Several methodologies have been developed to capture lncRNAs associated with chromatin at a genome-wide level. Analysis of RNA-DNA contacts can be combined with epigenetic and RNA-seq data to define potential lncRNAs involved in the regulation of gene expression.Long non-coding RNAs (lncRNAs) are defined as transcribed molecules longer than 200 nucleotides with little to no protein-coding potential. LncRNAs can regulate gene expression of nearby genes , a mark connected to actively transcribed chromatin in lizards. We successfully identified three trans-acting lncRNAs significantly associated with the H4K16ac signal, which are likely involved in the regulation of gene expression in A. carolinensis.We performed Chromatin Associated RNA sequencing (ChAR-seq) in We show that the ChAR-seq method is a powerful tool to explore the RNA-DNA map of interactions. Moreover, in combination with epigenetic data, ChAR-seq can be applied in non-model species to establish potential roles for predicted lncRNAs that lack functional annotations.The online version contains supplementary material available at 10.1186/s12864-023-09545-5. The eukaryotic cell is home to a plethora of non-coding RNAs, among which ribosomal RNAs, small nuclear RNAs, and small nucleolar RNAs are the most abundant. Ribosomal RNAs play a crucial role in translating messenger RNAs, while small nuclear RNAs are essential for gene splicing, and small nucleolar RNAs guide chemical modifications of other RNA molecules. The most enigmatic group of non-coding RNAs is long-non coding RNAs (lncRNAs) , encompaCis-acting lncRNAs regulate gene expression on the same chromosome from which they are transcribed, whereas trans-acting lncRNAs regulate gene transcription on different chromosomes. Some extensively studied cis-acting lncRNAs in vertebrates include XIST [RSX [ROX2 [trans-acting lncRNAs have been experimentally studied, their number remains limited. Notably, HOTAIR [HOXD gene by recruiting the Polycomb Repressive Complex 2 has faced challenges regarding its trans-activity following a recent study that analyzed HOTAIR knockout mice [LncRNAs can be categorized into two groups based on their regulatory activity. ude XIST –14, RSX IST [RSX , and ROXSX [ROX2 , which rSX [ROX2 , recruitSX [ROX2 , or inflSX [ROX2 . Althoug, HOTAIR , 21, a lout mice . Anotherout mice .For more than two decades, hybridization capture methods have been the standard technique for identifying the DNA and proteins associated with specific lncRNA . These mXIST exhibits long-range binding sites along the female X chromosome [Drosophila to unveil the detailed map of RNA-DNA contacts of ROX2 along the X chromosome of males [Recently, four all-to-all approaches have emerged to capture all possible interactions between RNA molecules and chromatin. These methodologies are designed to provide comprehensive insights into RNA-genome interactions. The four methods are MARGI (Mapping RNA–Genome Interactions) , ChAR-seromosome . Similarof males .Drosophila, RNA-DNA contact maps have yet to be explored in other species. In recent years, the number of reptile genomes deposited in public databases has increased by over 600% (from 17 genomes before 2018 to 123 genomes between 2018 and 2023). However, gene annotations in these genomes typically rely on automated modeling of gene predictions based on protein-coding genes from species with curated annotations. In some cases, such as the reference genome of the green anole lizard, Anolis carolinensis, transcriptomic data was utilized to enhance the annotations of coding and non-coding genes [A. carolinensis contains 3,176 lncRNAs (https://www.ensembl.org/Anolis_carolinensis/Info/Annotation), yet most of them lack functional information. In this study, we hypothesized that ChAR-seq-like methods could aid in identifying potential regulatory lncRNAs in genomes where they have been predicted. It should be noted that the ChAR-seq method can provide information about RNA molecules that interact with DNA but is unable to report interactions with other molecules, such as proteins. Therefore, we applied the ChAR-seq method in A. carolinensis to investigate the overall map of interactions between RNA molecules and chromatin. We characterized the frequencies of contact for different classes of RNAs and annotate cis- and trans-acting lncRNAs. By correlating the ChAR-seq results with ChIP-seq data for the acetylation of lysine 16 on histone H4 (H4K16ac) epigenetic mark, we identified three lncRNAs with trans-activity that likely play a role in gene expression regulation in A. carolinensis.While these all-to-all techniques have proven successful in model species such as humans, mice, and ng genes . The curA. carolinensis, we employed the ChAR-seq method on two adult liver samples. By utilizing a specialized short linker, we captured RNA molecules in contact with chromatin and sequenced a total of 1,020,074,230 and 9,96,050,284 reads from the two biological replicates. The paired reads were then mapped to generate a comprehensive genome-wide map of RNA-chromatin interactions.To investigate the interactions between RNA molecules and chromatin in A. carolinenesis, we found that ribosomal RNAs (rRNAs) accounted for 70% of the interactions, whereas long non-coding RNAs (lncRNAs) represented 14%, messenger RNAs (mRNAs) 13%, small nuclear RNAs (snRNAs) 2%, the metazoan signal recognition particle RNA (metazoan srpRNA) 1%, and small nucleolar RNAs (snoRNAs) 0.04% , were overrepresented in our results. Based on the gene annotations available for cis and trans, we focused on genes located on the assembled macro-chromosomes (1 to 6) and discarded the short fragments (scaffolds) in the A. carolinensis reference genome.Subsequently, we determined whether the RNA-chromatin contacts were localized within the same chromosomes  or involved contacts across different chromosomes . To assess this, we introduced a Contact Distribution Index (CDI), which was calculated by dividing the number of contacts on the chromosome with the highest interaction count by the total number of contacts across all chromosomes; CDI values around 0.2–0.3 indicated interactions spread across many chromosomes, while CDI > 0.4 indicated a bias toward fewer chromosomes. To ensure the reliability of the interactions in A. carolinensis genome.Our analysis revealed that rRNAs, metazoan srpRNAs, snRNAs, and snoRNAs displayed lower CDI values Fig. c,d, indicis-proximal) or distantly  on the same chromosome from which they are transcribed, as well as lncRNAs that have interactions with other chromosomes (trans-acting). To examine these types of interactions, we analyzed the lncRNAs on chromosomes 1 to 6 and estimated the percentage of cis-proximal, cis-distant, and trans-acting contacts. Notably, lncRNAs exhibited a gradient distribution between cis-proximal and trans-acting interactions, with a substantial bias towards cis-proximal interactions  supports the notion that our findings represent general active processes in A. carolinensis. Three lncRNAs are of particular interest due to their significant enrichment in chromatin contacts with the H4K16ac epigenetic mark. ENSACAG00000036367 may be involved in gene activation, while ENSACAG00000045045 and ENSACAG00000044053 could play a role in gene silencing. Further work using gene-specific techniques could reveal associated proteins, such as acetyltransferases or methyltransferases complexes.Despite the methodological difficulties encountered in working with A. carolinensis. Our data is limited to lncRNAs with broad expression patterns or specifically expressed in the liver. Currently, the liver is the most suitable tissue for ChAR-seq in lizards due to the considerable amount of starting material required and the small size of the organs. Ideally, future studies will integrate ChAR-seq, ChIP-seq, and RNA-seq analyses using the same sample. To further elucidate the functional characterization of annotated lncRNAs, additional data encompassing various tissues and developmental stages, along with other epigenetic modifications, would be necessary. It is worth noting that many lncRNAs are tissue-specific [The results presented in this study provide a partial glimpse into the interaction map between RNA-DNA molecules in specific –37, and A. carolinensis individuals, one male and one female, were captured in Tampico, Tamaulipas, Mexico . The animals were housed under controlled conditions, with an ambient temperature of 22 ± 2 °C, relative humidity of 55 ± 15%, and a day/night cycle of 12 h/12 h with live food and water ad libitum. The animals were housed together in a large terrarium  equipped with UV light. Prior to the experimental procedure, animals were euthanized using a guillotine. The procedure was performed by an experienced technician. All animal procedures were conducted in accordance with the ethical guidelines of the Bioethical Committee of the Universidad Nacional Autónoma de México. The livers were immediately flash-frozen in liquid nitrogen and stored in 1.5 ml tubes at -80 °C until use. Due to the requirements in starting material, the liver was the most suitable option for the study because it is the largest organ in lizards. The inclusion/exclusion criteria, randomization, blinding/masking, and outcome measures do not apply to this study since the experiment was carried out with two individuals as biological replicates.Two adult https://www.idtdna.com/site/home/) as follows: /5rApp/AANNNAAACCGGCGTCCAA GGATCTTTAATTAAGTCGCAG/3SpC3/. The bottom strand is a biotinylated ssDNA, HPLC purified, ordered at IDT as follows: /5Phos/GATCTGCGACTTAATTA AAGATCCTTGGACGCCGG/iBiodT/T). RNA molecules were reverse transcribed , the genomic DNA was cut with a restriction enzyme , and the 5’ of the adjacent genomic DNA was ligated to the other end of the linker using a DNA ligase . Proteins were removed using Proteinase K  and the 2nd strand of the cDNA-linker-DNA molecules was synthesized with Escherichia coli DNA polymerase I . cDNA-linker-DNA molecules were purified with magnetic beads coated with streptavidin  and prepared for sequencing. The biotinylated linker, which is the key to a successful ChAR-seq experiment, has a 5’ end that contains an adenylated single-stranded sequence that can bind RNA and a 3’ end that contains a recognition site for adjacent DNA fragments that have been digested by DpnII. The linker’s short sequence serves as a molecular tool to control the ligation of RNA and DNA molecules that are in direct contact or in close proximity. More details about the experimental protocol can be found in [ChAR-seq is a recent capture method that traps RNA/chromatin interactions , 30. We found in .A. carolinensis reference genome  using Bowtie2  [A. carolinensis transcriptome  using Bowtie2  [de novo male/female transcriptome generated using Trinity  [de novo transcriptome, and the genome using HISAT2. When a fragment was mapped to the same location in the different databases, we chose the coordinates from the Ensembl transcriptome. IDs from the paired-end reads were used to match the RNA and DNA mapping positions. Valid RNA-DNA contacts were defined as fragments that mapped a single time to their respective databases. We obtained 15,520,949 and 45,816,652 valid contacts for the two replicates. We assigned the contacts to specific genes using the annotations from A. carolinensis genome . We reorganized the contacts based on the different classes of RNA molecules. We plotted the number of contacts against the type of RNA molecules and using dot plots we plotted the specific positions of the RNA contacts mapped to the transcriptome against the positions of the DNA contacts mapped to the genome using R [Cis-acting lncRNAs were defined as those having > 50% of their contacts on the same chromosome from which they are transcribed. Trans-acting lncRNAs were defined as those having > 50% of their interactions in other chromosomes. We calculated the distance from the locus for the cis-acting lncRNA as the absolute difference between the middle point of the genomic position of a lncRNA and all the genomic positions of the start of the contacts on the same chromosome. We also estimated the difference between the middle point of the genomic position of a lncRNA and the genomic positions of its contacts restricted to 20 Kb, 40 Kb, 100 Kb, 200 Kb, and 300 Kb around the lncRNA locus. For trans-acting lncRNAs, we limited the analysis to those annotated on the assembled chromosomes 1 to 6 . We mapped the frequency of contacts along the genome, using the positions of the concatenated genome.One male and one female Illumina TruSeq stranded RNA library were sequenced using an Illumina NovaSeq 6000 machine in Novogene, California. We sequenced 1,020,074,230  and 9,96,050,284  150 nucleotides long paired-end reads. Reads were trimmed using the list of adaptors used in the experimental protocol with trimmomatic  . Valid r--no-sq) . RNA fra--no-sq) . Since try 150G) . Finallyry 150G) . RNA or  using R . For plo using R . Cis-actA. carolinensis from the Ensembl database . The results regarding the validity and robustness of the ChIP-seq data were published previously in [https://github.com/FelixKrueger/TrimGalore). ChIP-seq data were mapped to the A. carolinensis reference genome using Bowtie2 (v2.3.4.1) [trans-acting lncRNAs using R [P-values were corrected using the Benjamin Hochberg correction. Statistical analyzes were conducted in R [We downloaded the reference genome and transcriptome of ously in . We down2.3.4.1) . BAM fil2.3.4.1) . We then using R . Signifited in R .https://www.ncbi.nlm.nih.gov/sra; PRJNA381064). The results regarding the validity and robustness of the RNA-seq data were published previously in [https://github.com/FelixKrueger/TrimGalore). RNA-seq data were aligned to the reference transcriptome using Kallisto [http://www.webgestalt.org/). We used chicken as reference species, and we performed over-representation analyses of geneontology, focusing on biological processes (no-redundant) and pathways (KEGG [https://string-db.org/), standard settings, to explore potential interactions among selected genes. We obtained the TPM values of these target genes and plotted their TPMs from the 15 embryonic and 14 adult tissues using R [We downloaded RNA-seq data for 15 embryonic and 14 adult tissues from females (including liver samples) and 32 embryonic and 14 adult tissues from males (including liver samples) from the NCBI-SRA database . We use using R . Signifi using R .Below is the link to the electronic supplementary material.Supplementary Material 1


TDP-43 is a major pathological protein in sporadic and familial amyotrophic lateral sclerosis (ALS) and mediates mRNA fate. TDP-43 dysfunction leads to causes progressive degeneration of motor neurons, the details of which remain elusive. Elucidation of the molecular mechanisms of RNA binding could enhance our understanding of this devastating disease. We observed the involvement of the glycine-rich (GR) region of TDP-43 in the initial recognition and binding of G-quadruplex (G4)-RNA in conjunction with its RNA recognition motifs (RRM). We performed a molecular dissection of these intramolecular RNA-binding modules in this study. We confirmed that the ALS-linked mutations in the GR region lead to alteration in the G4 structure. In contrast, amino acid substitutions in the GR region alter the protein structure but do not void the interaction with G4-RNA. Based on these observations, we concluded that the structural distortion of G4 caused by these mutations interferes with RRM recruitment and leads to TDP-43 dysfunction. This intramolecular organization between RRM and GR regions modulates the overall G4-binding properties. Approximately 10% of the cases involve genetic mutations, whereas the remaining cases are considered sporadic due to unknown causes3. Currently, more than 50 causative or potentially related genes have been identified, of which the majority encode RNAs and RNA-binding proteins (RBPs)5. Therefore, identifying and analyzing the state of RNA molecules common to all is essential. Indeed, without exception, all these RNAs and RBPs are associated with G-quadruplexes (G4) and are components of ribonucleoprotein (RNP) granules assembled through liquid–liquid phase separation (LLPS)6  is a progressive neurodegenerative disorder characterized by the selective death of motor neurons in the motor cortex, brainstem, and spinal cord)6 Table . This isTARDBP) is an ALS-linked RBP4. The well-studied TDP-43 is a major disease-related protein involved in the pathogenesis of ALS, leading to form abnormal intraneuronal inclusions detected in more than 90% of patients10. Mutations in the TDP-43 gene have also been identified in familial and sporadic ALS patients11. Previously, we identified that TDP-43 binds to G4-containing mRNAs and transports them to distal neurites for local translation12. G4 is a higher-order DNA/RNA structure consisting of two or more guanine tetrads originating from four guanine bases assembled in a square planar arrangement via a Hoogsteen hydrogen bond scheme13. G4-binding RBPs may play essential roles in the regulation of mRNA transport and function, and the dysregulation of RBPs is feared to cause neurological disorders15. However, no detectable destabilization was observed for mutant TDP-43 proteins18, thus dysfunction of TDP-43 might be the cause of the disease onset.TDP-43  and a C-terminal glycine-rich (GR) region and forms a homodimer under physiological cellular conditionsTo clarify the roles of each RRM and GR region in the recognition of G4-RNA, we investigated the influence of ALS-linked TDP-43 mutations on the G4-RNA binding activity using these RNA-binding module segments. The mutations in GR segment alone induced the conformation alteration of G4-RNA while it showed the effects on G4-RNA binding only in the simultaneous presence of the RRM and GR regions. We proposed the participation of GR region in the recognition and binding of G4-RNA, thereby inducing the recruitment of RRM.25. However, the RRM of TDP-43 is not the only module that participates in RNA recognition25. Therefore, we purified a full-length untagged TDP-43 natural dimer and performed a more accurate RNA binding analysis21. Using PSD-95 (postsynaptic density protein 95) and CaMKIIα  20. In this study, we used these G4-RNAs and TDP-43 proteins that had the G287S or M337V mutations and the lowest G4-RNA binding properties  Table , two denhttps://bestsel.elte.hu/index.php)28. TDP-43G287S has a more helical content, whereas TDP-43M337V showed a decrease and an increase in β-turn. We, therefore, hypothesized that the G287S and M337V mutations induced changes in the secondary and tertiary structures.First, to confirm the effects of ALS-linked mutations on the overall structure, far-UV circular dichroism (CD) spectroscopy was used Fig. B,C. Seco6. As a result, this attempt was effective. By adding 2.5, 5, and 10 times the amount of protein to G4-RNA, we confirmed a dose-dependent over-shift in the G4-RNA mobility. The two mutant proteins showed a reduction in shifting bands, consistent with the SPR and secondary structure prediction data. The smeared bands of intermediate mobility might be dissociated RNAs during migration because the maximum signal is detected at this location when run at inappropriate pH condition for binding SPR Fig. C. It was33. Therefore, we analyzed the conformational changes in G4-RNA caused by disease-related amino acid substitutions in the GR region.These unexpected results suggested that the mutations in the GR region do not affect GR-G4-RNA binding but influence other molecular characteristics of GR such as the modulation of G4-RNA structures and/or intramolecular signal transmission to the RRM for modulation of its RNA-binding activity. Indeed, mutations or deletions of G4 binding proteins have been reported to alter the G4 conformation20. TDP-43 binds to cover the entire G4-RNA and maintains a stable conformation20. A positive peak near 265 nm and a negative peak near 240 nm characterized the parallel-stranded G4 conformation As Figs. , which i38. The wild-type GST-GR protein increased G4-dependent fluorescence enhancement, but the ALS-linked mutations G287S and M337V inhibited the increase 24, showed conditional reduction of binding at low concentrations, but when 10 times the amount was added, a binding signal almost equivalent to that of the wild-type RRM segment was detected. The results obtained corroborated our previous report12; therefore, we analyzed the interaction between RRM and G4-RNA.RRM is one of the most abundant RNA-binding modulesdy Figs. D and 4A.er Table . On the i.e., higher for PSD-95 G4 harboring A-loop  screening, we first identified a parallel-stranded G4 structure as a specific binding target, and proposed G4-containing RNAs as the initial selection signal of mRNAs to be transported into neurites for local translation by TDP-43We hypothesized that G4-RNA binding affinity exists independently on the RRM and GR regions, and ALS-linked amino acid substitutions in the GR region of TDP-43 simply resulted in reduced binding of the GR region. Unexpectedly, the GR region with the two mutations herein analyzed showed the same G4-RNA binding ability as the wild-type. Hence, it is interesting that the CD spectra and TO1B turn-on assay showed the two amino acid mutations affected the G4 conformation. A subsequent ternary complex formation experiment suggested that the conformational change of G4-RNA interferes with the recruitment of RRM and reduces the binding affinity of TDP-43.25. Furthermore, a compound that targeting NTD inhibits RRM-RNA binding via allosteric modulation42. However, we have so far failed to detect inhibition of the binding between RRM and UG-rich RNA by an isolated GR region or NTD.Regarding binding specificity, the essential fact is that the RNA-binding module, the RRM segment, bind the UG-rich sequences, whereas the full-length TDP-43 completely loses this binding capacity. We interpreted that the loss of strong RNA binding affinity may be the result of a perturbation by another intramolecular module such as the GR region, thereby inhibiting the binding to targets other than parallel-stranded G4-RNA. Another possibility is the involvement of the N-terminal domain (NTD). It has been reported that NTD behaves as a scaffold for nucleic acid binding and contributes to binding specificity46. Most of these amino acid substitutions were in the GR region. It remains unclear whether all of them interfere with RRM recruitment via the conformational changes in the G4-RNA structure without affecting G4 binding, as determined in this study. Further detailed analysis will be carried out to determine whether there are amino acid substitutions that affect direct interaction with G4. In addition, we recently found that G4-RNA promotes LLPS during FUS RNP granule formation47. The GR region of TDP-43 was involved in LLPS49, and interaction with G4-RNA may have complex effects. The insights provide essential information for the studying G4-binding proteins involved in ALS pathogenesis.Over 50 missense mutations have been identified in the TDP-43 cording gene in familial and sporadic patientsE. coli expression plasmid pGEX-GRWT was described previously20. pGEX-GRG287S and pGEX-GRM337V were constructed by PCR-based mutagenesis method using several primer pairs20. E. coli expression plasmid pET-His-RRM was constructed by replacing the cDNA fragment between the EcoRI and BamHI sites of pGEX-RRM (101-261aa) between the same sites of pET28a ( +) 12.WT, TDP-43G287S and TDP-43M337V were described previously20. Glutathione S-transferase (GST) and GST fusion proteins, GST-GRWT, GST-GRG287S and GST-GRM337V were purified as described20. His-RRM was overproduced in E. coli BL21(DE3) carrying a pET-His-RRM plasmid as noted above. The transformants were grown at 30 °C up to OD600 = 0.4, and then Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM. After 4 h of culture at 30 °C, the cells were harvested by centrifugation and suspended in lysis buffer  containing 1.5 mg/ml lysozyme, stored for 30 min on ice, and then sonicated. After centrifugation, the supernatant was loaded onto the Ni–NTA agarose  open column. The column was washed with lysis buffer and the bound protein was eluted with a buffer containing 250 mM imidazole.Full-length, untagged natural dimer proteins TDP-43All proteins were further purified by Superdex 75 10/300 GL  -1-piperazineethanesulfonic acid]-NaOH, pH 6.8, 300 mM NaCl and 0.1 mM EDTA) and the peak fractions were collected with use of ÄKTA explorer 10 S/100 system . The purified proteins were concentrated by a centrifugal filter  down to 0.5 mg/ml and stored frozen at −80 °C.647, Cy3-CaMKII-Ax647 and UG10- Ax647 (Table 2 and 150 mM NaCl) containing 0.1 mg/ml bovine serum albumin (BSA) and 0.1 mg/ml poly-dI-dC at 25 °C for 30 min. The samples were loaded onto a 1% agarose gel run at 50 V for 60 min at 25 °C. Although the buffer was 0.5 × TBE (pH 6.8), only the dissociation assay was performed under the condition of pH 8.3  using a BIAcoreJ instrument  at 25 °C. In this method, simultaneous analysis was performed using two flow cells. The dT16 oligomer was immobilized onto flow cell 2 and flow cell 1 was left blank to serve as an in-line reference surface. The analyte protein was injected into the flow cells-1 and -2 of the sensor chip. The plasmon resonance values  were obtained from the flow cell-2 data after subtracting the flow cell-1 data.SPR analysis was carried out as described previously47. The estimated G4 structure of the synthesized RNAs, PSD-95 and CaMKIIα  for 30 min at 25 °C. The scan was repeated five times, and Jasco Spectro Manager compiled the mean values. The x-axis represents the wavelength, whereas the y-axis represents the molar CD (Δε). The content of secondary structure was estimated by using the BeStSel online deconvolution webserver28.For conformational analysis of the wild-type and mutant TDP-43 proteins (0.5 mg/ml), the purified protein samples were diluted in PBS  to make a 0.2 μM solution, and then subjected to CD analysis with by using a spectropolarimeter Iα Table  was conf647 and Cy3-CaMKII-Ax647 (Table 2 and 150 mM NaCl) containing 0.1 mg/ml BSA. The mixtures of G4-RNAs and proteins were subjected to cross-linking upon exposure to 254 nm UV at 200 mJ/cm2 using a UV cross-linker, FS-1500 . The cross-linked samples were separated by 10% SDS-PAGE and the fluorescent signal  was scanned by Typhoon 9410 imaging system 21.The fluorescence-labeled oligonucleotides, Cy3-PSD-Ax47 Table   for 10 min at 25 °C. Twenty microliters of 1 μM thiazole orange-3PEG-Biotin, TO1B  was mixed in the same buffer and stored for 5 min at 25 °C. After 5 min, the ligand-enhanced fluorescence spectra were obtained using a fluorescence spectrometer (Shimadzu RF-5300PC) with an excitation wavelength of 505 nm and a scanning emission wavelength of 520–600 nm. The fluorescence intensities at 535 nm were used to plot the comparison graphs. The oligonucleotides used in this study were obtained from GeneDesign, Inc. (Osaka Japan) (Table The oligonucleotide (20 pmol) was incubated with the protein (40 pmol) in 180 μl buffer  Table .2 1.8 mM CaCl2 and 145 mM NaCl, 5.4 mM KCl and 0.05% Tween-20) containing 0.1 mg/ml BSA. These beads were washed two times and incubated with two μg of His-RRM in a 300 μl binding buffer containing 0.1 mg/ml BSA at 4° C for 2 h. These beads were washed thrice, and interacting proteins were eluted with 12.5 μl of 1 × SDS sample buffer. The samples were separated by 12.5% SDS-PAGE, transferred onto a porvinilidendifluoride membrane , and anti-TDP-43 antibodies  were used for detection as described previously20.Glutathione sepharose beads (5 μl)  immobilized with 4 μg of GST or GST fusion protein were incubated with G4-RNA, PSD-95 or CaMKIIα Table  (800 pmoSupplementary Information.


Triptorelin, a synthetic gonadotropin-releasing hormone (GnRH) agonist, has garnered increasing attention for its profound effects on endocrine profiles across diverse populations. This review article explores triptorelin’s impact on women’s health by examining its effects on healthy individuals, those with polycystic ovary syndrome (PCOS), and those experiencing hypothalamic amenorrhea (HA). The mechanism of triptorelin involves a transient surge in gonadotropin release, followed by receptor desensitization, leading to downregulation of the hypothalamus-pituitary-gonadal (HPG) axis. In healthy women, triptorelin’s controlled modulation of the HPG axis is a foundation for assisted reproduction techniques. In PCOS, it offers promise in restoring ovulatory function and mitigating hyperandrogenism. For HA individuals, triptorelin’s potential to restore proper GnRH pulsatility emerges as a therapeutic avenue. This review emphasizes the importance of personalized approaches based on specific health conditions, highlighting triptorelin’s versatility and potential applications beyond its current scope. As research progresses, triptorelin’s role in endocrine management is poised to reshape women’s health by optimizing hormonal equilibrium and overall well-being. The intricate dance of hormones within the human body orchestrates a symphony of physiological processes, including reproduction, metabolism, growth, and overall well-being. The hypothalamus-pituitary-gonadal (HPG) axis is central to this hormonal symphony, a complex network responsible for regulating the endocrine system’s key players. One notable component of this axis is triptorelin, a synthetic peptide analog of gonadotropin-releasing hormone (GnRH). In recent years, triptorelin has garnered significant attention for its ability to modulate endocrine profiles, affecting various health conditions. This review delves into the multifaceted effects of triptorelin on endocrine profiles, shedding light on its implications for healthy women, those with polycystic ovary syndrome (PCOS), and those experiencing hypothalamic amenorrhea (HA) ,2.Triptorelin, classified as a GnRH agonist, exerts its effects by binding to and activating GnRH receptors in the pituitary gland. This leads to a transient surge in gonadotropin release, desensitizing these receptors and causing downregulation of gonadotropin secretion. By manipulating the pulsatile release of GnRH, triptorelin can effectively suppress the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This suppression holds therapeutic potential in various contexts, including infertility treatment, sex hormone-related cancers, and hormone-dependent conditions ,4.Endocrine profiles serve as windows into the intricate balance of hormones within the body. Deviations from these profiles can herald a wide array of health concerns. Hormones are pivotal in maintaining menstrual regularity, promoting ovulation, regulating metabolic processes, and modulating mood and cognition. Therefore, disturbances in endocrine profiles can profoundly affect reproductive health, metabolic homeostasis, and overall quality of life. Understanding these profiles facilitates diagnosis and monitoring and enables the development of targeted interventions to restore equilibrium -7.Studying the effects of triptorelin on endocrine profiles across different populations provides valuable insights into its potential applications and mechanisms of action. Healthy women with regular menstrual cycles are a baseline for assessing triptorelin’s impact on normal endocrine regulation. Investigating triptorelin’s effects on women with PCOS is of paramount importance due to the syndrome’s intricate hormonal imbalances and associated reproductive challenges. Furthermore, women experiencing HA, characterized by disrupted GnRH pulsatility, can benefit from triptorelin’s potential to restore endocrine balance. By elucidating the nuances of triptorelin’s effects on these distinct groups, researchers and clinicians can tailor its use for optimal therapeutic outcomes ,9.Endocrine profiles in healthy womenNormal Menstrual Cycle and Hormone LevelsThe average menstrual cycle orchestrates an intricately timed sequence of hormonal fluctuations, spanning an average of 28 days, that serves as the cornerstone of female reproductive physiology. This rhythmic choreography is characterized by distinct phases governed by a coordinated interplay of hormones. The cycle commences with the follicular phase, an initial stage marked by the orchestrated orchestration of hormonal cues. At this juncture, the FSH takes center stage, propelling the growth and maturation of ovarian follicles. Within the ovaries, granulosa cells are spurred into action, synthesizing and secreting estradiol, a vital estrogen hormone. This surge in estradiol levels, akin to a crescendo, sets the stage for a profound surge in LH release ,11.The culmination of this hormonal symphony is ovulation, a climactic event where a matured ovarian follicle achieves its zenith and ultimately releases an egg, the potential harbinger of new life. The orchestrated surge of LH plays a pivotal role in this process, precisely timed to prompt the rupture of the follicle and release of the egg from the ovary. This carefully choreographed ballet of hormonal cues ensures that the ovulated egg is poised for potential fertilization, in line with the intricacies of the female reproductive cycle .As the ovulatory curtain closes, the luteal phase takes the spotlight. During this interval, the remnants of the ovulated follicle metamorphose into the corpus luteum, an endocrine structure responsible for progesterone production. Progesterone, aptly called the “hormone of pregnancy,” is instrumental in orchestrating the uterine environment to implant a fertilized egg potentially. The uterine lining, known as the endometrium, is meticulously primed and thickened, creating a nurturing milieu that could foster the development of an embryo .This intricately woven narrative of hormonal symphony underscores the awe-inspiring complexity of the normal menstrual cycle. Every hormonal cue, every surge and dip, is a meticulously timed note in this symphonic composition that orchestrates the potential for life. The follicular, ovulation, and luteal phases harmoniously collaborate to create the cyclical melody central to the rhythms of female fertility and reproductive health .Role of the Hypothalamus-Pituitary-Gonadal AxisThe HPG axis is a cornerstone of the intricate hormonal symphony orchestrating reproductive processes. This complex axis, which comprises the hypothalamus, pituitary gland, and gonads, serves as a finely tuned regulatory system that governs the cyclic release of crucial sex hormones and holds paramount importance in maintaining reproductive function. The orchestration begins with the hypothalamus, a small yet vital brain region that operates as the conductor of this hormonal symphony .The hypothalamus executes its role by precisely releasing GnRH, a potent signaling molecule that guides the pituitary gland’s actions. The release of GnRH is marked by its characteristic pulsatile pattern, emphasizing the intricacy and precision of this orchestration. This rhythmic release pattern of GnRH plays a pivotal role in maintaining the delicate balance of hormonal feedback loops .Upon reaching the pituitary gland, GnRH acts as a catalyst, prompting the release of two key gonadotropins: LH and FSH. LH and FSH, collectively known as gonadotropins, venture to the ovaries, the gonadal powerhouse in females. These gonadotropins interface with their respective receptors within the ovaries, effectively stirring the production and release of sex hormones .Estradiol and progesterone, the primary sex hormones in females, are the crescendos of this harmonious hormonal symphony. Estradiol, a potent estrogen, is pivotal in promoting the growth and maturation of ovarian follicles, which are sacs containing the developing eggs. It reaches its peak levels during the follicular phase of the menstrual cycle, playing a central role in stimulating the LH surge that triggers ovulation .Impact of Triptorelin on Healthy Women’s Endocrine ProfilesTriptorelin’s synthetic analog nature enables precise manipulation of the HPG axis. Triptorelin’s administration initially causes a paradoxical surge in LH and FSH in healthy women, mimicking the natural GnRH stimulation. However, this effect is transient, as continued administration leads to receptor desensitization and downregulation of LH and FSH release. This suppression reduces estradiol and progesterone levels, leading to anovulation and menstrual cycle disruption .Triptorelin’s controlled suppression of gonadotropin release finds application in assisted reproductive technologies, where controlled ovarian hyperstimulation is necessary for optimizing oocyte retrieval. Furthermore, it offers therapeutic potential in conditions requiring hormone-sensitive suppression, such as endometriosis or uterine fibroids. Understanding the nuances of triptorelin’s impact on healthy endocrine profiles contributes to its broader applicability in clinical practice .Polycystic ovary syndrome and endocrine disruptionsIntroduction to Polycystic Ovary Syndrome and Diagnostic CriteriaPCOS is a multifaceted endocrine disorder with profound implications for reproductive-age women. It is a condition marked by a complex interplay of hormonal imbalances and reproductive irregularities that transcend mere gynecological concerns. The intertwining triad of hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology is central to PCOS’s intricate manifestation .Hyperandrogenism, the elevation of male hormone levels such as testosterone, contributes to the clinical androgenic manifestations observed in PCOS. This can encompass hirsutism, characterized by excessive facial and body hair growth, acne, and alopecia. Ovulatory dysfunction, a core feature of PCOS, leads to irregular menstrual cycles or even anovulation, where ovulation fails to occur. This irregularity disrupts fertility and contributes to the complex clinical picture of PCOS .One of the defining characteristics of PCOS, often visible through ultrasound imaging, is the presence of polycystic ovaries. These ovaries are characterized by multiple small follicles that appear like a “string of pearls” on ultrasound. However, it is important to note that not all individuals with PCOS will exhibit classic ovarian morphology, and the diagnostic criteria extend beyond this hallmark feature .To standardize the diagnosis of PCOS, the Rotterdam criteria have gained widespread acceptance. These criteria stipulate that for a diagnosis of PCOS to be established, at least two of the following must be present: irregular menstrual cycles, clinical or biochemical signs of hyperandrogenism, and polycystic ovaries on ultrasound. This approach acknowledges the heterogeneous nature of PCOS presentations and highlights the importance of assessing multiple aspects of the syndrome to reach a comprehensive diagnosis .Altered Hormone Levels and Menstrual Irregularities in Polycystic Ovary SyndromePCOS is a complex endocrine disorder that intricately impacts hormone levels and disrupts the delicate balance within the reproductive system. One of the hallmark features of PCOS is the presence of elevated androgen levels, particularly testosterone. This androgen excess contributes to physical symptoms such as hirsutism (excessive hair growth) and acne, affecting individuals’ appearance and emotional well-being .Central to the hormonal dysregulation in PCOS is the HPG axis dysfunction. This axis orchestrates the menstrual cycle by coordinating the release of crucial hormones that stimulate the ovaries and regulate ovulation. In PCOS, the intricate dance of this axis becomes disrupted, leading to anovulatory cycles in which ovulation does not occur. The ovaries’ sensitivity to gonadotropins such as LH and FSH is reduced, further compounding the irregularities in hormone levels and ovulation .Another pivotal aspect of PCOS is the frequently coexisting insulin resistance, wherein the responsiveness of the body’s cells to insulin actions diminishes. This physiological state gives rise to hyperinsulinemia, characterized by elevated insulin levels in the bloodstream. This, in turn, plays a significant role in the augmentation of ovarian androgen production, specifically testosterone synthesis. The intricate interaction between insulin resistance and the surplus of androgens establishes a self-perpetuating cycle. The heightened levels of insulin act as a stimulant for the ovaries to enhance the generation of androgens, thereby sustaining the hormonal imbalances that define PCOS .Effects of Triptorelin on Endocrine Markers in Polycystic Ovary SyndromeTriptorelin’s potential impact on individuals with PCOS holds particular intrigue due to its unique ability to modulate the intricate web of the HPG axis and the associated hormone secretions. Research has shed light on the intricate mechanisms through which triptorelin administration could alleviate the multifaceted hormonal disruptions characteristic of PCOS .Triptorelin’s administration in PCOS patients has shown promising results in normalizing LH and FSH levels. This normalization represents a pivotal step in restoring the delicate equilibrium between these two gonadotropins, which are often dysregulated in PCOS. Restoring LH and FSH balance has significant implications for ovarian function, potentially fostering a more favorable environment for ovulation. By fine-tuning the pulsatile release of these hormones, triptorelin may act as a catalyst in reestablishing the natural ovulatory processes often compromised in PCOS .Moreover, triptorelin’s effects extend to androgen regulation, the cornerstone of PCOS pathology. By suppressing the release of LH and FSH, triptorelin indirectly dampens the ovarian production of androgens, such as testosterone. This reduction in androgen levels can potentially mitigate the hyperandrogenic symptoms that frequently plague individuals with PCOS, including hirsutism and acne. The interconnected nature of the endocrine system means that triptorelin’s impact on gonadotropin suppression can indirectly address the androgen excess that is a hallmark of PCOS .Potential Therapeutic Implications of Triptorelin in Polycystic Ovary Syndrome ManagementThe intricate hormonal imbalances characteristic of PCOS have spurred interest in triptorelin as a potential therapeutic intervention. Triptorelin’s controlled manipulation of endocrine profiles offers a promising avenue for addressing the multifaceted challenges posed by PCOS .PCOS, marked by hyperandrogenism, anovulation, and insulin resistance, underpins various reproductive and metabolic dysfunctions. Triptorelin’s mechanism of action, involving transient gonadotropin release followed by downregulation, holds the potential to restore ovulatory cycles, a central concern for PCOS patients desiring conception. By orchestrating a regulated pattern of gonadotropin release, triptorelin may help normalize follicular development and ovulation, enhancing the chances of a successful pregnancy .Moreover, triptorelin’s impact on androgen levels presents an intriguing dimension for PCOS management. Elevated androgens contribute to hirsutism, acne, and alopecia, significantly affecting the quality of life. Triptorelin’s suppression of gonadotropins indirectly leads to reduced ovarian androgen production, potentially alleviating these cosmetic concerns. Additionally, as insulin resistance exacerbates PCOS symptoms, triptorelin’s role in regulating hormonal balance might contribute to metabolic improvements .However, while the potential therapeutic implications of triptorelin in PCOS management are promising, several aspects require further elucidation. Establishing optimal dosing regimens and treatment durations specific to PCOS is paramount to balancing achieving desirable outcomes and minimizing potential side effects. Furthermore, a comprehensive investigation into the long-term effects of triptorelin in PCOS is essential, ensuring that its benefits are sustained over time and potential risks are thoroughly understood .Hypothalamic amenorrhea and endocrine dysregulationHypothalamic Amenorrhea: Causes and ConsequencesHA is a perplexing disorder characterized by the conspicuous absence of menstrual periods due to intricate disruptions within the delicate HPG axis. Often targeting women with reduced energy availability, strenuous exercise regimes, or elevated stress levels, HA unveils the remarkable sensitivity of the female reproductive system to physiological stressors. The origins of HA are frequently woven from a complex tapestry of interrelated factors, with a convergence of inadequate caloric intake, intense physical exertion, and psychosocial stressors driving the disorder’s manifestation .The repercussions of HA extend far beyond the realm of fertility, echoing through multiple facets of a woman’s physiological well-being. The absence of menstruation reflects the underlying disarray within the HPG axis, where diminished GnRH pulsatility engenders hormonal dysregulation. Amidst this turmoil, the impaired secretion of LH and FSH disrupts the orchestrated dance of ovarian follicle development, eventually leading to anovulation .Yet, HA’s impact reaches beyond reproductive concerns, leaving an indelible imprint on broader health domains. The compromised estrogen production stemming from the anovulatory state strikes at the heart of bone health, rendering women vulnerable to decreased bone mineral density and osteoporosis, a condition fraught with long-term implications for skeletal integrity. Furthermore, the muted estrogen milieu in HA reverberates through cardiovascular corridors, potentially heightening the risk of adverse cardiac events. The repercussions even stretch into the landscape of psychological well-being, potentially fostering mood disturbances and altered cognitive function .In the tapestry of women’s health, HA emerges as a complex thread, intertwining intricate hormonal disruptions with sweeping consequences for diverse physiological systems. The recognition of HA’s multifaceted nature underscores the imperative for a holistic approach that restores reproductive function and attends to the broader canvas of well-being .Disrupted Hypothalamus-Pituitary-Gonadal Axis and Hormone Imbalances in Hypothalamic AmenorrheaHA is characterized by an intricate interplay of endocrine disruptions that reverberate across the HPG axis, exerting far-reaching effects on hormonal balance and reproductive function. Within this context, the HPG axis, which orchestrates the pulsatile release of GnRH and its downstream effects, becomes dysregulated in HA, giving rise to intricate hormone imbalances .Central to this disruption is the diminished GnRH pulsatility, a hallmark of HA. The hypothalamus, responsible for generating these pulsatile signals, experiences a blunting of its GnRH release pattern, leading to reduced frequency and amplitude of GnRH pulses. This dampened GnRH output translates into diminished LH and FSH secretion from the pituitary gland. This hormonal derangement results in a cascade of downstream effects reverberating through the entire reproductive system .In the context of ovarian function, the suppressed LH and FSH levels hinder the growth and maturation of ovarian follicles. The reduced stimulation from these hormones results in insufficient development of follicles, thwarting the ovulatory process. This anovulatory state deprives women of the potential to release mature eggs, contributing to infertility .Moreover, the decreased ovarian activity translates into inadequate estrogen production, a hormone pivotal for many physiological functions beyond reproduction. The implications of estrogen deficiency extend to bone health, where reduced estrogen levels can precipitate a decline in bone mineral density. Compromised bone strength elevates the risk of long-term osteoporosis and fractures .The suppression of reproductive hormones in HA reverberates beyond reproductive and bone health. It impacts various physiological systems, including the cardiovascular, metabolic, and psychological. The absence of estrogen’s protective effects on cardiovascular health may contribute to increased cardiovascular risk factors. Metabolic perturbations such as altered glucose metabolism and lipid profiles may also arise. The hormonal imbalance could also manifest as psychological disturbances, including mood fluctuations and cognitive changes .Exploring the Role of Triptorelin in Restoring Endocrine Balance in Hypothalamic AmenorrheaThe mechanism underlying HA is intricate, involving the disruption of the HPG axis and subsequently leading to the absence of menstrual cycles. This multifaceted condition frequently emerges due to factors such as energy deficiency, excessive exercise, or psychosocial stress. These triggers set off a complex series of endocrine imbalances that have extensive ramifications. In light of this, the investigation into triptorelin’s potential role in addressing HA has gained significant momentum. This is attributed to triptorelin’s unique and meticulously orchestrated mechanisms , which oTriptorelin’s initial paradoxical effect on gonadotropin release, followed by downregulation, is particularly intriguing when applied to HA. The transient simulation of LH and FSH release, akin to natural GnRH pulsatility, holds the promise of restoring the disrupted hormonal rhythms observed in HA. By initiating these pulses, triptorelin may trigger a cascade of events, including reinstating proper GnRH signaling. This, in turn, could promote more regular menstrual cycles, a crucial aspect of reproductive health beyond fertility concerns .One of the paramount implications of triptorelin’s potential role in HA is the enhancement of estrogen production. The suppressed gonadal activity in HA results in diminished estrogen levels, posing adverse effects on bone health, cardiovascular function, and overall well-being. If successful, triptorelin’s modulation of the HPG axis could lead to the restoration of estrogen production. This, in turn, may alleviate the adverse consequences of estrogen deficiency, particularly the heightened risk of osteoporosis and other associated health issues .The ramifications of triptorelin’s application in HA extend beyond reproductive health, encapsulating broader well-being. While the primary focus may be restoring ovulation and menstrual regularity, the potential impact on hormonal balance reverberates through various physiological systems. If successful, triptorelin could emerge as a tool for fertility enhancement and holistic health improvement in HA individuals .Considerations for Triptorelin Use in Hypothalamic Amenorrhea TreatmentTriptorelin presents a promising avenue for HA treatment, potentially restoring the disrupted GnRH pulsatility and consequently reestablishing menstrual regularity. However, a thoughtful assessment of various considerations is imperative before embracing triptorelin as a therapeutic solution .Addressing the underlying causes of HA is paramount. HA often arises from factors such as energy deficiency, excessive exercise, and psychosocial stress. These factors are pivotal in suppressing GnRH pulsatility and disrupting the HPG axis. Therefore, while triptorelin can potentially restore the endocrine balance, a comprehensive approach encompassing lifestyle modifications, nutritional counseling, and stress reduction strategies should be integrated alongside pharmacological interventions. Treating the root causes with triptorelin ensures a holistic and sustainable outcome .The optimal dosing and duration of triptorelin treatment necessitate careful calibration. Balancing the objective of reinstating menstrual regularity with the potential risks is critical. Overstimulation or extended suppression of the HPG axis could lead to unintended consequences or inadequate outcomes. Tailoring triptorelin dosing regimens to individual needs, considering factors such as age, the severity of HA, and responsiveness to treatment, is essential to strike an optimal balance between restoration of fertility and minimizing potential adverse effects .In treating HA with triptorelin, vigilance regarding potential side effects is paramount. As with any pharmaceutical intervention, there is a spectrum of potential reactions. Monitoring bone health is of particular concern, as HA’s disruption of the hormonal milieu may lead to decreased bone density. Triptorelin’s impact on estrogen levels and its subsequent influence on bone health warrants close observation. Additionally, side effects such as hot flashes and mood changes accompanying hormonal therapies should be monitored and managed effectively to ensure a positive treatment experience .Comparative analysis of triptorelin effectsComparing Endocrine Responses in Healthy, Polycystic Ovary Syndrome, and Hypothalamic Amenorrhea WomenTriptorelin’s multifaceted impact on endocrine profiles is magnified when considering its effects across distinct populations: healthy women, those with PCOS, and individuals experiencing HA. These three groups represent a spectrum of endocrine regulation, allowing for a comprehensive assessment of triptorelin’s capabilities and limitations. By delving into these diverse responses, a more nuanced understanding of the drug’s effects emerges, illuminating its potential applications and highlighting its adaptability .In healthy women, triptorelin’s introduction triggers a brief yet intense surge in LH and FSH. This surge, akin to the natural pulsatile pattern of GnRH release, is swiftly followed by receptor desensitization and subsequent downregulation of the HPG axis. This effect, observed in response to triptorelin, mirrors the fine-tuned hormonal fluctuations of a healthy menstrual cycle .Unlike the typical endocrine profile, individuals with PCOS exhibit a distinctive hormonal landscape marked by heightened androgen levels and irregular menstrual patterns. This specific endocrinological aberration characterizes PCOS. The role of triptorelin in this context is notably profound. While its primary mechanism entails the inhibition of LH and FSH release, it also engenders the normalization of these pivotal hormones. This restorative normalization can potentially reestablish ovulatory function and mitigate the hyperandrogenic symptoms observed in PCOS. This multifaceted triptorelin action directly addresses the disorder’s fundamental aspects , underscIndividuals grappling with HA present yet another complex endocrine scenario. HA is marked by disruptions in GnRH pulsatility, suppressing LH and FSH release, and ovulatory dysfunction. Triptorelin’s role in this population lies in its potential to restore proper GnRH pulsatility, a process crucial for reinstating regular menstrual cycles and fertility. By leveraging its ability to transiently stimulate LH and FSH release, triptorelin may facilitate the re-establishment of healthy hormonal rhythms, offering hope for HA individuals seeking to regain their reproductive health .Identifying Commonalities and Disparities in Triptorelin’s ImpactDelving into the effects of triptorelin across diverse populations, healthy individuals, those with PCOS, and individuals experiencing HA, unveils a complex tapestry of commonalities and disparities. This comparative analysis is a powerful lens to peer into the intricate mechanisms of triptorelin’s actions and extrapolate its clinical implications .Commonalities resonate at the core of triptorelin’s influence. The universal thread woven through these populations is the suppression of gonadotropin release, primarily LH and FSH. This suppression cascades downward, leading to decreased production of sex hormones, such as estrogen and progesterone. This shared effect underpins triptorelin’s utility as a modulator of endocrine balance, irrespective of the underlying health condition .Disparities emerge as distinct brushstrokes across the canvas of triptorelin’s impact. While the fundamental outcome, reduction in sex hormone levels, may be consistent, the nuances of this impact vary. In the context of PCOS, triptorelin offers the prospect of restoring ovulatory function milestones unattainable for healthy individuals. Furthermore, restoring proper hypothalamic function and subsequent GnRH pulsatility in HA is a unique hallmark of triptorelin’s potential .These disparities extend to the degree of suppression and the duration of effects. Healthy individuals may experience a transient yet significant gonadotropin surge, whereas the degree of suppression might be more pronounced in conditions characterized by excess hormone production, such as PCOS. Additionally, the duration of triptorelin’s effects could span from the duration of administration to more extended periods, presenting both therapeutic windows and challenges .The implications of identifying these commonalities and disparities are far-reaching. Researchers and clinicians gain insights into these effects’ underlying mechanisms by deciphering the intricate web of triptorelin’s actions. Moreover, this understanding lays the groundwork for developing targeted interventions. Tailoring triptorelin’s use to the specific needs of each group promotes ovulation in PCOS, restoring GnRH pulsatility in HA, or achieving controlled hyperstimulation in assisted reproduction revolutionizes endocrine management .Implications for Personalized Treatment ApproachesThe intricate analysis of triptorelin’s effects across distinct populations enhances our understanding of its mechanisms and lays the foundation for tailoring treatment strategies to individual health conditions. This paradigm shift toward personalized approaches holds significant implications for optimizing therapeutic outcomes .For healthy women seeking assisted reproduction, the controlled ovarian hyperstimulation induced by triptorelin becomes a strategic tool. By synchronizing follicular development and enhancing the chances of multiple viable oocytes, triptorelin offers a targeted approach to maximize the success of assisted reproductive techniques. This individualized intervention aligns with this group’s needs and goals, contributing to more effective fertility treatments .In PCOS, a condition characterized by complex hormonal imbalances, triptorelin’s potential to restore hormonal equilibrium and promote ovulation emerges as a beacon of hope. Tailoring treatment for PCOS patients using triptorelin addresses the root causes of the disorder, thereby improving menstrual regularity and reducing the detrimental effects of hyperandrogenism. This individualized approach in PCOS management enhances reproductive prospects and alleviates the associated metabolic and cosmetic concerns .Mechanisms of triptorelin action on endocrine profilesMechanism of Gonadotropin-Releasing Hormone AgonistsGnRH agonists, with triptorelin as a prominent example, constitute a class of pharmacological agents meticulously designed to intricately modulate the HPG axis. These synthetic analogs are strategically developed to leverage structural similarities with the native GnRH, the pivotal regulator of reproductive hormonal pathways. Notably, while these synthetic counterparts retain structural parallels, they exhibit divergent pharmacokinetic profiles, which underlie their distinctive impacts on the endocrine system . The nuaThe frequency and administration route of pulsatile stimulation in GnRH agonists are crucial to address. This mechanism involves a precise interplay between controlled suppression and pulsatile stimulation. These agonists are administered in a pulsatile manner by mimicking the natural pulsatile secretion pattern of GnRH in the hypothalamus. This carefully orchestrated approach initiates the activation of GnRH receptors, leading to a surge in the release of LH and FSH from the pituitary gland. This, in turn, replicates the physiological gonadotropin surge necessary for facilitating ovulation within a healthy menstrual cycle .However, the intrigue deepens when considering prolonged exposure to GnRH agonists in dosage and duration. Upon continuous administration, an intriguing paradox emerges, receptor desensitization. The sustained stimulation of GnRH receptors leads to their desensitization, reducing the pituitary gland’s responsiveness to GnRH agonists. This finely orchestrated process results in a gradual decrease in LH and FSH release over time. Given the pivotal roles of LH and FSH in driving gonadal activity, their dampened effects due to GnRH agonists ultimately suppress gonadal function .This controlled suppression and desensitization mechanism underpins the therapeutic potential of GnRH agonists such as triptorelin. Their capacity to temporarily mimic the physiological surge in gonadotropins, followed by the subsequent downregulation, allows for precise manipulation of endocrine profiles. This versatility has profound implications, from fertility enhancement in assisted reproduction techniques to ameliorating hormone-driven conditions such as hormone-sensitive cancers. Thus, the mechanism of GnRH agonists unveils a delicate balance between stimulation and suppression, presenting a strategic tool for modulating the intricate web of hormonal regulation within the HPG axis .Triptorelin’s Impact on Gonadotropin Release and Gonadal FunctionTriptorelin’s influence on gonadotropin release and gonadal function is a pivotal aspect of its mechanism of action. This influence is mediated through its intricate interaction with GnRH receptors in the hypothalamus and pituitary gland. Upon initial administration, triptorelin initiates a complex cascade of events that temporarily mirrors the natural pulses of GnRH secretion. This paradoxical effect rapidly increases LH and FSH, mimicking the characteristic preovulatory hormonal surge .However, this surge is not sustained. With the continuation of triptorelin administration, a shift occurs in the responsiveness of pituitary receptors. The prolonged exposure to triptorelin ultimately triggers the desensitization of these receptors. As a result, the pituitary gland enters a refractory state, rendering it less sensitive to GnRH signals. This, in turn, leads to a gradual reduction in LH and FSH secretion, ultimately dampening the ovarian response and suppressing gonadal activity .The controlled downregulation of the HPG axis detailed in this section bears profound therapeutic implications. The precision with which hormonal release can be modulated holds substantial promise across various clinical scenarios. Notably, with its ability to effectively lower gonadotropin levels, triptorelin assumes a pivotal role in treating hormone-dependent cancers. By diminishing the supply of sex hormones that drive the proliferation of these malignancies, triptorelin exerts a therapeutic effect that has been increasingly recognized. Furthermore, the controlled ovarian hyperstimulation facilitated by triptorelin’s transient surge of gonadotropins has a significant role in assisted reproduction. This process lays the groundwork for optimizing procedures such as oocyte retrieval and in vitro fertilization, as highlighted in prior research . Adding Modulation of Hormonal Signaling Pathways by TriptorelinTriptorelin’s impact on endocrine profiles extends beyond its effects on the HPG axis, encompassing a range of downstream hormonal signaling pathways. Notably, this peptide exerts its influence by meticulously modulating specific pathways, yielding diverse physiological outcomes. In the context of hormone-related disorders such as PCOS, triptorelin’s suppression of gonadotropin release and subsequent reduction in sex hormone levels play a pivotal role. This orchestrated intervention mitigates the repercussions of excessive hormone production commonly observed in such conditions. Moreover, triptorelin’s intricate adjustments to estrogen and progesterone levels carry systemic implications, reverberating through domains such as bone health, cardiovascular function, and metabolic equilibrium .Beyond its implications for reproductive health, triptorelin’s remarkable capacity to manipulate hormonal signaling pathways holds promise for broader therapeutic applications. An illustrative example lies in its potential to curtail the progression of hormone-sensitive cancers. By limiting the availability of hormones through suppressing gonadotropins, triptorelin emerges as a strategic contender in impeding cancer growth. Furthermore, triptorelin’s nuanced regulation of estrogen levels can yield therapeutic advantages in managing conditions intricately linked to hormone balance . This muFuture directions and implicationsUnanswered Questions and Research GapsDespite significant progress in understanding triptorelin’s effects on endocrine profiles, several questions and research gaps still need to be answered. One key area is the long-term impact of triptorelin use on bone health and cardiovascular function, particularly in populations prone to these concerns, such as women with HA. Additionally, optimal dosing regimens and treatment durations for specific conditions such as PCOS require further investigation. The interplay of triptorelin with other therapeutic interventions, such as lifestyle modifications and hormonal supplementation, also warrants exploration.Potential Applications Beyond Polycystic Ovary Syndrome and Hypothalamic AmenorrheaWhile triptorelin’s effects on PCOS and HA have garnered attention, its potential extends to other conditions involving endocrine dysregulation. For instance, in endometriosis, where hormonal imbalances contribute to symptomatology, triptorelin’s capacity to suppress gonadotropins could offer relief. Similarly, its role in managing hormone-sensitive cancers, such as breast or prostate cancer, is an area ripe for exploration. These potential applications highlight the versatility of triptorelin across a spectrum of endocrine-related conditions.Integrating Triptorelin Into Comprehensive Endocrine ManagementAs research unfolds, integrating triptorelin into comprehensive endocrine management strategies holds promise. Tailoring its use to individual patient profiles, including age, hormonal levels, and underlying conditions, is essential for optimizing outcomes. Combining triptorelin with other modalities, such as nutritional counseling, exercise regimens, or hormonal therapies, could yield synergistic effects, particularly in complex conditions such as PCOS and HA. Collaborative efforts between endocrinologists, reproductive specialists, oncologists, and other relevant disciplines will be instrumental in developing holistic treatment approaches.In conclusion, triptorelin emerges as a dynamic tool that can shape the intricate landscape of endocrine regulation. Its precise mechanism of transient stimulation and subsequent downregulation of the HPG axis provides a nuanced approach to manipulating hormonal balance. Triptorelin’s effects unfold uniquely across diverse populations, ranging from healthy individuals to those grappling with conditions such as PCOS and HA. This underscores the significance of tailoring treatment strategies to the specific health condition, accounting for the variations in endocrine responses. Numerous questions and research gaps beckon as we traverse uncharted territories, inviting exploration into triptorelin’s long-term implications, optimal dosing regimens, and potential applications beyond its current scope. The promise of triptorelin, however, is undeniable. It can potentially revolutionize women’s health by restoring ovulatory function, mitigating hormonal imbalances, and fostering a comprehensive approach to endocrine management. With every investigation, we move closer to unlocking triptorelin’s full potential and its role in reshaping the landscape of hormonal equilibrium, thereby elevating the realms of reproductive health and well-being for women.


One of the main drawbacks faced by the dental implant surgeons is to assess the healing of the tissues and implant success for patients who are smokers. It is of interest to evaluate inflammatory biomarkers to understand the soft and hard tissuehealing between smokers and non-smokers based on levels of IL-6 and STAT-3. This study included totally 20 patients (Group 1 : smokers (n=10) and Group 2: non-smokers (n=10)) undergoing stage-1 implant surgery and collected a tissue sample for thepatients to assess the levels of IL-6 and STAT-3. The results indicated that there is a pronounced increase in the biomarkers in patients who are smokers in comparison to non-smokers. Dental implants are artificial tooth roots that are surgically placed into the jawbone to provide a foundation for replacement teeth . They arTissue sample collection of smokers and non-smokers: Tissue samples are harvested during implant stage 1 surgery  using ti[1] Homogenization or lysis of the sample to release the RNA and other cellular components ,15. [2] Addition of a denaturant or chaotropic agent, which disrupts the cellular structure and facilitates RNA binding to a silica-based column or resin. [3] Binding of the RNA to the column or resin, and removal of other cellular components by washing with various buffers. [4] Elution of the RNA from the column or resin in a low-salt buffer, and quantification of the RNA using a spectrophotometer or fluorometer. The Nanodrop instrument passes a beam of UV-Vis light through the RNA sample, which causes the RNA molecules to absorb the light in a characteristic manner. The amount of light absorbed by the RNA molecules is directly proportional to theirconcentration in the sample. The Nanodrop software uses this information to calculate the concentration of RNA in the sample, which is reported in units of ng/µl. In addition to quantifying RNA concentration, Nanodrop can also measure thepurity of the RNA sample by calculating the A260/A280 ratio. This ratio is an indicator of the presence of contaminants, such as proteins or other organic compounds, in the RNA sample.Thermal cycler is a commonly used technique in medical research to convert RNA molecules into complementary DNA (cDNA) molecules. The process involves the reverse transcription of RNA into cDNA using a thermal cycler machine. Here are the generalsteps involved in cDNA synthesis by thermal cycler .RNA isolation: The RNA is first isolated from the biological sample using standard RNA extraction protocols. Reverse transcription reaction: The RNA is then converted into cDNA using a reverse transcriptase enzyme and primers that are specific tothe gene of interest. The reaction mix contains reverse transcriptase enzyme, random hexamer primers or gene-specific primers, and nucleotides. The mixture is incubated at an appropriate temperature and time to allow the reverse transcription to occur.PCR amplification: After cDNA synthesis, PCR amplification is performed using specific primers to amplify the gene of interest. The PCR reaction is carried out using a thermal cycler machine which allows for temperature control and specific cyclingconditions that are optimized for the primers and the PCR enzyme .The qRT-PCR reaction is carried out using a thermal cycler machine, which allows for temperature control and specific cycling conditions. The reaction mix contains the cDNA template, gene-specific primers, fluorescent probes, and a qPCR enzyme.The reaction mixture is amplified by cycling through different temperatures, and the fluorescent signal is monitored in real-time. Data analysis: The data obtained from qRT-PCR is analyzed using various software programs that allow for the quantificationof the amount of RNA transcript present in the sample ,Figure 3The results of samples collected show high risk in smokers compared to non-smokers. Smoking has been known to have detrimental effects on oral health, including the success rate of dental implants. Recent studies have shown that smokers have higherlevels of inflammatory biomarkers, such as STAT-3 and IL-6, compared to non-smokers . These iPeri-implantitis is a serious condition that can lead to implant failure and significant patient morbidity . Recent


ABCB1 locus in humans), has taken center stage as a novel player in immune regulation. Although MDR1 remains widely viewed as a simple drug efflux pump in tumor cells, recent evidence suggests that this transporter fills key endogenous roles in enforcing metabolic fitness of activated CD4 and CD8 T cells. Here, we summarize current understanding of the physiological functions of ABC transporters in immune regulation, with a focus on the anti-oxidant functions of MDR1 that may shape both the magnitude and repertoires of antigen-specific effector and memory T cell compartments. While much remains to be learned about the functions of ABC transporters in immunobiology, it is already clear that they represent fertile new ground, both for the definition of novel immunometabolic pathways, and for the discovery of new drug targets that could be leveraged to optimize immune responses to vaccines and cancer immunotherapies.Nearly 50 ATP-binding cassette (ABC) transporters are encoded by mammalian genomes. These transporters are characterized by conserved nucleotide-binding and hydrolysis  domains, and power directional transport of diverse substrate classes – ions, small molecule metabolites, xenobiotics, hydrophobic drugs, and even polypeptides – into or out of cells or subcellular organelles. Although immunological functions of ABC transporters are only beginning to be unraveled, emerging literature suggests these proteins have under-appreciated roles in the development and function of T lymphocytes, including many of the key effector, memory and regulatory subsets that arise during responses to infection, inflammation or cancers. One transporter in particular, MDR1 (Multidrug resistance-1; encoded by the  Among many notable discoveries, it is now understood that the activation and differentiation of immune cells – including T cells – involves rapid and profound metabolic reprogramming. On one hand, antigen- and TCR-mediated increases in aerobic glycolysis and mitochondrial respiration allows primed T cells to meet the bioenergetic and biosynthetic demands of cell growth and proliferation. On the other, differences in the magnitude and type of metabolic activities an antigen-primed T cell undertakes can play instructive roles in the commitment of emergent effector, memory and/or regulatory T cell subsets –5. CD8 eFlux through growth-supporting metabolic pathways requires active transport of numerous organic and inorganic molecules across biological membranes. Adenosine triphosphate (ATP)-binding cassette (ABC) transporters constitute one of the largest super-families of transmembrane proteins encoded by the human genome. These transporters utilize ATP hydrolysis to power directional translocation of diverse substrates, against chemical gradients, and across lipid membranes, including the plasma membrane and membranes of intracellular organelles . ABC traMore recent studies have begun to highlight direct, important and endogenous functions of ABC transporters in adaptive immune regulation generally, and development and function of T cells specifically –25. Here248 human genes encode ABC transport proteins, most of which have direct orthologs in mice and lower vertebrates. In the early 2000’s, ABC transporters were renamed and reclassified into seven sub-families (ABCA to ABCG), based on phylogenetic analysis and sequence/structural similarity , 26–28. X-ray crystallographic studies of several ABC transporters have provided atomic-level resolution of the canonical structure of ABC transporters, and specifically the organization of four main functional domains — two nucleotide binding domains  and two transmembrane domains . Most eukaryotic ABC transporters are expressed as either a single polypeptide containing all four functional domains, or as half-transporters capable of homo- or hetero-dimerization . The NBD3a.k.a., cystic fibrosis transmembrane conductance regulator (CFTR)], which functions as an apical chloride channel on lung epithelium, and whose loss-of-function leads to chronic bacterial infections of the lung (The first described and arguably most famous ABC transporters within the immune system are the transporters associated with antigen processing (TAP1/2), which transport cytosolic peptides into the endoplasmic reticulum (ER) for loading onto MHC Class I molecules . Anotherthe lung . Howeverthe lung , 24. In the lung Table 1.3.1As many as 20 human ABC transporters are thought to transport discrete lipids or lipid metabolites. Among these, ABCA1 and ABCG1 are perhaps the best characterized for the roles they play in cholesterol transport and homeostasis, and for regulating TCR signaling intensity , 25, 37.-/-Abcg1 mice display larger thymuses than wild-type controls due to thymocyte hyperproliferation and an increase in total thymic cellularity , reduces T cell proliferation  are incapable of becoming productively activated, of accumulating efficiently in response to acute viral or bacterial infections, and of forming functional memory cells , which upon being emptied, activate extracellular Ca2+ influx across the plasma membrane  (2•−) that “leak” from complexes I and III of the mitochondrial electron transport chain (ETC)  . This inin (ETC) .ROS function as key secondary messengers in T cells and are required, at moderate levels, to promote proliferation, differentiation and survival. The activation of extracellular signal-regulated kinase (ERK) 1/2, for example, is highly ROS-dependent, and promotes activation and translocation of several transcription factors, including AP-1 family members, that are necessary for T cell growth and proliferation , 63. ROSGpx4 impairs CD8 T cell responses to viral infections due to accumulation of lipid peroxides, leading to death by ferroptosis , catalases, glutathione peroxidases (GPXs), glutathione reductases, etc.) to convert ROS into less reactive products, or to increase production of ROS scavenging molecules, such as glutathione, ascorbate, pyruvate, α-ketoglutarate, and oxaloacetate . Disruptroptosis . Thus, p5Considering that both the developmental trajectories and functions of individual T cell clones involve unique TCR signaling dynamics, and thus discrete redox demands, it becomes increasingly important to decipher whether and how ABC transporters not only regulate the magnitude of monoclonal  T cell responses, but also the selection of T cell clones specific for high- vs. low-affinity antigens. Given the preponderance of available evidence, it is reasonable to expect that MDR1, and perhaps other lipid and multidrug transporters, preferentially regulate T cell responses to abundant and high-affinity  antigens, in which the highest levels of intracellular ROS are generated. If true, these pathways (and ABC transporters) may prove vital for advancing the next generation of medicines that can improve T cell-mediated immunity to infectious diseases and cancers, where the survival of T cells specific for immunodominant antigens are paramount for efficacy.in vitro studies, but it is becoming increasingly apparent that these transporters have important and context-dependent functions that need to be evaluated in vivo, during both normal- and patho-physiology. This is especially true for transporters, like MDR1, which remain classified as ‘multi-drug’ efflux pumps, despite continued elaboration of new and potent endogenous functions in in vivo mouse and ex vivo human systems. For these, the key unanswered question is why would an ABC transporter be conserved throughout evolution unless it serves a core endogenous function? The continued pursuit of basic science, and the advancement of next-generation medicines, await answers.Despite being one of the largest families of human transmembrane proteins, a comprehensive understanding of endogenous ABC transporter functions remains lacking. Historically, this has been due to the inherent difficulty of working with membrane proteins and transporters, as well as a dearth of contemporary research reagents . While significant strides have been made in recent decades with regard to the structural organization of these transporters, meaningful insights into the physiological and immunological functions of these transporters have lagged. Mechanistic understanding of these transporters remains largely based on data from pharmacological and MS: Conceptualization, Funding acquisition, Supervision, Writing – review & editing. AB: Conceptualization, Writing – original draft, Writing – review & editing.