The Sonic hedgehog (Shh) signaling pathway is an essential pathway in the human body that plays an important role in embryogenesis and tissue homeostasis. Aberrant activation of this pathway has been linked to the development of different diseases, ranging from cancer to immune dysregulation and infections.Uncontrolled activation of the pathway through sporadic mutations or other mechanisms is associated with cancer development and progression in various malignancies, such as basal cell carcinoma, medulloblastoma, pancreatic cancer, breast cancer and small-cell lung carcinoma. Targeted inhibition of the pathway components has therefore emerged as an attractive and validated therapeutic strategy for the treatment of a wide range of cancers. Currently, two main components of the pathway, the smoothened receptor and the glioma-associated oncogene homolog transcriptional factors, have been investigated for the development of targeted drugs, leading to the marketing authorization of three smoothened receptor inhibitors for the treatment of basal cell carcinoma and acute myeloid leukemia.The Shh pathway also seems to be involved in regulating the immune response, possibly playing a role in immune system evasions by tumors, development of autoimmune diseases, such as rheumatoid arthritis and Crohn’s disease, airway inflammation, and diseases related to aberrant activation of T-helper 2 cellular response, such as allergy, atopic dermatitis, and asthma.Finally, the Shh pathway is involved in pathogen-mediated infection, including influenza-A and, more recently, SARS-CoV-2 viruses. Therefore, agents that inhibit the Shh signaling pathway might be used to treat pathogenic infections, shifting the therapeutic approach from strain-specific treatments to host-based strategies that target highly conserved host targets. The Sonic hedgehog (Shh) signaling pathway plays a key role in regulating the growth, differentiation and proliferation of several cells and tissues.The Shh pathway is highly complex; it involves multiple effectors and regulators and experiences central compartmentalized coordination in the primary cilium.12Both SMO and the GLI family of zinc finger transcription factors are regarded as important targets for cancer therapy. To date, two SMO receptor inhibitors (sonidegib and vismodegib) have received Food and Drug Administration (FDA) and European Medicines Agency (EMA) approval for treating basal cell carcinoma4–6While most efforts have been devoted to pharmacologically targeting SMO, developing the GLI-targeted approach also has its merit. GLI proteins can be activated by both Shh ligand-dependent and Shh ligand-independent mechanisms. Moreover, it has recently been recognized that partial inhibition of multiple targets is more productive than a single hit, as it may reduce the chances of developing drug resistance.15The development of agents targeting other components of the pathway has proven challenging so far. For example, the upstream protein Shh, which is overexpressed in many cancers, has been considered a promising antitumor target, but only a few small molecules have been identified that can disrupt the Shh-Patched 1 protein (Shh-PTCH) interaction.14 16 17Evidence has recently emerged that Shh signaling is also involved in the inflammatory and immune responses of different diseases. Some pathogens use this pathway to control the local infected environment. Among the human inflammatory diseases where Shh pathway is considered to play a role are an influenza-A infection, cigarette-induced airway inflammation, arthritis, inflammatory bowel diseases, pancreatitis, colitis-associated cancer, asthma, allergy, atopic dermatitis and T-cell polarization.1 2 18–20This commentary will examine the current knowledge on the pathophysiological interactions of Shh signaling that make this pathway a potential target for future treatments in a wide range of human diseases besides various cancers.The Shh signaling pathway plays an important role in normal embryonic tissue development by modulating the epithelial–mesenchymal interactions, which regulate cell proliferation and differentiation.Aberrant activation of the Shh/GLI signaling pathway by various mechanisms may be responsible for the development of various human cancers. Targeting Shh/GLI remains a promising treatment strategy with the potential for curative effects by eradicating cancer stem cells involved in tumor initiation, metastasis and drug resistance. Upregulation of the Shh ligand in autocrine or paracrine fashion has been reported in pancreatic, gastrointestinal, breast, lung, brain and prostate cancers.3–6The Shh/GLI signaling modulates inflammation in cancer by several mechanisms, as summarized in Box 1Cancer cells release CCL2/3 in response to oncogenic Shh/GLI signallingsignaling, thereby recruiting tumor-associated macrophages (TAMs) and immunosuppressive myeloid-derived suppressor cells.Shh/GLI-induced PD-L1 expression in cancer and dendritic cells inhibits tumor specific cytotoxic T-cells via binding to PD-1.GLI2 drives production of immunosuppressive cytokines and growth factors (IL-10 and transforming growth factor-β), which results in the inactivation of tumor-specific CD8 +T cells.Shh/GLI-induced IL-10 from stromal cells promotes FoxP3 expression in regulatory T-cells.Proinflammatory signals such as IL-6/signal transducer and activator of transcription 3 (STAT3) interact with Shh/GLI signallingsignaling; Shh/GLI-induced autocrine IL-6 signallingsignaling and/or proinflammatory IL-6 from TAM and stromal cells activate STAT3 signallingsignaling in cancer cells, thereby promoting malignant growth .In basal-cell carcinoma, the interaction of Shh/GLI and proinflammatory IL-6/STAT3 signaling synergistically regulates common GLI-STAT3 target genes and promotes cancer proliferation.On the one hand, the activation of Shh/GLI generates an immune-suppressive response through the release of immune-suppressive molecules or by activating specific anti-inflammatory pathways in cancer and immune cells. Shh/GLI induces, for example, the expression of CCL-2/3 on cancer cells, thus recruiting tumor-associated macrophages (TAMs) and immunosuppressive myeloid-derived suppressor cells to the cancer lesion. Moreover, it stimulates cancer cells and dendritic cells to produce PD-L1, which inhibits tumor-specific cytotoxic T cells (CD8+) via the PD-1 receptor. Furthermore, the release of immune-suppressive cytokines and growth factors, such as interleukin-(IL)-10 and transforming growth factor-beta reduces the number of effector lymphocytes in the tumor tissue and enhance the polarization of T cells towards the formation of regulatory T-cell (Treg) formation and an anti-inflammatory environment.4On the other hand, Shh/GLI can stimulate proinflammatory signals, which have been recognized as a potent promoter and enabler of malignant development when the activation of the immune system is persistent and inappropriate. Several studies have shown that during malignant development, Shh/GLI signaling and proinflammatory pathways, such as IL-6/signal transducer and activator of transcription-3 (STAT3) have reciprocal regulatory interactions, including tumor-promoting synergistic signal integration processes.4Shh/GLI-induced autocrine IL-6 signaling and/or proinflammatory IL-6 from TAM and stromal cells activate STAT3 signaling in cancer cells, thereby promoting malignant growth .GLI/STAT3 target genes and promotes cancer proliferation.7 8Moreover, in basal cell carcinoma, the interaction of Shh/GLI and proinflammatory IL-6/STAT3 signaling synergistically regulate common The importance of the Shh/GLI signaling in oncogenic development and immune cross-talk and modulation makes this pathway a promising target for new anticancer therapeutic strategies.In a rat model of colitis-associated cancer and human colon cancer cells, Shh inhibitors suppressed TNF-α-induced inflammatory signaling, especially IL-6/IL-6R/gp130 pathway.The signaling cascade identifies the transcription factor GLI1 as a central mediator of the IL-6 signaling network initiated in the tumor microenvironment, regulating the progression of pancreatic precursor lesions and tumor formation. When GLI1 is absent, IL-6 signaling from tumor-associated fibroblasts diminishes, and pancreatic precursor lesions do not progress to advanced stages.22Inhibition of Shh signaling pathway with small molecules targeting the smoothened (SMO) can successfully inhibit the growth of various tumors.29et al discovered that SMO is involved in T-cell activation and that administration of SMO inhibitors results in functional disruption of the immunological synapse and consequently loss of T-cell effector activity.4Despite the promising results obtained by Shh pathway inhibitors, some challenges and concerns have emerged regarding the use of Shh inhibitors as immune modulators; given the complex and opposite effects, this pathway may have in terms of proinflammatory and anti-inflammatory signals. Studies in mice showed the importance of the anti-inflammatory cytokine IL-10 and Treg expression, mediated by Shh/GLI, in dampening the inflammation and preventing inflammatory damage in pancreatitis and colitis.Given the complexity and diverse effects of the Shh/GLI pathway on the immune microenvironment of malignant and non-malignant tissue, it is crucial to understand how deregulation of this signaling axis precisely alters antitumor immunity and tumor-promoting inflammation in order to support the development of more sophisticated tumor therapies.NS1, the authors speculated that the hastened lethality caused by the mutant virus might be due, in part, to a Shh-dependent over-production of cytokines , which have been considered the cause of past influenza pandemics -γ, IL-1, IL-6, TNF-α, and IL-18. The beneficial effects of specific monoclonal antibodies in reducing symptoms and improving organ functions have confirmed the key role of excessive cytokine production.35The cytokine storm is considered the main mechanism underlying the devastating effects of SARS-CoV-2 pandemic named COVID-19.One of the earliest therapies targeting cytokine storm in COVID-19 patients was the anti-IL-6 receptor monoclonal antibody tocilizumab.Box 2Regulates immunosuppressive mechanisms, such as enhanced regulatory T-cell formation and production of immunosuppressive cytokines.In several cancer entities, aberrantly activated HH/GLI signaling drives tumor proliferation and growth, while simultaneously dampening inflammation and favors immunosuppression.IL-6 synergizes with HH/GLI in oncogenic transformation.The interaction of HH/GLI and proinflammatory IL-6/signal transducer and activator of transcription-3 (STAT3) signaling synergistically regulates common GLI–STAT3 target genes and promotes cancer promotion.Intestinal epithelial cells TNF-α stimulated IEC-6 cells exhibit increased levels of proinflammatory cytokines, whereas SHH inhibitors suppress TNF-α-induced inflammatory signaling, especially IL-6/IL-6R/gp130 pathway.SHH inhibitors significantly reduce tumor incidence and multiplicity, decrease the expression of IL-6, TNF-α, COX-2, STAT3 and NF-κB, and significantly induce apoptosis.Reduce paw swelling, serum levels of TNF-α, IL-1β, IL-6 and histological scores of joint damage in rats.Promotes cartilage extracellular matrix production with a potential clinical interest in rheumatoid arthritis treatment.The HH pathway inhibitor cyclopamine attenuates the nicotine-induced increase in IL-6, IL-8 and TNF-α, while IL-10 levels increase, suggesting that the HH signaling pathway may partly contribute to cigarette-induced airway inflammation through the regulation of inflammatory mediators.Plays a key role in cigarette-induced airway inflammation, via the regulation of inflammatory mediators.The hedgehog pathway seems to be involved in various diseases such as autoimmune diseases and airway inflammation.H1) or T helper 2 (TH2) or inducing cellular anergy.H2 differentiation of human CD4 +cells, thus suggesting that Shh signaling may be involved in the etiology of diseases mainly driven by this response, such as allergy, atopic dermatitis and asthma.19Different components of the Shh pathway are expressed in the thymus and seem to be involved in multiple stages of T-cell development. In fact, Shh signaling influences the differentiation and proliferation of early thymocyte progenitors and plays a role in repertoire selection, clonal deletion of autoreactive cells and T-cell peripheral activation. Moreover, hedgehog proteins modulate T-cell receptor signaling, influencing T-cell differentiation into T helper 1 (TSome studies also suggest the possible involvement of Hh signaling in autoimmune disorders such as rheumatoid arthritis and Crohn’s disease. Cyclopamine, a Hh inhibitor, attenuated inflammation and cartilage damage of adjuvant‐induced arthritis rats, as evidenced by reduced paw swelling, serum levels of TNF-α, IL-1β, IL-6 and histological scores of joint damage. GLI1 mRNA levels correlated negatively with type-II collagen and aggrecan mRNA levels, suggesting Hh signal inhibition was associated with promoting cartilage extracellular matrix production. These data suggest that inhibition of Hh signaling pathway might be of potential clinical interest in rheumatoid arthritis treatment.41In Crohn’s disease, Hh signaling provides negative feedback to the lamina propria, down-regulating inflammatory cytokines and inhibiting leukocyte migration and fibroblast proliferation while favoring fibroblast migration. Therefore, Hh signaling appears to be strongly implicated in the pathogenesis of intestinal inflammation, thus representing a novel therapeutic target in inflammatory bowel diseases.42Finally, in a study of human alveolar epithelial cells,Dysregulation of Shh/GLI signaling plays fundamental yet distinct roles in cancer and various chronic inflammatory diseases by exerting complex and diverse effects on the immune microenvironment of malignant and non-malignant tissues. Understanding the molecular rationale of how deregulation of the Shh/GLI signaling axis precisely alters immunity and inflammation will support the development of more sophisticated therapies. Shh signaling has also been shown to be a target for some pathogens that presumably use the pathway to control the local infected environment, thus justifying therapeutic uses of approved molecules that inhibit Hh signaling be expanded to treat pathogenic infections. In contrast to the currently available therapies, such as vaccines and antivirals, which target strain-specific and rapidly mutating viral proteins, treatments that target highly conserved host targets may ultimately provide superior and continual protection across a broader spectrum of strains. The emerging knowledge on the broad effects of the Shh signaling pathway may represent a rationale for their evaluation as a potential treatment of COVID-19.


Human T cell leukemia virus type 1 (HTLV-1) was identified as the first pathogenic human retrovirus and is estimated to infect 5 to 10 million individuals worldwide. Unlike other retroviruses, there is no effective therapy to prevent the onset of the most alarming diseases caused by HTLV-1, and the more severe cases manifest as the malignant phenotype of adult T cell leukemia (ATL). MicroRNA (miRNA) dysfunction is a common feature of leukemogenesis, and it is no different in ATL cases. Therefore, we sought to analyze studies that reported deregulated miRNA expression in HTLV-1 infected cells and patients’ samples to understand how this deregulation could induce malignancy. Through in silico analysis, we identified 12 miRNAs that stood out in the prediction of targets, and we performed functional annotation of the genes linked to these 12 miRNAs that appeared to have a major biological interaction. A total of 90 genes were enriched in 14 KEGG pathways with significant values, including TP53, WNT, MAPK, TGF-β, and Ras signaling pathways. These miRNAs and gene interactions are discussed in further detail for elucidation of how they may act as probable drivers for ATL onset, and while our data provide solid starting points for comprehension of miRNAs’ roles in HTLV-1 infection, continuous effort in oncologic research is still needed to improve our understanding of HTLV-1 induced leukemia. In 1979, human T cell leukemia virus type 1 (HTLV-1) was identified as the first pathogenic human retrovirus ,3. The SBrazil is possibly the country with the highest absolute number of HTLV cases in the world, although Japan is the country with the highest prevalence of HTLV-1 infection. Estimates from the Brazilian Ministry of Health indicate that between 700,000 and 2 million people are infected, and although the virus has been among the infectious agents in the country’s blood centers since 1993, there is no specific national policy for HTLV. Most carriers are asymptomatic, and there are more case reports of HTLV-2 infections in people who inject drugs and in the indigenous population ,5,6,7.Unlike other retroviruses, such as the Human Immunodeficiency Virus (HIV), there is no effective therapy to prevent the onset of the most alarming diseases caused by HTLV-1 . Outcome+ T cells. It is a retrovirus of the deltaretrovirus genus, and several subtypes of HTLV were discovered later—HTLV-2, HTLV-3, and HTLV-4. It has a structural organization like all retroviruses; two mature viral glycoproteins derived from a common precursor, an outer surface protein (SU) associated with a transmembrane (TM) protein, which is responsible for anchoring the SU-TM complex on the surface of the infected cell, an envelope consisting of a lipid bilayer of cellular origin, and the matrix protein located next to the membrane [HTLV-1 is the causative agent of ATL, a malignant tumor of CD4membrane ,11,12,13gag, pol, and env, and two regulatory genes, tax and rex. The tax and rex genes regulate the transactivation of viral replication, in addition to regulating the expression of viral proteins and the basic leucine zipper factor (HBZ) of HTLV-1, which are essential for the maintenance of viral persistence and pathogenesis, in addition to helping in the oncogenic process of ATL, stimulation of tumor growth and development [It is an enveloped double-stranded RNA virus, and its genome contains three structural genes: elopment ,13,14,15Viral integration into the host’s DNA happens preferably at transcriptionally active regions, specifically near transcription factor binding sites. HTLV-1 uses different strategies to induce neoplastic transformation, but the immunogenic profile of the host is associated with inflammatory responses, promoting or protecting against the development of HAM or ATL. Its replication occurs parallel to the cell during mitosis, and its infection can occur vertically, during childbirth or breastfeeding, or parenterally, which consists of transfusions, transplants, intravenous drug use, and unprotected sexual intercourse ,16,17,18Furthermore, HTLV infects mainly T lymphocytes but is also capable of infecting monocytes and dendritic cells due to its direct interaction with glucose transporter 1 (GLUT 1), which is ubiquitously expressed on cell surfaces  18,19,2,21.After infection and entry into the cell, viral RNA is reverse transcribed into viral DNA or provirus. After that, the virus can integrate into the host cell genome and exploit normal cell physiology. Viral integrase recognizes and binds to viral long terminal repeats (LTRs) in viral DNA, forming a pre-integration complex (PIC). PIC associates with the host enzyme, protein phosphatase 2A, and engages with host DNA in regions of open chromatin. In addition, HTLV-1 increases genomic instability by direct actions on the DNA, and, in turn, genomic instability can also alter the proviral genome that is often mutated or deleted, thus creating an escape from the host’s immune system ,24,25,26During infection, there is viral transmission by cell–cell contact due to an organizing center of polarized microtubules at the cell–cell junction and a virological synapse triggered by Tax protein. From there, the HTLV-1 Gag complex, viral RNAs, and enveloped HTLV-1 virions accumulate at the synapse and migrate into the uninfected cell ,28,29. AFirst reported in Japan in 1977, ATL is a lymphoproliferative disease that presents as an oligoclonal or monoclonal expansion of HTLV-1-infected T cells that occurs decades after infection. ATL onset is multifactorial, involving factors related to the virus and the host’s immune and inflammatory responses ,15.The disease is classified according to clinical characteristics into acute, chronic, lymphoma-like, and smoldering. Its manifestations include malaise, fever, weight loss, jaundice, skin lesions, adenomegaly, hypercalcemia, elevation of lactate dehydrogenase, and clinical alterations in the number of leukocytes in peripheral blood, with the presence of atypical lymphocytes ,30,31.In the acute phase, it is also possible to find lymphocytes with peculiar characteristics called floral cells in the peripheral blood smear, which is a characteristic clinical finding of ATL development. The diagnosis for HTLV-1 infection is performed by ELISA and confirmed by PCR and Western blot, and a worse prognosis may be expected in the acute and lymphoma forms, being more aggressive and with a median survival of one year ,27,32.ATL cells’ clonal expansion is promoted by the accessory proteins of HTLV-1 and the Tax protein. Tax utilizes several mechanisms for cellular transformation, including the creation of chromosomal instability, amplification of centrosomes, abrogation of DNA repair, activation of cyclin-dependent kinases and nuclear factor-kB (NF-kB), and AKT signaling, and even silencing of TP53 checkpoints. The maintenance of the ATL transformation occurs through HBZ protein activity, in addition to this protein being essential for the establishment of persistent viral infection ,28,33,34The molecular mechanisms of HTLV-1-mediated transformation and carcinogenesis are still unknown; however, there are studies that correlate HTLV carcinogenesis to microRNAs (miRNAs), which are endogenous 18–25 nucleotide long RNAs that play regulatory roles in cell metabolism, directing messenger RNAs (mRNA) for cleavage or translational repression ,36. TheyMiRNAs genes can be located within the introns and exons of protein-coding genes or in intergenic regions and are transcribed by RNA Polymerase II and processed by the Drosha and Dicer enzymes ,39,40. DDiscovering miRNAs, identifying their targets, and clarifying their functions has been a critical strategy for understanding normal biological processes and their roles in disease development . FurtherFor this reason, in this study, we sought to identify in previously published works which miRNAs were altered in ATL; in addition, we performed bioinformatics analysis in order to describe possible pathways of malignant transformation and biological interactions to predict the role of these miRNAs in HTLV-1-mediated leukemogenesis. The results are presented below in p > 0.95) for binding probability [In this study, a list of 42 miRNAs, pointed out in the studies above, was input to the miRWalk algorithm (version 3.0), and for prediction of individual target genes using TargetScan, (version 7.2), miRDB (version 6.0), and miRTarBase (version 8.0), generating three unique files for further analysis ,60,61. Tbability , preferebability .After that, we simulated miRNA-gene interaction networks that were performed with Cytoscape (version 3.9.1). The data generated by the prediction of the three files obtained with miRWalk were used to construct the three respective miRNA-gene interaction networks. Then, the three networks were merged according to Cytoscape’s manual merge procedure with an intersection operator to obtain a single miRNA-gene interaction network .p-value) < 0.05. An interactive network was built with Cytoscape to show all target genes enriched in KEGG pathways. The topologies of all networks were constructed with network density < 0.05.Lastly, we performed functional annotation of the genes linked to 12 miRNAs that showed a significant biological interaction between all described in the studies, using DAVID   to identMost miRNAs described in these studies were mentioned only once, and a correlation between authors and cited miRNAs can be found in The results of the bioinformatics analysis are shown in the figure below . The tarThere was a significant enrichment of miRNA target genes in important KEGG pathways for cancer, such as hsa05200: Pathways in cancer; hsa04310: WNT signaling pathway; hsa04010: MAPK signaling pathway; hsa04350: TGF-β signaling pathway; hsa04014: Ras signaling pathway, all shown at central interaction network in TP53 as a trending gene, appearing as part of most of the identified significantly deregulated pathways.While the deregulation of any signaling pathways is sure to disrupt cell metabolic homeostasis, some are more relevant for leukemogenesis than others and will be discussed further in this paper. Of importance to note is the presence of TP53 tumor suppressor gene is a very common genetic lesion in human cancer, so much so that the inheritance of a germline mutation in TP53 generates increased risks of developing neoplasms. The reason for this well-reported carcinogenesis is the tumor suppression functions performed by the protein that is commonly called the guardian of the genome, as it protects DNA integrity [The loss or mutation of the ntegrity ,68,69.TP53 gene is mutated in about 50% of human cancers, but there is also a biological inactivation of its pathway in the others. It can be altered directly or by targeting the oncoproteins MDM4 regulator of p53 (MDM4) and MDM2 proto-oncogene (MDM2) that regulate TP53 activity, thus being altered indirectly.According to Borrero and El-Deiry , the TP5In response to stress factors such as DNA damage, hypoxia, UV irradiation, oxidative free radicals, oncogenes, or growth factor deficiencies, the TP53 protein is activated and can modulate target genes that regulate cell cycle arrest, apoptosis, DNA repair, angiogenesis, and metastasis ,69,70,71TP53 importance in carcinogenesis is a well-established concept, and several different miRNAs have been reported to silence TP53 expression directly or indirectly, while TP53 also regulates miRNAs expression, making it clear that this regulation can be associated with malignant changes [ changes ,70. In A changes ,73.TP53 activity. There is, however, controversy when determining miRNA expression levels in HTLV-1 infection, with the former reporting an upregulated expression of miR-150-5p levels in HTLV-1 asymptomatic carriers (ACs) when compared to healthy controls (HC) and ATL patients, and the latter reporting a downregulation of miR-150-5p in HTLV-1 infected cells when compared to HC.Studies by Nascimento et al.  and RuggTP53 mutational profile. TP53 status as a tumor suppressor may be easily overridden even by single-nucleotide mutations that are able to induce gain of oncogenic functions (GOF) for TP53 protein and dominant-negative effects (DNE) over wild-type TP53. Oversimplifying the past statements, downregulating TP53 may actually be beneficial for the proper regulation of cell homeostasis if its mutations are inducing an oncogenic-protein phenotype and disrupting its physiological regulating activities [It is important to note that, while the downregulation of a miRNA that interferes with TP53 activity may seem beneficial for proper cell cycle control, the modulation of TP53 and ATL onset may also be correlated with tivities ,75.TP53, overexpression of miRNAs modulating genetic GOFs and DNEs is what keeps leukemogenesis in check [This difference in miRNA expression levels may be representative of proliferation and survival advantages that cells obtain in in vivo models during disease development that are not seen when compared to HTLV-1 infected cell lines in vitro. Nascimento et al.  describein check ,26,46.TP53 mutations and rearrangements are important prognostic factors and major predictors of response to treatment in ATL patients [Furthermore, the reported downregulation of miR-150-5p in HTLV-1 infected cell lines compared to samples of HC by Ruggero et al.  only corpatients ,77.TP53, miR-150-5-p validated targets include other proto-oncogenes, such as c-Myb and the NOTCH3 gene [c-Myb gene, which encodes transcription factors that regulate cell proliferation, differentiation, and apoptosis, is found to be overexpressed in a wide range of human cancers, and leukemia studies have associated its expression with a worse prognosis and its transcript physically binds to TP53 [Beyond CH3 gene . The c-M to TP53 ,79.NOTCH3 pathway, which regulates cell proliferation, differentiation, cell fate determination, and stem/progenitor cell self-renewal in adult and embryonic organs. Elevated expression is increasingly documented in cancers and is correlated with rapid malignant progression, abnormal differentiation, metastasis, and worse prognosis. There is also a known interaction of NOTCH signaling with other major signaling pathways such as WNT and TGF-β [Another validated target of miR-150-5p is the nd TGF-β ,81.2+ pathway that has Wnt5a as the main effector. This pathway mainly plays a pro-tumor role, and WNT/β-catenin signaling is activated in many types of cancer. Its primary receptors are frizzleds (FZD), which are transmembrane G-coupled proteins. WNT signaling is inhibited by endogenous inhibitors and secreted FZD-related proteins (sFRPs) that interact directly with WNTs [The WNT signaling pathway is associated with cell differentiation, polarization, and migration during development and is fundamental in cell growth control. Its main component is the family of WNT proteins that activate cell membrane receptors in a paracrine and autocrine manner. It is based on three signaling pathways: WNT canonical signaling pathway (CP), which is β-catenin-dependent, and two WNT non-CP that are β-catenin-independent: planar cell polarity (PCP) signaling pathway and a WNT/Caith WNTs ,83,84.2+ for tumorigenesis development while inhibiting the WNT CP, which seems to have inhibitory effects on ATL cell growth through HBZ mediated activity. The miRNA expression profiles identified in our analysis support these observations and corroborate an excessive signaling of Wnt5a.However, Ma et al.  previousAn overactivation of Wnt5a may be observed in ATL cells mediated by increased expression of c-myb, which is expected to happen at ATL onset after downregulation of miR-150-5p expression ,51,86. AOther miRNAs that we identified as interacting with the WNT pathway, however, are much more associated with HTLV-1 capability of inhibiting the CP. It is worth noting the upregulation of miR-34a-5p in HTLV-1 infected cell lines since we were able to identify its interaction with the transcription factor lymphoid enhancer-binding factor 1 (LEF1), which is a main effector of WNT CP and was already reported to be suppressed by HBZ in HTLV-1 infection ,85. In TThis particular profile of respective suppression and overactivation of canonical and non-canonical WNT pathways is not unexpected, even when disregarding miRNA activity since both pathways are activated by different ligands that interact in a reciprocal inhibitory fashion due to their competitive binding with the same FZD membrane receptors ,91.Transforming growth factor-β (TGF-β) signaling is essential during embryo development and associated with many important biological processes; however, TGF-β roles in cancer are diverse and dependent on other cell signaling pathways, being considered a tumor suppressor at early tumor stages and an enhancer of malignant phenotypes later in cancer development ,93,94.TGF-β achieves its role as a tumor suppressor due to the ability to regulate cell proliferation, differentiation, and immune cell modulation through the activity of its main signal transducers, the SMAD family member (SMAD) proteins. Downstream of TGF-β activation, SMADs interact with cyclin-dependent kinase inhibitors (CDKNs), inhibitors of DNA binding (IDs) proteins and other proliferation drivers to modulate genomic expression, cell cycle, and apoptosis induction ,96,97.The oncogenic role of TGF-β, however, is highlighted at later stages of tumor development, where overactivation of TGF-β signaling is associated with increased potential of invasion and metastasis, as well as with downregulation of immune cell activity in the tumor microenvironment ,98.SMAD2, zinc finger FYVE-type containing 16 (ZFYVE16), and Sp1 transcription factor (SP1) are signal transducers acting downstream of TGF-β that we identified to be regulated by the activity of miR-155-5p, miR-222-3p, and miR-150-5p, respectively. All of the three previously mentioned miRNAs were reported by Nascimento et al.  as upregZFYVE16 is a direct enhancer of SMAD2 activity, as well as other SMAD members, and was reported by Zhao et al.  as an inIt is also of note that, after HTLV-1 integration in the host genome, the integrated viral DNA harbors multiple binding sites for SP1 at viral gene promoters. While the roles of all of these binding sites as repressors or enhancers of gene expression are not fully elucidated, SP1 activity seems to be an important regulator for latent HTLV-1 activation in response to cell stressing factors ,103.Beyond the above-mentioned interactions, TGF-β inducing factor homologous box 2 (TGIF2) and E2F transcription factor 5 (E2F5), both of which act downstream of TGF-β signaling, have also had an identified interaction in our analyses. Mir-34a-5p, reported by Ruggero et al.  as upregTGIFs are known antagonists of TGF-β signaling due to their repressor effects over SMAD2/3 activity. By inhibiting the formation of the E1A binding protein p300 (p300)/SP1 transcriptional complex, which acts downstream of SMAD2/3 activity, TGIF2 negatively regulates p300/SP1-mediated cell cycle arrest and thus is implied as a cofactor for carcinogenesis in a variety of tumors ,108,109.Furthermore, Zhao et al.  describeRAS-related (RRAS) gene and platelet-derived growth factor receptor (PDGFR), specifically PDGFRA, a classic proto-oncogene encoding receptor tyrosine kinases [Another important interaction of miR-34a-5p was with RAS and MAPK pathways, through  kinases .Members of the RAS family that contains more than 100 proteins, the RRAS subfamily is composed of only 3 small proteins: RRAS, RRAS2, and MRAS, which are molecular switches that toggle between GTP-bound and GDP-bound conformations. It has been reported that RRAS is involved in several cellular functions, including enhancement of integrin function, regulation of cell adhesion, invasion, and migration, and related to different mechanisms that regulate its oncogenic potential, one of which is epigenetic regulation. The widespread prevalence of RAS mutations in human cancer has been recognized for many years. In cancer, members of the RRAS subfamily have been implicated in anchorage-independent growth, increased invasiveness, stimulation of angiogenesis, and tumorigenicity. In addition, RRASs have been linked to leukemias, and their overexpression can impair tumor-targeted lymphocytes, indirectly promoting tumor growth ,117,118.MAPK is, in its classical pathway, an extension of RAS signaling through the RAS-RAF-MEK-ERK signaling cascade, which regulates genes that control cell development, differentiation, proliferation, and apoptosis. It plays a central role in human cancer, and many of its elements have been identified as oncogenes, being overactivated in a wide variety of tumors. In addition, aberrant activations of MAPK signaling and its interactions with other cell signaling pathways in the pathogenesis of chronic lymphocytic leukemia have also been described ,120,121.Interestingly though, the upregulation of miR-34a-5p reported by Ruggero et al.  in HTLV-Still adjacent to MAPK signaling is the activity of the tumor necrosis factor (TNF) family and their ambiguous role in tumor development ,127. MiRThrough a bioinformatic approach, we were able to predict many interactions of miRNAs in important pathways in carcinogenesis that were correlated with HTLV-1 infection, and Several studies reveal deregulated miRNAs in HTLV-1 infection, and our bioinformatic analysis was able to predict several of these miRNAs’ interactions with important pathways in tumorigenesis, especially TP53, WNT, RRAS, TGF-β, and MAPK, thus being able to address them as probable drivers for ATL onset. While the data generated by our bioinformatics tools are able to provide solid starting points for further comprehension of miRNAs’ roles in HTLV-1 infection, the continuous effort in oncologic research to clarify still to be elucidated molecular pathways is needed to improve our understanding of HTLV-1 induced leukemia.


The growth and stress responses developed by the plant in virtue of the action of PGPR are dictated by the changes in hormone levels and related signaling pathways. Each plant possesses its specific type of microbiota that is shaped by the composition of root exudates and the signal molecules produced by the plant and microbes. Plants convey signals through diverse and complex signaling pathways. The signaling pathways are also controlled by phytohormones wherein they regulate and coordinate various defense responses and developmental stages. On account of improved growth and stress tolerance provided by the PGPR to plants, there exist crosstalk of signaling events between phytohormones and other signaling molecules secreted by the plants and the PGPR. This review discusses some of the important aspects related to the ambiguities of signaling events occurring in plants, allowing the interaction of PGPR with plants and providing stress tolerance to the plant. Plant growth-promoting rhizobacteria (PGPR) are known to positively stimulate the growth of plants by providing important nutrients and stress tolerance . In addition to MAPKs, CDPKs have also shown an important role in plant growth and development under abiotic stresses by modulating abscisic acid (ABA) signaling and regulating reactive oxygen species (ROS) accumulation  pathways and calcium-dependent protein kinase (CDPK) pathways working inside the plant  and effector-triggered immunity (ETI). PTI is triggered by conserved microbial signatures called pathogen/microbe-associated molecular patterns (PAMP/MAMP), which are perceived by pattern recognition receptor (PRR) at the cell surface, leading to a basal level of the immune response. The second level of immunity, ETI, is developed against effector molecules injected by pathogens inside the plant to counteract the PTI. ETI is also called the R gene-dependent resistance or gene for gene resistance , Gibberellic acid (GA), zeatin, ET, and ABA  are important hormones regulating seed germination, apical dominance, senescence, and interaction of the plant with microbes  deaminase controlling excessive ethylene production . BacteriBesides signaling molecules produced by PGPR, the plant secretes low molecular weight, high molecular weight, volatile, and non-volatile compounds in the root exudates, which influences the interaction of various microbes in the rhizosphere. Studies have shown that signaling molecules such as coumarins, triterpenes, flavonoids, and benzoxazinoids allow the growth and inhibition of specific microbes in rhizospheric regions of plants . The SLs are involved in many of the important aspects of plant development earlier defined as ex-planta signaling molecules secreted by plants' roots inducing germination of parasitic plants. Later, they were found to help in establishing a symbiotic relationship between plants and arbuscular mycorrhizal fungi  also called Nod factors are signaling molecules, originally studied to be produced by Rhizobia with leguminous plants is with the help of recognition of LCOs/Nod factors (NFs) by Nod factor receptors (NFRs) of leguminous plants. Two NFR receptors, NFR1 and NFR5, bind Nod factors directly , which shows the dual functions, i.e., in symbiosis as well as in plant defense , containing oligosaccharide-binding LysM domain. It is presumed that binding of LCOs with LysM receptors activates the signaling pathway, leading to the oscillation of nuclear calcium concentration, leading to the formation of nodules, and promoting the symbiotic association. The symbiosis established by Oldroyd, . A studyOldroyd, . SeveralXanthomonas campestris. The induction of nodulation by Bel 2-5 effector is through the expression of cytokinin-related genes involved in nodule organogenesis and repressing ethylene and defense-related genes  to deliver virulence factors (T3 effector proteins), certain rhizobial strains also produce effector protein called nodulation outer protein L (NopL), affecting the nodule formation in symbiosis. NopL bypass the NFR for the formation of the nodule in the legume. NopL affects the process of nodule formation by interacting with plant signaling pathways. A study was performed to show that phosphorylation of NopL was inhibited by Ser/Thr kinase inhibitor and MAPKK inhibitor, suggesting NopL to be a substrate of MAPK  , late embryogenesis abundant (RAB18), and also the important players of MAPK signaling cascade MPK3 and MPK6, thus increasing the ROS scavenging activity  by the enzyme phosphoethanolamine-lipid-A transferase (EptA)  controls the Na+ import and overexpression of this transporter fails to impart salt tolerance, making plants more susceptible  was reported to improve salt and drought tolerance by reprogramming the biosynthesis of stress hormones, such as salicylic acid and abscisic acid, chlorophyll content, and expression of antioxidants  has been reported to act as a biocontrol agent apart from its beneficial role in imparting salt tolerance. This bacterium exerts its effect by modulating phytohormone signaling, producing secondary metabolites, osmoprotectants, and scavenging ROS  from University Grant Commission, Govt. of India, and DST-SERB start-up grant (File no. SRG/2021/001830-G) Govt. of India.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ursolic acid (UA) is a pentacyclic triterpene isolated from a large variety of vegetables, fruits and many traditional medicinal plants. It is a structural isomer of Oleanolic Acid. The medicinal application of UA has been explored extensively over the last two decades. The diverse pharmacological properties of UA include anti-inflammatory, antimicrobial, antiviral, antioxidant, anti-proliferative, etc. Especially, UA holds a promising position, potentially, as a cancer preventive and therapeutic agent due to its relatively non-toxic properties against normal cells but its antioxidant and antiproliferative activities against cancer cells. Cell culture studies have shown interference of UA with multiple pharmacological and molecular targets that play a critical role in many cells signaling pathways. Although UA is considered a privileged natural product, its clinical applications are limited due to its low absorption through the gastro-intestinal track and rapid elimination. The low bioavailability of UA limits its use as a therapeutic drug. To overcome these drawbacks and utilize the importance of the scaffold, many researchers have been engaged in designing and developing synthetic analogs of UA via structural modifications. This present review summarizes the synthetic UA analogs and their cytotoxic antiproliferative properties reported in the last two decades. Cancer is the first or the second leading cause of death worldwide, accounting for 19.2 million new cases and 9.6 million deaths, in 2020 ..43a–d)  [UA derivatives were prepared with a modification at the positions C3 and C28 of UA. Compounds 44a  and 44b ctively) .45   in comparison to the parent UA [Zhao et al. synthesized UA analog (46)  by modifarent UA .47  and 1.7 µM (for A2780 ovarian carcinoma) [Kahnt et al. reported several UA-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugates with various oligo-methylene diamines as linkers. From them, compound 47  was founrcinoma) .48)  pathway has been implicated in tumor angiogenesis, growth, and metastasis. In the continuous effort to develop potential chemotherapeutics, another potent HIF-1α inhibitor (48)  was repo potency .UA analogs generated upon modification of both the C3 and C28 positions, reduced cell cycle arrest and induced apoptosis by activating p53, Bax and caspases-8, -9, and -3, and also by reducing Bcl2 and survivin. Apoptosis is involved in maintaining homeostasis of cell numbers by removing damaged or unwanted cells; therefore, UA derivatives are important chemotherapeutic compounds to reduce tumor growth. UA derivatives also inhibit NF−κB, which in turn, reduces cell growth arrest. NF−κB signaling plays an important role in cell growth and the inhibition of NF−κB signaling will reduce cell growth by inducing cell cycle arrest .49 (50 of 1.9 µM against the pancreatic cancer (AsPC1) cell line. Further investigation confirms 49 arrests the cell cycle in the G1 phase and induces apoptosis in AsPC1 cells with the up-regulation of p53, p21waf1 and NOXA protein levels [50, which is showing anticancer properties against various cancer cell lines at significantly low concentrations. Compound 50 also arrests the cell cycle at the G1 phase with the up-regulation of p21waf1. Apoptosis was induced at an inhibitor concentration of 8 µm with up-regulation of NOXA and down-regulation of c-FLIP [In addition to C3 and C28 modifications, several structural changes are reported that help enhance anticancer properties. Leal et al. synthesized a series of heterocyclic derivatives of UA as therapeutics for pancreatic cancer. Compound 49 , which in levels . The samf c-FLIP .51 , respectively, which are comparable to doxorubicin. The reduced cytotoxicity against noncancerous LO2 cells  makes 52 a potential lead molecule [In an attempt to develop potent anticancer agents, a set of carbazole derivatives of UA were prepared. Among the various carbazoles, compound 52  showed pmolecule .53 showed IC50 values of 0.48 ± 0.11 and 0.74 ± 0.13 mM for MCF7 and Hela cells, respectively, and significantly low cytotoxicity to LO2 cells. A further molecular mechanistic study revealed that compound 53 inhibits cell migration, induces cell cycle arrest at the S phase, and triggers mitochondrial-related apoptosis by increasing the generation of intracellular ROS and decreasing the mitochondrial membrane potential (MMP). In addition, it participates in the up-regulation of the protein expression level of Bax and the downregulation of the level of Bcl2. Furthermore, molecular docking indicates that MEK1 kinase could be one of the possible targets for 53 [Similarly, Wang et al. synthesized a set of 2-amino-4-aryl-pyrimidine derivatives of UA and tested their anticancer potential against MCF7 and Hela cells. Compound s for 53 .54 . Compound 54  was founG1 phase .55 (50 = 4.0 µM). Further investigation indicated the down-regulation of HIF-1α protein expression, possibly by suppressing its synthesis, reducing the production of vascular endothelial growth factor, and inhibiting the proliferation of the cancer cells [As we discussed earlier, an aminoguanidine moiety scaffold plays an important role in the development of potential drug candidates. Here, Wu et al. introduced the scaffold at the C3 position of the UA and synthesized compound 55 . The comer cells .56 was found to be the most active with IC50 4.28 μM against HT29. An additional bioassay explained that this compound induced cell cycle arrest at the G1 phase and apoptosis in a dose-dependent manner. In addition, compound 56 (57) was developed, which showed a 3- to 9-fold higher cytotoxicity as compared with the parent drug in A549, MCF7, HepG2, HT29, and HeLa cells. Further analysis concluded that 57 arrested the MCF7 cell cycle in the G1 phase, which was associated with caspase activation and a decrease in the Bcl2/Bax ratio. A molecular docking study unveiled that the nitroxyethyl moiety of 57 possibly establishes hydrogen bonds with caspase-8 amino acid residues (SER256 and HIS255) [Nitric oxide (NO) is an important messenger molecule in the human body that plays a key role in many physiological processes. High levels of NO can inhibit the proliferation of cancer cells, but unfortunately, the in vivo half-life of NO is very short. Recently, NO-donating derivatives, which can transport and release NO within the body, were used to explore the development of chemotherapeutic agents ,122. Thepound 56  was foun HIS255) .58  level, and decrease mitochondrial membrane potential. In addition, the Western blot analysis confirmed that compound 58 up-regulated Bax, cleaved caspases-3 and -9, cleaved PARP levels, and down-regulated the Bcl2 level of MCF7 cells. Meanwhile, compound 58 markedly decreased p-AKT and p-mTOR expression, which revealed that compound 58 probably exerts its cytotoxicity by targeting the PI3K/AKT/mTOR signaling pathway [A series of indolequinone derivatives of UA having ester, hydrazide, or amide moieties were prepared and tested for their in vitro antiproliferative properties against MCF-7, Hela, and HepG2 cell lines and a normal gastric mucosal cell line (Ges1). From the series, compound 58  was iden pathway .59  or in human volunteers, will move these compounds from the laboratory bench to the clinic. These studies are economically challenging for most laboratories engaged in creating analogs. However, select compounds, such as compounds


Drosophila melanogaster. Later, it has been found that the HH system is highly conserved across species and essential for organogenesis. HH signaling pathways play key roles in the development of the brain, face, skeleton, musculature, lungs, and gastrointestinal tract. While the sonic HH (SHH) pathway plays a major role in the development of the central nervous system, the desert HH (DHH) regulates the development of the gonads, and the indian HH (IHH) acts on the development of bones and joints. There are also overlapping roles among the HH molecules. In addition to the developmental role of HH signaling in embryonic life, the pathways possess vital physiological roles in testes and ovaries during adult life. Disruption of DHH and/or IHH signaling results in ineffective gonadal steroidogenesis and gametogenesis. While DHH regulates the male gonadal functions, ovarian functions are regulated by both DHH and IHH. This review article focuses on the roles of HH signaling in gonadal development and reproductive functions with an emphasis on ovarian functions. We have acknowledged the original research work that initially reported the findings and discussed the subsequent studies that have further analyzed the role of HH signaling in testes and ovaries.Three distinct hedgehog (HH) molecules, , two HH receptors (PTCH1 and PTCH2), a membrane bound activator (SMO), and downstream three transcription factors  are the major components of the HH signaling. These signaling molecules were initially identified in  Drosophila melanogaster [Drosophila larvae’s spiked phenotype in the cuticle, which appears like the HH animal’s spikes [The hedgehog (HH) signaling was identified for their roles in the body segmentation of nogaster . The nams spikes ,3. Subses spikes . The HH s spikes . As the s spikes ,6.The major role of HH signaling is limited to embryonic development, particularly in organogenesis. SHH plays an essential role in neuronal development, DHH acts on the gonadal development and steroidogenesis, and IHH regulates various developmental functions including bone development, as well as ovarian steroidogenesis and folliculogenesis ,8. DysreHH signaling is primarily known for its essential roles during embryonic development . It alsoShh gene (KOShh) in the adrenal cortex leads to adrenocortical hypoplasia, indicating that SHH is essential for the development of the adrenal gland [SHH is widely expressed in different organs and tissues, and it is the most potent HH ligand among the three . It is kal gland .Dhh gene knockout (KODhh) male mice are infertile due to the absence of mature sperms [KODhh male mice is associated with pseudo-hermaphroditism, characterized by incomplete masculinization of the testes and the male genital tract [DHH is primarily involved in the development of the gonads ,20,28,42e sperms ,44. Expre sperms . Defectial tract . Disruptal tract .Ihh gene knockout (KOIhh) mice suffer from a lack of mineralization of bones and fails to form the osteoblasts in endochondral bones, which is required for skeletal growth [In contrast to SHH and DHH, IHH plays a predominant role in the development of bones and cartilages . It is el growth ,49. IHH l growth . While Dl growth   before they are secreted in an active form . Such PTOne of the distinctive characteristics in vertebrate HH signaling is the relationship between primary cilia and HH signaling . The priThe major target molecules of HH proteins are PTCH1, PTCH2, SMO, GLI1, GLI2, and GLI3. The initial step is mediated by two transmembrane receptors: one is either PTCH1 or PTCH2, and the other is SMO  Figure C. In a cHH signaling can be impacted by several interacting molecules . KIF7 anIn contrast to the canonical HH signaling, the non-canonical HH signaling can be mediated by two distinct mechanisms. The type I non-canonical HH signaling pathway is SMO-independent but GLI dependent ,94 FiguA,B. WherWhile DHH regulates the development and functions of male gonads, the development and functions of female gonads are dependent on both DHH and IHH. DHH is expressed in the SCs of developing testes starting from a mid-embryonic stage, whereas DHH and IHH are expressed in the GCs of activated follicles. While SC-derived DHH acts on LCs and spermatocytes, GC-derived DHH and IHH act on TCs.Both SCs and the male germ cells are enclosed in the seminiferous tubules by a basement membrane formed by the peritubular myoid cells (PTMCs). SRY induces the expression of SOX9 that differentiates the SCs ,97. DiffGCs in dormant primordial follicles (PdFs) do not express the HH molecules; both DHH and IHH are induced in the GCs of activated follicles starting from the primary follicle stage (PrF) stage ,100 FigC. HoweveDhh in SCs [Sox9 gene exhibit the sex reversal of the XY chromosome background [During the development of the gonads, WT1, GATA4, GATA6, SOX9, and SRY are involved in regulating the transcription of h in SCs ,101. Amoh in SCs ,101. Patckground ,103. SRYckground .The expression of DHH and IHH remain undetectable in ovarian PdFs and is induced in the GCs of activated follicles. The expression of both HHs remain higher in the GCs of early-stage follicles but downregulated in more developed preovulatory follicles ,74. StudSexually dimorphic features are prominent between the male and female gonadal systems . HH signThe sexually dimorphic structures of the male and female reproductive system develop in three distinct steps. In the first step, sex determination occurs when X or Y chromosome-carrying sperm fertilizes an X chromosome-carrying oocyte, the XX gametes develop to females, and the XY gamete develop to males . FollowiThe final step of sex differentiation occurs with the development of the reproductive tracts and external genitalia. The testes express the anti-Müllerian hormone (AMH) and androgens, which induces Müllerian duct regression and Wolffian duct differentiation into the epididymis, vas deferens, and seminal vesicles in male embryos ,107 Fig. As the DHH plays a key role in the development of male gonads ,109. DevKO mice, which leads to the disorganized structure of the corda testes [KO mouse testes [KO mouse testes possess the undifferentiated LCs, resulting in testosterone deficiency [KO XY mice may develop a variety of testicular phenotypes depending on the genetic background; ultimately, they become infertile due to the lack of mature sperms [In the absence of DHH, the development of PTMCs and FLCs is defective in Dhha testes ,42,109. e testes . DhhKO mficiency ,42. Howee sperms ,42,109.Interactions between the oocytes, GCs, and TCs are essential for the development and maturation of ovarian follicles . HH signDuring follicle development, the mesenchymal compartment appears to be a predominant target of HH ligands . ActivatDHH regulates spermatogenesis and the maturation of sperms . DHH relKO XY gonads lose the expression of both PTCH1 and CYP11A1 [It has been demonstrated that DHH signaling initiates the development and maturation of FLCs in mice by upregulating the expression of SF1 ,117. In  CYP11A1 ,28,118.  CYP11A1 . Express CYP11A1 . HoweverKO or IhhKO female mice were found to be fertile and demonstrated the presence of corpora lutea in their ovaries [KO and IhhKO resulted in defective TC development, follicular arrest at preantral phases, and failure of ovulation [Ovary-specific either Dhh ovaries . Howevervulation . TC diffvulation .KO; IhhKO) fail to develop the TC cells, and suffer from impaired steroidogenesis and infertility due to failure of ovulation [KO or IhhKO mice [KO female mice, but not the DhhKO mice, showed progressively lower levels of dehydroepiandrosterone, testosterone, and progesterone [GC-derived DHH and IHH activate GLI TFs, which translocate to the nucleus of activated TC precursor cells. Activated GLIs induce the expression of target genes that mediate the proliferation and differentiation of the TC percussor cells to mature TCs  Figure . Mice lavulation . HoweverhKO mice . In the hKO mice . Remarkaesterone . It was esterone .KO mice are the direct effects of DHH deficiency or an indirect effect of androgen deficiency due to abnormal development and functions of LCs. It is also suggested that DHH signaling has distinct roles on the development of LCs, and PTMCs that are independent of androgens. While the male gonadal functions are regulated by DHH alone, steroidogenesis and folliculogenesis in the female gonad are regulated by both DHH and IHH signaling. We observed that while the expression of IHH in GCs is regulated by ERβ, the expression of DHH is independent of the ERβ signaling. We suspect that IHH and DHH may execute a differential regulatory role in ovarian steroidogenesis and/or folliculogenesis. Further studies are required to distinguish between the roles of IHH and DHH in the ovarian follicle development and ovarian functions.It remains unknown whether the phenotypic consequences on spermatogenesis in Dhh


The long lapse between the presumptive origin of schizophrenia (SCZ) during early development and its diagnosis in late adolescence has hindered the study of crucial neurodevelopmental processes directly in living patients. Dopamine, a neurotransmitter consistently associated with the pathophysiology of SCZ, participates in several aspects of brain development including pruning of neuronal extensions. Excessive pruning is considered the cause of the most consistent finding in SCZ, namely decreased brain volume. It is therefore possible that patients with SCZ carry an increased susceptibility to dopamine’s pruning effects and that this susceptibility would be more obvious in the early stages of neuronal development when dopamine pruning effects appear to be more prominent. Obtaining developing neurons from living patients is not feasible. Instead, we used Monocyte-Derived-Neuronal-like Cells (MDNCs) as these cells can be generated in only 20 days and deliver reproducible results. In this study, we expanded the number of individuals in whom we tested the reproducibility of MDNCs. We also deepened the characterization of MDNCs by comparing its neurostructure to that of human developing neurons. Moreover, we studied MDNCs from 12 controls and 13 patients with SCZ. Patients’ cells differentiate more efficiently, extend longer secondary neurites and grow more primary neurites. In addition, MDNCs from medicated patients expresses less D1R and prune more primary neurites when exposed to dopamine. Haloperidol did not influence our results but the role of other antipsychotics was not examined and thus, needs to be considered as a confounder. Although schizophrenia (SCZ) is typically diagnosed in late adolescence or early adulthood, this illness is commonly considered as a neurodevelopmental disorder that originates from a combination of genetic predisposition and environmental factors . This loNot surprisingly, the neurodevelopmental process at fault in SCZ remains unidentified. However, evidence collected from adult patients provides some insights. The most consistent finding in SCZ is decreased brain volume , 3 which1 receptors (D1R) in living, unmedicated individuals with SCZ found it decreased [2 receptors (D2R) are commonly encountered [A second consistent pathophysiological aspect in SCZ, but rarely approached from a neurodevelopmental perspective, is its association with dopamine. This relationship relies on several lines of evidence. For instance, at high levels, dopamine produces psychotic symptoms in healthy individuals  and exacecreased . Follow ecreased , 37. In ountered . Overallountered . In vivoountered –42. In lountered –46.Evidence of shorter neuronal extensions in patients with SCZ, together with the regularity in which dopamine has been associated with this psychotic disorder, and the role of dopamine in retracting neuronal extensions, compelled us to propose the following hypothesis: patients with SCZ carry an increased susceptibility to dopamine’s pruning effects. This susceptibility would be more obvious in the early stages of neuronal development when dopamine pruning effects appear to be more prominent. The main challenge to test this hypothesis is that obtaining developing neurons from living patients is not feasible. To circumvent this problem, we used Monocyte-Derived-Neuronal-like Cells (MDNCs).We have recently developed a protocol to transdifferentiate human circulating monocytes into neuronal-like cells without the need for reprograming . These MIn this manuscript we expanded the number of individuals and serial samples in which we tested the reproducibility of MDNCs’ results. We then compared if the neurostructure of MDNCs behaves similarly to that of human developing neurons in vitro. The neurostructure of MDNCs from 12 controls and 13 patients with schizophrenia was thoroughly studied. Dopamine pruning effects on MDNCs were assessed, as well as the expression of D1R. The potential influence of haloperidol on our results was also examined.All participants, after receiving full description of the study, gave their informed and written consent. All study procedures were approved by local ethics committees and were in accordance with the Helsinki declaration. Experiments pertaining to the cohort of patients and controls were approved by the ethics committee Ile de France II while experiments on the characterization of MDNCs involving only control individuals were approved by the Institutional Review Board at Penn State University (Study #00006911).Patients were diagnosed using criteria described in the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV). Diagnosis was based on clinical interviews and clinical records. Patients were recruited from the Department of Psychiatry at Sainte-Anne Hospital in Paris, France. Healthy controls were recruited through local advertisements. Controls were screened to rule out any past or present history of DSM-IV axis 1 disorders. Only individuals older than 18 years old were recruited.Thirty-five subjects, 16 controls and 19 patients with SCZ, were recruited for this study. In the SCZ group one patient was diagnosed with schizoaffective disorder and another with pervasive developmental disorder while one control had hemochromatosis  and dopamine , involved three concentrations for colchicine  and two for dopamine (4 mM and 5 mM). Detail information about dopamine preparation as well as preincubations with a D1-like receptor antagonist is provided in supplementary materials and methods. Tracing and cell characterization were done blinded.Human neurons were obtained from ScienCell Research laboratories (1520-10) and cultured following the manufacturer’s instructions. Further information about the procedures followed using human neurons is listed in Supplementary Materials and Methods.Flow cytometry was only used to measure levels of immunofluorescence for CD14 or to determine differences in the percentage of cells expressing nestin or D1R. The protocol we followed has been previously described  includint tests. The data at picture- or sample-level were also analyzed with a mixed model analysis to account for correlations of repeated measures within subjects. To compare pruning effects of experimental conditions between study groups, we adjusted for baseline retraction (response under control conditions), differentiation efficiency, and structure at baseline because these factors may affect the pruning effects of each treatment condition (dopamine & colchicine). Since different samples were used to measure these factors, we were unable to perform analysis with picture level data. Therefore, we ran linear regression analysis based on subject-level averaged data adjusting for these factors in the models as covariates. Least square means and standard errors were estimated from the models for each study group. Other statistical tests used are described in supplementary materials and methods.We conducted a descriptive analysis to examine the distributions of measures from MDNCs for healthy controls and SCZ patients. Subject-level measurements for MDNCs were obtained by first averaging the picture-level data for each sample and then averaging the sample-level data within each subject. Differentiation efficiency and structural parameters were summarized at the subject-level and compared between study groups using analysis of variance (ANOVA) or two-sample We have previously shown that the neuronal structure of MDNCs transdifferentiated from two serial blood samples from four healthy men were not statistically different . Here weDifferentiation percentage established by cellular phenotype yield significant differences between serial samples for subjects 1, 2, 3 and 6 but not for the other individuals, as shown in Fig. Different neurostructural parameters of MDNCs were assessed: longest primary neurite (LPN), longest secondary neurite (LSN), number of primary neurites, number of secondary neurites, and number of all neurites . There are no statistical differences in LPN in any of the 8 subjects Fig. . For LSNIn a previous publication we showed that the structure of MDNCs is comparable to the structure of Human Developing Neurons (HDNs) after 5 days in culture  Fig. 1B1B. Here Under controlled conditions both HDNs and MDNCs retract their LPN slightly after one hour of incubation. Treatment with colchicine 0.5 µM for one hour leads to additional retraction of LPN that is comparable in both cell types. HDNs present a net LPN retraction of 14%, which is significantly different when compared to the level of retraction under control conditions. Likewise, MDNCs present an LPN net retraction of 19% when incubated with colchicine  and 12 patients with SCZ plus one patient with pervasive developmental disorder and psychosis were included in the analysis. Two patients with SCZ were not taking any medications. The other 11 patients were receiving antipsychotics as well as other psychotropic medications Table . We ran After isolating monocytes from blood samples, we followed our 20-day transdifferentiation protocol . As prevMeans and standard deviations were summarized at subject level by study groups. Repeated ANOVAs based on the mixed model framework were performed to examine group differences for each day. Random effect was specified in the models to account for correlations between microphotographs within each subject or variation among subjects. With this approach, the structural path to transdifferentiation between patients and controls shows that there are no statistical differences on days 4, 10 and 13 on any of the structural stages. On day 7, patients evidence less uncharacterized cells while the rest of the structural stages do not differ from controls Fig. . Analysit test. This approach reveals that the percentage of cells that acquire a neuronal morphology is higher in patients with SCZ than in CTL. Such statistical difference is still present when only medicated patients (MED) are included in the analysis .After twenty days in culture following our transdifferentiation protocol, around 13% of monocytes acquire a neuronal morphology characterized by the presence of a well-defined soma and long thin neurites  Fig. 1B1B. We hasis Fig. . If a paIn a prior publication we showed that MDNCs reduce or even completely abolish its expression of CD14 , a surfaSince transdifferentiation occurs after 20 days in culture, the vast majority of MDNCs structural analyses were conducted between days 20 and 21. We analyzed whether there are structural differences between MDNCs at day 20 (D20) versus day 21 (D21). Only those individuals in each group for whom we have microphotographs of MDNCs at D20 and D21 were included in the analysis , differentiation efficiency, and structure at baseline since these factors may affect the pruning effects of each treatment condition. After accounting for these variables, we found that treatment with colchicine 0.4 µM retracts LPN and LSN in all groups Fig. . In contMDNCs from the three groups incubated with dopamine 4 mM retract its LPN and LSN comparably. Likewise, pruning of primary, secondary and total number of neurites reveals no statistical differences Fig. . There aActivation of D1R has been consistently associated with pruning of neuronal extensions during early stages of development in vivo  and in vWe then tested whether preincubation with SCH-23390, a D1R antagonist, prevents any of the structural changes elicited by dopamine 4 mM. SCH-23390 decreases pruning in LPN by 10% which is statistically significant. In contrast, pruning of LSN is not significantly affected by SCH-23390. No primary neurites were pruned either with dopamine or SCH-23390 + dopamine, while the number of secondary neurites and total number of neurites tends to decrease in the presence of SCH-23390 but without reaching statistical significance Fig. .In order to determine whether antipsychotics impact levels of differentiation or the structure of MDNCs, we incubated monocytes from day 4 to 7 of the transdifferentiation process with haloperidol at known circulating levels (20 ng/ml) . HaloperA non-parametric one-way ANOVA on the percentage of differentiated MDNCs established by phenotype, indicates CTL cells are not statistically different to those incubated with either vehicle (VEH) or haloperidol (HAL) Fig. . The strWe also analyzed whether haloperidol treatment impacts pruning. LPN from MDNCs cultured under CTL conditions, VEH or HAL retract equally. Likewise, the amount of retraction in LSN between the three culture conditions is similar. Similarly, the number of primary, secondary and total number of pruned neurites is comparable between the three groups Fig. .The pruning effects of dopamine 5 mM on MDNCs was also investigated after haloperidol treatment from day 4 to 7. Pruning of LPN and LSN is equivalent between the three treatment conditions. The same is true for number of primary, secondary and total number of pruned neurites Fig. . FinallyDespite intense research during the last several decades, the pathophysiology of SCZ remains obscure. One of the main obstacles to study this mental illness is the difficulty of accessing neurons directly from patients. The extended lapse between SCZ putative origin during neurodevelopment and its clinical emergence makes this challenge even harder. In order to overcome this obstacle, we used MDNCs that allow us to test in vitro whether some of the very early neurodevelopmental steps involving the neuronal structure are deficient in SCZ. Particularly intriguing is the possibility that patients with SCZ carry an increased susceptibility to the pruning effects of dopamine. This susceptibility is likely to be more obvious in the early stages of neuronal development when dopamine pruning effects appear to be more prominent –46. In fIn a prior publication we showed that the structure of MDNCs is comparable to that of HDNs maintained in culture for 5 days . As a fuConcerns have been raised about the reproducibility of results obtained with other cellular models , 50, 62.We also tested the reproducibility of several neurostructural parameters and found different degrees of consistency Fig. . ResultsAfter determining MDNCs’ reproducibility and its similarities with HDNs, we compared MDNCs from patients with SCZ versus cells from CTL. Patients were diagnosed using clinical interviews by experienced psychiatrists as well as medical records but no standardized scales were utilized to conclude patients had SCZ. Such approach should be considered as a limitation. We also included one patient with pervasive developmental disorder that presented psychotic symptoms. The cohort of patients and controls was matched by age and gender Table . The numWhile the path to differentiation is similar between groups Fig. , MDNCs fIn a previous publication we showed via genetic, immunofluorescence and flow cytometry that MDNCs decrease its expression of CD14 when compared to macrophages from the same individual . A drop After accounting in our statistical analysis for differences between SCZ and CTL in differentiation efficiency, size of secondary neurites and number of primary neurites, we challenged the structure of MDNCs’ with colchicine and dopamine. Both compounds are capable of pruning neuronal extensions through different mechanisms of action. Colchicine acts by directly depolymerizing microtubules  and thusTwo different concentrations of dopamine did not reveal differences in LPN, LSN, number of secondary neurites or total number of neurites between groups Fig. . NonetheMonocytes are exposed to medications during the 2–3 days they remain in blood circulation . After mAnother potential alternative to explain why we found increased susceptibility to dopamine pruning effects only in medicated patients could be patient heterogeneity. In this scenario, only a subset of patients with SCZ carries increased susceptibility to dopamine pruning actions. Further studies are needed to confirm this statement. In the meantime, the effects of medications on any of our comparisons between SCZ and CTL should be considered a confounding factor.There is additional information that needs to be highlighted to properly appraise our results. While our cohort is among the largest in the field of SCZ and stem cells, only a subset of individuals were included when we tested MDNCs structural responses to colchicine and dopamine as described in Supplementary Table In conclusion, our results suggest that a subset of patients with SCZ could carry an increased susceptibility to the pruning effects of dopamine during early stages of neurodevelopment but the possibility that antipsychotics, except for haloperidol, could have influenced our results through epigenetic mechanisms cannot be excluded.Supplementary Table S1Supplementary Tables S2 to S8Supplementary Table S9Supplementary Table S10Supplementary Table S11Supplementary Table S12Supplementary Table S13Supplementary Table S14Supplementary Table S15Supplementary Table S16Supplementary Table S17Supplementary Table S18Supplementary Table S19Supplementary Figure S1Supplementary Figure Legend


Infertility, defined as the inability to conceive after one year of intercourse without the use of contraception, affects 15% of couples. Many factors, such as genitourinary tract infections, endocrine disorders, immunological factors and drug-related injuries, affect the male reproductive system and cause infertility. Due to men's fear of infertility, it is very important to pay attention to medicinal plants that are effective in male fertility. Therefore, the aim of this study was to evaluate the medicinal plants that affect sperm and testosterone production in men.In this review, we used the following search terms, consisting of herbal medicine, traditional medicine, traditional therapies, sperm, testosterone, testicles and Iran were used to retrieve the relevant articles published in the journals indexed in the Information Sciences Institute, Science Direct, PubMed, Scopus, PubMed Central and Scientific Information Databases. We searched and used papers published between 2000 and 2020. Then, we analyzed the eligible papers. We collected and analyzed 35 papers from the databases. We selected only the articles about herbs that affect sperm and testosterone production.Apium graveolens, Cinnamomum camphora, Cornus mas, Satureja khuzestanica, Withania somnifera, Fumaria parviflora, zingiber officinale, cinnamomum zeylanicum and Phoenix dactylifera are used in the male reproductive system.Based on the results, herbs Plants can probably be useful in increasing fertility due to their antioxidant power and low side effects. Infertility, defined as the inability to conceive after one year of intercourse without the use of contraception, affects 15% of couples , reducing gonadotropins and testosterone, increasing the affinity for sex-specific proteins, thickening the basement membrane, reducing germinal epithelial cells and reducing the irregular placement of these cells, reducing sperm count, motility, and sperm viability, but causing side effects such as infertility, at certain doses . Garcinie stress . Flavonoe stress . In Irane stress . The effPlants can probably be useful in increasing fertility due to their antioxidant power and lower side effects. The use of medicinal plants with the property of enhancing male fertility can be used as a substitute or supplement to chemical drugs that affect male fertility. On the other hand, it is recommended that plants with fertility reduction properties be used less or not at all in men with infertility disorders.