Because brain inflammation in epilepsy is not merely a pathological phenomenon but may also be related to the mechanisms of neuronal hyperexcitability, it can potentially be considered a biomarker of disease development and severity. 43 Several studies have revealed increased levels of inflammatory mediators (eg, cytokines such as IL-6 and TNF-α) in epilepsy. 44 TNF-α and IL-6 may increase the excitability of neural TNF-α and IL-6 expression in PTZ-kindled rats, but these increases were ameliorated by Sema7A knockdown, indicating that Sema7A may mediate seizure activity by increasing neuroinflammation in the brain during epilepsy.
As we described earlier, Sema7A exerts an important effect via the MAPKS pathway. In neuronal cells, Sema7A leads to increased expression of the inflammatory cytokines IL-6 and TNF-α by activating ERK1/2 phosphorylation, while the production of cytokines induced by Sema7A is inhibited by the MEK1/2 inhibitor U0126. 26 The MAPK pathway is a crucial signaling pathway that transduces seizure activity, 49  Many studies have shown that Sema7A is not only an immunoregulatory molecule but also a classical axon guidance cue that can promote axon growth. 27,54,55 Sema7A promotes axon outgrowth by activating integrin receptors and MAPK signaling pathways. 29 Mossy fibers are the most studied axons in epilepsy. [56][57][58] In the hippocampus, mossy fibers converge in the hilus, branch out and connect with dendrites of hilar neurons, including mossy cells and pyramidal basket cells, and then project to the CA3 region. 59 Aberrant synapses are formed by mossy fibers and dendrites of abnormally growing granule cells, leading to recurrent excitatory circuitry. 60 In this study, we qualitatively observed the effect of Sema7A on mossy fiber F I G U R E 1 0 U0126 suppresses cytokines IL-6 and TNF-α after Sema7A overexpression in epileptic rats. Western images A, and bars B, C, of IL-6 and TNF-α in the Sema7A + DMSO + PTZ and Sema7A + U0126 + PTZ groups. The mean intensity ratios showed significantly decreased expression of IL-6 (B) and TNF-α (C) in the Sema7A + U0126 + PTZ group. (*P < 0.05, compared with the DMSO-treated group, unpaired t test, n = 5) F I G U R E 11 U0126 inhibits mossy fiber (mf) projections. Rats in Sema7A + DMSO + PTZ group exhibited numerous mossy fibers projecting to the CA3 field, extending deeply into the SP and growing beyond into the SO (A). Furthermore, compared with those in the Sema7A + DMSO + PTZ group, rats in Sema7A + U0126 + PTZ group exhibited a reduced number of mossy fibers, which projected into the CA3 but did not extend deeply ( These results are consistent with previous studies showing that Sema7A promotes the growth of brain axons via the ERK pathway, leading to abnormal neural circuits. Knocking out Sema7A causes miniature excitatory postsynaptic current (mEPSCs), suggesting that Sema7A promotes excitatory inputs. 54 In CNS, Sema7A promotes the growth of neurites and neuroinflammation by binding to β1-Integrin receptor, which activated Ca2 + currents through effects on voltage-sensitive calcium channels and excitatory NMDA currents in neurons. 61,62 These changes could enhance the glutamate excitability and burst activities in hippocampus, cause hyperexcitability in neurons, and could further lead to the development of the epilepsy.
This study is the first to explore the relationship between Sema7A and epilepsy and to further explore the inflammatory and morphological mechanisms of Sema7A-mediated seizures in epilepsy. However, more studies on the mechanistic relationship between Sema7A and excitatory or inhibitory receptors are also needed.
In conclusion, our study demonstrates that Sema7A is upregulated in the brain tissues of refractory TLE patients and PTZ-kindled model rats and plays an important role in epilepsy. In PTZ-kindled rats, Sema7A may promote seizure activity, ERK phosphorylation, IL-6 and TNF-α production, and hippocampal mossy fiber projections. Sema7A may mediate seizure activity by regulating the ERKmediated inflammatory response and mossy fiber growth in epilepsy.
Hence, we speculate that Sema7A may be a potential therapeutic target and provide possibilities for future clinical treatment.

ACK N OWLED G M ENTS
We sincerely thank the patients and their families for their participation in this study. We also thank Xinqiao Hospital of the Third Military Medical University, Chongqing, China, for providing the human brain tissues.

CO N FLI C T S O F I NTE R E S T
The authors have no conflict of interest.