While these seminal events are critical for the establishment of interneuron developmental milestones, recent studies on cellular signaling cascades have begun to elucidate some potential contributions of cell signaling during development. Here, we review studies underlying three broad signaling families, mTOR, MAPK, and Wnt/beta-catenin in cortical interneuron development. Notably, each pathway harbors signaling factors that regulate a breadth of interneuron developmental milestones and properties. Together, these events may work in conjunction with transcriptional mechanisms and other events to direct the complex diversity that emerges during cortical interneuron development and maturation.The development and maturation of cortical GABAergic interneurons has been extensively studied, with much focus on nuclear regulation  The processes by which cortical GABAergic interneurons (CINs) develop, differentiate and acquire their unique properties have been studied for many years. Insights into the transcriptional cascades that guide these events have been well investigated, especially in early developmental stages, when CIN progenitors are first programmed and start to diverge into unique trajectories. In recent years, further stages during development and maturation have been explored, including how the local environment impacts their molecular and electrophysiological properties. Moreover, cellular signaling events may be involved in facilitating transcription factor recruitment, linking extracellular cues, and orchestrating neuronal activity, which may be translated downstream into nuclear events or other signaling intermediates.Multiple ventral telencephalic regions, including the medial ganglionic eminence (MGE), caudal ganglionic eminence (CGE), and preoptic area (POA) generate CINs . The provia a sonic hedgehog (SHH) morphogen gradient, which induces the expression of transcription factors (TFs) such as Nkx2.1, Dlx2, and Gsx that promote the early development of MGE and CGE  signaling pathway is critical for cellular growth, proliferation and synaptic properties . Activatase, AKT . AKT thehibition . In the ard Rheb , haltingard Rheb . While tTsc1 using the pan GABAergic Dlx5/6-Cre led to a loss of GABAergic lineages and increased sensitivity to a seizure inducing drug /beta-catenin and related non-canonical signaling events. This pathway has early effects on MGE progenitors GE cells . One exaelopment .via the RYK receptor emerged as a potential moderator of SST expression in some caudal MGE populations when active RYK protein signaling was modulated. However, RYK’s role during development is complicated, as loss of function in MGE progenitors leads to several unidentified lineages with no observable ratio change in SST and PV CINs, suggesting a further role in MGE progenitors. Moving forward, this story may help bridge other work on Ryk and CINs (WNT ligands are also enriched closer to caudal regions of the MGE and the MGE responds in turn to WNT signaling in these regions, which predominantly generate SST + CINs . While tand CINs .Mafb and c-Maf are involved in GSK3-beta signaling; phosphorylation of MAF proteins by GSK3-beta leads to increased protein activity downstream of MAFs (Mafb and c-Maf using Nkx2.1-Cre result in an increase in SST + CINs at the expense of fated PV + progenitors in addition to gross loss of CINs (Mafs, including Mef2c (Mef2c was initially identified as a TF involved in PV CIN fate and development (The TFs  of MAFs . Deletio of CINs . Later sng Mef2c , as a prelopment  and its elopment . While telopment .The unique roles of cellular signaling proteins in the development and function of CINs is just beginning to be uncovered. Here, we focused on three critical pathways with broad applications for many CIN subgroups. Their role in brain development and specific CIN milestones reveal distinct and shared functions that will have major implications for the development field as well as neurological/neuropsychiatric disorders caused by protein disruptions. While the mTOR, MAPK, and Wnt/Beta-catenin pathways are just some of the cellular signaling families that could modulate CIN development, future studies are likely to uncover more signaling implications. When combined with the instruction of TFs and activity-dependent maturation, it suggests the potential for complex regulation of CIN development.An interesting finding from these initial studies is that within the core signaling families, distinct proteins can exhibit unique functions in CINs. For example, both PTEN and TSC proteins are inhibitors of the mTOR pathway, yet uniquely impact SST CIN numbers, PV axon outgrowth, PV expression and electrophysiological properties . Some ofNf1 and Mek that led to hyperactivity of the MAPK pathway and subsequent similar losses of PV + CINs (Distinct proteins in a cascade may have common phenotypes during CIN development, as was noted for manipulations of V + CINs . This laV + CINs . As a faNf1 loss of function/hyperactive MAPK mutants (Maf TFs, which regulate SST/PV cell properties (Other considerations include the possibility that each signaling protein within a family may crosstalk with other distinct family members. For example, in cancer cells it is common that the mTOR and RAS/MAPK proteins interact . Also, t mutants . Moreoveoperties , 2020, aoperties , suggestAll authors planned and wrote the minireview and contributed to the article and approved the submitted version.


The Li-enriched oilfield brine is a very important lithium resource. It has gained much attention and become the target of active Li surveys with the growing global demand for Li. However, only little is known about their feature and nature. In the study, hydrochemical data from 155 oil wells tapping the Eocene to Lower Oligocene Qianjiang Formation of the Jianghan Basin, central China indicate that the brines are of the Na–Cl or Na–Ca–Cl type and are characterized by highly variable Li contents of 7.56 to 150 mg/L, with Mg/Li ratios less than 11.65. High Na/Cl and Cl/Br molar ratios indicate distinct contributions from halite dissolution. The Ca excess, Na deficit and Ca/Mg and Ca/Sr molar ratios in the brines imply multiple diagenetic processes, including halite dissolution, dolomitization, albitization and calcite or anhydrite cementation. The lithium contents of these brines have a weak relationship with the salinity and a negative correlation with Cl/Br ratios, possibly indicating that these Qianjiang oilfield brines have been diluted by secondary brines derived from halite dissolution. The spatial distribution patterns for Li and B concentrations of the brines are different from those for salinity and Br contents and show a geographic pattern, indicating that Li enrichment in the Qianjiang brines is likely connected with geothermal sources associated with volcanic activity. The principle lithium resources include Li-rich hard rocks (pegmatites and volcanogenic clays) and continental brines3, among which the latter accounts for about 65% of the global lithium occurrences4. These Li-rich brines with average Li grades > 600 mg/L are distributed in the salt pans of the South American Altiplano-Puna7, in the salt lakes of the Qinghai-Tibetan plateau10, and in the Great Basin of the western USA13.The success of applications of Li batteries in electric and hybrid vehicles, in recent years, has spurred an increased interest in Li as a strategic resource, to the point that it has been served as one of the most critical metal minerals14. The known oilfield brine deposits include those from the Jurassic Smackover Formation in the Gulf Coast Basin of America15, the Fox Creek area in the Alberta Basin of Canada16 and the Paleogene and Neogene formation in the western Qaidam Basin of China17.As previously mentioned, the growing global demand for Li driven by its high electrochemical potential in emerging technologies and the rising price in the market greatly encourage the development of mining projects. As such, less-rich brine deposits such as oilfield brines and geothermal brines, with Li grades between 50 and 400 mg/L, gained much attention and became the target of active Li surveys throughout the world. Most petroliferous basins contain abundant Li-enriched oilfield brines19. Li enrichment in brines and evolution of brine hydrochemistry, however, remain unclear, except that potassium in these oilfield brines and reservoir characteristics have been reported by Yu et al.20.The Jianghan Basin is an important Late Cretaceous to Oligocene petroliferous basin in central China. Large quantities of Li-rich oilfield brines are stored in the Upper Eocene to Lower Oligocene Qianjiang Formation of the basin. The brine deposits at mean Li grades of 60.6 mg/L hold LiCl resources of ~ 4 MtThis paper presents the first broad hydrochemical data of oilfield brines from the Qianjiang Formation of the Jianghan Basin, China and aims to investigate the water chemistry characteristics and temporal and spatial distribution of Li in brines and determine its potential enrichment mechanism. In addition, this study also offers an overview of the potential of unconventional Li survey and contributes to knowledge of the origin of Li-enriched oilfield brines in the continental petroliferous basins.22. The basin underwent two salt-forming stages: Late Cretaceous to Early Eocene and Middle Eocene to Oligocene23. The Middle Eocene to Late Oligocene Qianjiang Depression is the subsidence and deposition center of the basin  of water samples were analyzed by inductively coupled plasma optical emission spectrometer (ICP-OES). Cl−, SO42− and Br− were measured by ion chromatography (IC). HCO3− and CO32− were determined by 0.05 M HCl titration (AT). The analytical errors for ICP-OES, IC and AT were 2‰, 5‰ and 3‰, respectively. The contour diagrams of TDS, Br, Li, and B were performed using Surfer 12.0 software.The main cations . The brine samples from the Qianjiang Depression have Na/Cl molar ratios ranging from 0.83 to 1.21 , indicating the influence of multiple water–rock interactions.The trends between Ca/Mg and Ca/Sr molar ratios Fig. b suggestses Fig. . Data fo17. A negative correlation is shown in the Fig. Variations in the grades of Li in the Qianjiang brines can be revealed by plotting Li contents against salinity and Cl/Br molar ratios. Figure Based on the contour diagrams of Li, B and Br contents and TDS of these brines in the Qianjiang Depression, some spatial distribution patterns seem to be revealed. The high-salinity brines are mainly distributed in the Haokou, Zhongshi, Wangchang and Xiongkou areas Fig. a. The hi38. The geographic distribution pattern for Li concentrations of these brines in the Qianjiang Depression, in combination with the widely developed volcanic rocks in the Jianghan Basin, indicates that Li enrichment in brines is more likely to be associated with geothermal sources during volcanic activity40.Weathering of igneous rocks and hydrothermal supplies associated with volcanic activity have been suggested to interpret the sources of Li-enriched brinesThis study reports the results of the hydrochemical analysis of oilfield brines from the Qianjiang Formation of the Jianghan Basin, central China. The brines are of the Na–Cl or Na–Ca–Cl type and have Li contents ranging from 7.56 to 150 mg/L, with Mg/Li ratios less than 11.65.High Na/Cl and Cl/Br molar ratios of these brines suggest contributions from halite dissolution. Plotting of Ca excess vs. Na deficit and Ca/Mg vs. Ca/Sr molar ratios indicates multiple diagenetic processes, including dolomitization, calcite or anhydrite cementation, halite dissolution and albitization. Variations in the grades of Li in brines have a weak correlation with the salinity. The Li contents of these brines may be diluted by the mixing of secondary fluids derived from halite dissolution. The spatial distribution for the concentrations of Li and B is different from those of TDS and Br and shows a geographic pattern, which is likely associated with geothermal supplies associated with volcanic activity.Supplementary Table S1.


The precise execution of various cellular functions relies on the maintenance of signaling specificity from input detection to cellular outputs. However, diverse signaling pathways share similar or identical intermediate components. A well‐conserved intermediate, the Mitogen‐Activated Protein Kinase (MAPK) cascade, participates in a myriad of signaling pathways, regulating signal transduction from input to output. This typifies the “hourglass conundrum”, where a multitude of inputs and outputs all operate through a limited number of common intermediates. Therefore, understanding how MAPK cascades regulate a variety of outputs with specificity is a fundamental question in biology. This review highlights four major insulating mechanisms that improve signaling specificity: selective activation, compartmentalization, combinatorial signaling, and cross‐pathway inhibition. We focus on plant pathways that share MAPK cascade components and compare mechanisms with those of animals and yeast. We hope this conceptual overview will aid future studies to better understand plant signaling specificity. Diverse signaling pathways share similar or identical intermediate components. The MAPK cascade is a well‐conserved intermediate, participating in myriad signaling pathways. The precise execution of various cellular functions relies on signaling specificity from input detection to cellular outputs. Comparing plant and animal pathways, this review highlights four major insulating mechanisms that improve signaling specificity. Please refer to Glossary section at the end of the article The ability to respond to external stimuli is a fundamental characteristic of all living organisms. The smallest response unit, a cell, converts external signals into requisite cellular responses by activating appropriate signaling pathways. The evolution of multicellularity in eukaryotes followed by functional diversification of the cells has drastically increased the complexity and intricacy of signaling pathways. The execution of a myriad of cellular functions thus relies on these pathways to maintain specificity from signal input to cellular output. These outputs range from basic growth and proliferation to highly specialized functions such as development of xylem tissues for structural support and transport in vascular plants, and the adaptive immune response to a specific pathogen in mammals.). The resulting high interconnectivity of signaling pathways raises an important challenge to understand how output specificity is maintained, and more precisely: How do common signaling modules regulate a variety of outputs? Relative specificity is achieved when one pathway's input can activate more of its own output than another pathway's output. Absolute specificity means one pathway can only activate its own output and cannot activate another pathway.However, it is a widespread phenomenon for diverse signaling pathways to share similar or identical intermediate components. One key intermediate module, the Mitogen‐Activated Protein Kinase (MAPK) cascade, is activated by a wealth of known stimuli in plants, animals, and fungi , 2, 3, 4), making it important to understand the mechanisms whereby undesirable crosstalk is avoided and signaling specificity maintained.Likely as an adaptation to their sessile lifestyle, land plants have dramatically expanded their receptor kinase repertoire , 6. For The MAPK cascades are universal signaling modules present in all eukaryotes. They serve as the nexus of diverse signaling pathways, regulating fundamental aspects of biology. A typical cascade comprises three tiers of sequentially activating kinases: MAPK kinase kinase (MKKK), MAPK kinases (MKK), and the terminal MAPK Fig. . Plants selective activation, compartmentalization, combinatorial signaling, and cross‐pathway inhibition. We focus on plant signaling pathways that share MAPK cascade components and compare them with those of animals and yeast upon which specificity models were built . Th. Th27]. MAPK [et al. [vice versa, indicating that these docking interactions provide intrinsic selectivity at MKK‐MAPK level  to specific tissues or subcellular localizations Fig. . For botK family , 53. TheK family . How dynK family , 54 and Arabidopsis MKK4/5‐MPK3/6 cascade can regulate distinct biological processes by phosphorylating different MAPK substrates in different tissues [GPT1, a gene important for lipid body accumulation during pollen development [MKKK‐MKK4/5‐MPK3 to upregulate genes important for embryogenesis [With immobile cells and well‐defined tissue organization in plants, substrate compartmentalization in different tissues could contribute to signaling specificity. The same  tissues . Many MA tissues , a large tissues , 56. MKK tissues , but act tissues . WRKY34  tissues , where ielopment . WRKY33 elopment , 61. Difogenesis , 63, 64.MKKK‐MKK6‐MPK4 cascade that controls cytokinesis in Arabidopsis [Clustering of plant MAPK cascade genes from all tiers by expression similarities broadly reflects tissue‐specific patterns . These “bidopsis , 68. ThiMKKK, and they activate MPK3/6, but show distinct activities during different stomatal development stages. Subcellular localization of autoactive MKK7/9, but not MKK4/5, to the mitochondria determines the former's ability to promote stomatal clustering during the FAMA stage [MKKK activation at plasma membrane, MPK6 deactivation in the nucleus) was shown to control stomatal fate [Different subcellular localization of MAPK pathway elements can provide specificity. As the example above, both MKK6 and its downstream MPK4 and MPK6 accumulate at the equatorial plane of the phragmoplast—a microtubule structure formed between the two daughter cells before division , 69. So,MA stage . InteresMA stage , 71. Rectal fate .Furthermore, plant receptor complexes are recruited to distinctive microdomains that could compartmentalize signals near the plasma membrane . This reMAPK is a shared component central to signaling pathways that determine cell migration, division, and survival. To evoke the specific output of epithelial cell survival, ERK requires the combined inputs from growth factor signaling and cell adhesion [MKKK‐MEKMKK‐ERKMAPK cascade, while the cell adhesion elicits regulators that could modulate cascade activation and nuclear translocation of ERK [Combinatorial signaling occurs when the concomitant action of two or more independent signals are required to evoke a particular output, which effectively acts as a “molecular AND gate”  Fig. 5A5A. Even adhesion . The epin of ERK , 76.Arabidopsis, the induction of the defensin gene PDF1.2 requires simultaneous activation of jasmonic acid (JA) and ethylene (ET) pathways [PDF1.2 expression thus serves as the “AND gate” that integrates two separate inputs from JA and ET: lacking either pathway would abolish the output  versus the invasive growth pathway (Kss1MAPK) in yeast. Though the mating pheromone could activate both Fus3 and Kss1, the mating specific Fus3 prevents Kss1 from activating Tec1 (a transcription regulator for invasive growth) by suppressing Kss1 activity [When a downstream component of pathway A inhibits a downstream component of pathway B, it could diverge pathway outputs even with shared components: Input A only produces output A, as it prevents output B; and input B activates output B only when input A is absent Fig. . An examactivity  and by pactivity , 91  and inhibits defense‐related genes  [Bilateral inhibition of two pathways could improve mutual insulation. In Arabidopsis, downstream of ET signaling, MKK9‐MPK6 activates ERF1 , a trans as VSP2 , 93. Con PDF1.2) , 92. Thi PDF1.2) . In effe PDF1.2) , 95. Dis PDF1.2) , 97. ThiSignaling networks often employ more than one insulating mechanism to improve specificity. Fine‐tuning of signaling activity at different levels could also contribute to specificity: modulation of the amplitude and/or duration of cascade inputs, regulation of activation and deactivation kinetics, positive and negative feedback loops.Input modulation occurs at multiple levels such as ligand processing/diffusion and receptor complex assembly/stability. Recent findings show that mechanical signals modulate input intensity via regulating receptor expression in both plants and animals. Plant tissue damage increases pathogen‐pattern recognition receptor FLS2 expression to enable perception of damage‐inducing invaders , while sMAPK are sufficient to dictate the outcomes of differentiation versus proliferation in mammalian PC12 cells [et al. [Sustained versus transient signals of ERK12 cells . Sözen e [et al.  suggeste [et al. .MAPKs are deactivated faster in the nucleus than the cytoplasm, thus requiring continuous shuttling of activated proteins into the nucleus. Indeed, the slower nuclear trafficking of ERK1 compared to ERK2 reduces its capacity to produce signaling output [Activation versus deactivation rates of kinases control signal amplitude and duration. Many of the insulating mechanisms described here are conditioned by these rates. For example, compartmentalization can be further tuned by differential activation/deactivation rates in distinct compartments. ERK1/2g output . MAPK deg output . MAPK trg output .MAPK [Positive and negative feedback mechanisms are common in MAPK cascades to amplify or tightly control outputs. For example, the yeast scaffold Ste5 contains binding domains for both activation and downregulation of the target Fus3MAPK , and actMAPK . These dMAPK , 93, 102MAPK . The posMAPK .Plant MAPK cascade signaling shows more complexity compared to yeast and mammals, likely as an adaptation to the enormous expansion at receptor/ligand input level. The animal/yeast models appear more clearcut due to two reasons: (a) less crosstalk, (b) distinct/unambiguous readouts for outputs. In plants, many crosstalks are perhaps desirable. This is evident by the well‐known extensive interconnections among plant hormonal pathways  and largThe frequent crosstalks raise the challenge to distinguish common from unique outputs when assessing pathway specificity. Current readouts for pathway outputs in the plant field rely heavily on fast and highly responsive gene/protein markers and sometimes include signatures that are common among multiple pathways . It is often not known (not tested) if a particular pathway's marker could be as responsive if not more responsive to other inputs. Given the nature of complex signaling crosstalk in plants, many of these pathway markers are likely to respond to inputs other than the authentic ones. Furthermore, output assessments using collective functional traits , which can mislead functional interpretation. Furthermore, some members of MAPK cascade signaling are known to be guarded by the plant immune system, and thus perturbation of their function  leads to autoimmune phenotypes  that mask their function in other processes [Studying MAPK cascade signaling in plants has relied on functional analysis, typically gain or loss of function analyses. Given these multifunctional kinases often have discrete functions in different cell types, the major limitation is that constitutive global perturbation of these widely expressed, multifunctional proteins often lead to pleiotropic phenotypes  belongs to the heterotrimeric G proteins complex in Arabidopsis, which comprise one Gα (GPA1), one Gβ (AGB1), and three Gγ subunits .ANPs: Arabidopsis Nucleus and Phragmoplast localized protein kinases  belong to the MKKK family.At + Gene name: Genes or proteins from Arabidopsis thaliana (At).BASL: Breaking of Asymmetry in the Stomatal Lineage (BASL) is a polarity protein leading to the asymmetric division during stomatal formation.BSL phosphatases: The BSU1 (bri1 suppressor 1)‐Like (BSL) phosphatase family play key roles in stomatal development. Three BSL phosphatases  directly interact with BASL to enable asymmetric cell division. At the plasma membrane, all four members of BSL contribute to positive regulation of YDA to promote stomatal differentiation, whereas in the nucleus, BSL2, BSL3 and BSU1 impose negative regulation on MPK6 that suppresses stomatal cell fate.CDPKs: Calcium‐Dependent Protein Kinases play important roles including growth, development, stress responses and hormonal signalling in plants.Crosstalk: Originally defined as unwanted signals in a communication channel caused by leakage from another circuit. In this review, it refers to the interferences between the pathways , it can be either desirable or undesirable.ERF1: Ethylene response factor 1 is a bZIP transcription factor. Its promoter is targeted by the EIN3 (Ethylene insensitive 3) transcription factor.FAMA stage: Three sequential stages of stomatal differentiation are defined by three different bHLH transcription factors: the SPCH stage initiates the transition of a cell to start division and stomatal lineage, the MUTE stage specifies guard mother cell, which will give rise to the guard cells, and the FAMA stage starts the final differentiation of the guard cells [rd cells .Fus3: Cell Fusion 3 is a yeast MAPK involved in mating decision. It is only activated during the mating pathway and remains inactive in other situations.GPT1: Gluocose‐6‐Phosphate Translocator 1 is a gene important for lipid body accumulation during pollen development.Heterotrimeric G proteins: Transmit diverse extracellular cues by coupling with the plasma membrane‐localized receptors and different signalling proteins inside the cells. It consists of three subunits: Gα, Gβ and Gγ.KSR1: Kinase Suppressor of Ras‐1 is a mammalian scaffold protein. The Ras protein family belongs to the small GTPases.Kss1: Kinase suppressor of Sst2 mutations‐1 is a yeast MAPK homologous to Fus3. It regulates filamentation and invasive growth.Mammalian MAPK families  including JNK1/2/3, are also involved in stress responses, T cell differentiation and inflammation; ERK5 is specifically activated by MEK5MKK, and is involved in endothelial cell function.MAPK (or MPK): Mitogen‐Activated Protein Kinase. MPK is used when a specific MPK in plants is mentioned.MAPK cascade: Commonly refers to the sequential phosphorylating cascades of MKKK‐MKK‐MAPK. This review does not mention MAP4Ks that act upstream of MKKKs. For easy recognition of cascade elements with noncanonical names, superscripts of MAPK, MKK or MKKK are used.MKK : Mitogen‐activated protein Kinase Kinase.MKKK : Mitogen‐activated protein Kinase Kinase Kinase.MYC2: A bHLH transcription factor that acts as a master regulator of JA signalled plant immune responses.PDF1.2: Plant Defensin 1.2 contributes to broad spectrum disease resistance and is ET and JA responsive.RACK1: Receptor for Activated C‐Kinase 1 is a member of the WD40 repeat family of b‐propeller proteins. It was discovered through its ability to function as a scaffold protein, stabilizing signalling complexes involving protein kinase C.RLK/Pelle: The plant Receptor Like Kinase with its kinase domain sharing homology to a cytoplasmic protein kinase (Pelle) that establishes dorsoventral polarity in Drosophila embryos. Pelle genes are also involved in immune response. Due to kinase homology, RLK/Pelle is also sometimes called IRAK (interleukin‐1 receptor‐associated kinase) which plays a central role in inflammatory responses in mammalian immune cells.ROS production: Reactive Oxygen Species is a normal product of plant cellular metabolism and increased production is triggered by various environmental stresses.SCRM: SCREAM is a basic helix‐loop‐helix (bHLH) transcription factor important for stomatal development. SCRM functions as a scaffold to bring MPK3/6 in proximity with SPEECHLESS (SPCH), thereby allowing SPCH phosphorylation and downregulation to inhibit stomatal cell fate.Ste5: Sterility 5 is a yeast MAPK scaffold protein. It is recruited to the membrane upon induction of mating by appropriate mating pheromones.SUMM2: A nucleotide‐binding leucine‐rich repeat (NLR) protein that guards the MKKK1‐MKK1/2‐MPK4 cascade, which triggers cell death when the cascade activity is disrupted.Triple response: Typical ethylene response phenotypes observed in dark grown seedlings: shortening and thickening of hypocotyls and roots and exaggerated apical hook curvature.VIP1: VirE2‐interacting protein 1 is a bZIP transcription factor.YDA: YODA is a MKKK that functions for stomatal development, embryo development and immune responses.ZAR1: Zygotic arrest 1 is a leucine‐rich repeat receptor‐like kinase that controls zygote elongation and asymmetric division via the ZAR1‐YDA‐MKK4/5‐MPK3/6 cascade.The authors declare no conflict of interest.YM conceived the idea. YM and JN wrote the manuscript and made figures.


Despite the development of therapeutic agents that selectively target cancer cells, relapse driven by acquired drug resistance and resulting treatment failure remains a significant issue. The highly conserved Hedgehog (HH) signaling pathway performs multiple roles in both development and tissue homeostasis, and its aberrant regulation is known to drive the pathogenesis of numerous human malignancies. However, the role of HH signaling in mediating disease progression and drug resistance remains unclear. This is especially true for myeloid malignancies. The HH pathway, and in particular the protein Smoothened (SMO), has been shown to be essential for regulating stem cell fate in chronic myeloid leukemia (CML). Evidence suggests that HH pathway activity is critical for maintaining the drug-resistant properties and survival of CML leukemic stem cells (LSCs), and that dual inhibition of BCR-ABL1 and SMO may comprise an effective therapeutic strategy for the eradication of these cells in patients. This review will explore the evolutionary origins of HH signaling, highlighting its roles in development and disease, which are mediated by canonical and non-canonical HH signaling. Development of small molecule inhibitors of HH signaling and clinical trials using these inhibitors as therapeutic agents in cancer and their potential resistance mechanisms, are also discussed, with a focus on CML. Drosophila melanogaster .,44.43,44Numerous small molecule inhibitors of HH signaling have been developed for the treatment of a variety of human cancers. Many of these are direct SMO antagonists, binding to the SMO receptor and blocking downstream activation of GLI and other HH effector proteins ,52. ThesCML is a myeloproliferative disease characterized by the presence of a constitutively active BCR-ABL1 tyrosine kinase fusion gene that plays a key role in driving the oncogenic signaling pathways of the disease. While the advent of tyrosine kinase inhibitors (TKI) has been effective in managing the disease, functional cure is challenging to attain due to BCR-ABL1-independent resistance mechanisms and various treatment escape mechanisms available to leukemic stem cells (LSC) ,55,56,57−/− HSCs retrovirally transduced with BCR-ABL1/GFP were less able to induce leukemia when injected into immunodeficient mice; disease latency was extended by more than three months in this group compared to the control group, and only 60% of recipients developed lethal disease . Ot. Ot26]. As discussed above, non-canonical HH signaling or SMO-independent mechanisms are also drivers of resistance to SMO inhibitors. For example, in gastrointestinal cancer, HH signaling increases gene expression of ATP-binding cassette subfamily G member 2 (ABCG2) via GL1, enhancing drug efflux and reducing drug concentrations in cancer cells, which could generate chemoresistance . In pancAn important question raised by these studies of resistance to SMO inhibitors relates to the origin of HH pathway activation in CML. Canonical HH signaling commences with the binding of HH ligands to the transmembrane protein PTCH. HH ligands can be supplied to target cells in an autocrine manner, or from surrounding stromal cells or CML cells in a paracrine manner B–D. HoweSince the characterization of the HH gene in 1980, significant advancements have been made in establishing its importance both in the development and in driving the pathogenesis of cancer. Despite the development of several HH pathway inhibitors that have been tested in clinical studies for a range of different solid tumor pathologies, clinical studies of these inhibitors in hematological malignancies, especially CML, remain limited. Even for existing SMO inhibitors, toxicity and resistance remain significant issues. Thus, complementary therapeutic strategies including the use of next-generation SMO inhibitors, combination therapies, and genetic suppression of HH pathway proteins using shRNA or CRISPR-Cas9, are urgently required.


Cancer treatment, in particular with gonadotoxic potential, may affect the fertility of cancer patients and cause temporary or permanent damage to the reproductive organs and glands that control fertility. Taking into account that some patients ultimately do not lose fertility during treatment, some do not survive cancer therapy, and some do not decide to use cryopreserved reproductive material, the review analysed the percentage of usage of cryopreserved reproductive material collected before treatment to preserve the fertility of patients after cancer treatment. The obtained review results indicate a low return/usage rate of cryopreserved reproductive material among both women and men. This review highlights potential organizational issues related to storage costs, space needed, and the use or disposal of stored material. Considering the increase in the number of cancer patients, the scale of this problem may turn out to be significant in the coming years.Background: Many cancer treatment methods can affect fertility by damaging the reproductive organs and glands that control fertility. Changes can be temporary or permanent. In order to preserve the fertility of cancer patients and protect the genital organs against gonadotoxicity, methods of fertility preservation are increasingly used. Considering that some patients ultimately decide not to use cryopreserved reproductive material, this review analysed the percentage of post-cancer patients using cryopreserved reproductive material, collected before treatment as part of fertility preservation. Methods: A systematic search of studies was carried out in accordance with the Cochrane Collaboration guidelines, based on a previously prepared research protocol. The search was conducted in Medline (via PubMed), Embase (via OVID), and the Cochrane Library. In addition, a manual search was performed for recommendations/clinical practice guidelines regarding fertility preservation in cancer patients. Results: Twenty-six studies met the inclusion criteria. The studies included in the review discussed the results of cryopreservation of oocytes, embryos, ovarian tissue, and semen. In 10 studies, the usage rate of cryopreserved semen ranged from 2.6% to 21.5%. In the case of cryopreserved female reproductive material, the return/usage rate ranged from 3.1% to 8.7% for oocytes, approx. 9% to 22.4% for embryos, and 6.9% to 30.3% for ovarian tissue. In studies analysing patients’ decisions about unused reproductive material, continuation of material storage was most often indicated. Recovering fertility or death of the patient were the main reasons for rejecting cryopreserved semen in the case of men. Conclusion: Fertility preservation before gonadotoxic treatment is widely recommended and increasingly used in cancer patients. The usage rate is an important indicator for monitoring the efficacy of these methods. In all of the methods described in the literature, this indicator did not exceed 31%. It is necessary to create legal and organizational solutions regulating material collection and storage and to create clear paths for its usage in the future, including by other recipients. According to the definition by the International Classification of Diseases 11th Revision, female infertility (ICD-11: GA31) is a “disease of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse” [According to the data of the International Agency for Research on Cancer (based on the GLOBOCAN estimates), in 2020 there were approximately 3.1 million new cases of cancer among people of reproductive age aged 15–49) [9 4,5,6,,6,7,8.In 2006, Dr. Teresa K. Woodruff of Northwestern University launched a new field of medicine that combines oncology with reproductive health, calling it “oncofertility”. The interdisciplinary initiative “Oncofertility Consortium”, established by her, focuses on problems related to healthcare and the quality of life of young cancer patients, in particular on issues related to fertility after cancer .In order to preserve the fertility of cancer patients and protect the genitals against gonadotoxic effects (chemotherapy and/or radiotherapy), the following are used in women: oocyte cryopreservation, embryo cryopreservation, ovarian tissue cryopreservation, gonadal shielding, ovarian transposition (oophoropexy), surgical techniques (trachelectomy), and treatment with gonadotropin-releasing hormone agonist (GnRHa) ,13,14,15Factors affecting the patient’s decision regarding the usage of the above-mentioned methods may include personal beliefs, religious and cultural limitations, prognosis, the patient’s knowledge of fertility preservation methods, as well as the method of disseminating information about the possibility of fertility preservation by medical personnel ,21,22,23Due to the increasing usage of fertility preservation methods and emerging discussions on the ethics of the above-mentioned procedures (in terms of embryo freezing) and subsequent usage of cryopreserved materials, a review of scientific evidence describing the reproductive results obtained as a result of using fertility preservation procedures among cancer patients was performed. Considering that some cancer patients do not lose fertility during treatment, some do not survive cancer therapy, and some ultimately do not decide to use cryopreserved reproductive material, this review analyses the percentage of patients using cryopreserved reproductive material collected before treatment to preserve fertility.Bearing in mind the above, the main objective of the article is to analyse the usage of cryopreserved reproductive material collected from patients before cancer treatment as part of fertility preservation.The analysis was carried out based on the results of available studies. The search was based on a detailed protocol developed prior to the commencement of this work. It takes into consideration the criteria for including studies in the review, the search strategy, the method of selecting studies, and the planned methodology for conducting the analysis. The review was performed according to the Cochrane Collaboration guidelines .The analysis included clinical trials that met the criteria regarding:Population: people diagnosed with cancer;Interventions: fertility preservation ;Alternative technologies (comparators): not restricted;Outcome: return/usage rate, decisions/disposal of unused reproductive material;Types of studies: systematic reviews, experimental or observational studies.The review did not include studies that analysed fertility preservation methods in patients undergoing cancer treatment that did not require the collection and storage of reproductive material, e.g., gonadal protection, hormone therapy, conserving treatment, and ovarian transposition.The following sources of medical information were searched for studies published in the last 10 years : Medline (via PubMed), Embase (via Ovid), and The Cochrane Library. The search of the databases was carried out on 12 June 2023 in accordance with the search strategies presented in the At all stages of the review, studies were selected by two analysts working independently (K.W. and J.Ś.). Inconsistencies were resolved by consensus, with the participation of a third independent analyst (W.M.).The review included publications that clarified the presented methodology, were of high quality, and had a low risk of error.Based on the included publications, data from each publication were summarized in terms of two main outcomes . In terms of the usage of cryopreserved reproductive material in cancer patients, the results were presented in tabular and descriptive form (separately for women and men). Reproductive material that was cryopreserved was analysed and the number of used materials/return for materials, number of patients who cryopreserved material, and the return/usage rate were indicated. In the case of the decisions/disposal of unused reproductive material outcome, information was presented regarding the number of patients who cryopreserved material, reproductive material, disposal of unused cryopreserved material, and, additionally, usage rate.The study selection stages are presented in The most common reasons for the exclusion of studies from the analysis were issues related to methodology  and intervention (preserving fertility among people without cancer).As part of this review, sixteen publications were found indicating the percentage of female cancer patients who underwent fertility preservation procedures, who after their treatment decided to use cryopreserved reproductive material ,38,39,40According to the results of the Fraison 2023 meta-analysis, 4.6% of women used cryopreserved oocytes for fertilization under assisted reproductive technology (ART), and approx. 9% of women decided to get pregnant using cryopreserved embryos . In turnAs part of the studies analysed in the found reviews, the return rate was 3.1% to 7.5% for cryopreserved oocytes ,35,38, 1In a 25-year prospective cohort study, 8.7% of women returned to attempt pregnancy using their stored oocytes . MoreoveBelow are the individual results of the studies regarding the percentage of women who used reproductive material after completing cancer treatment as part of fertility preservation .As part of the analysis of publications found in the review, studies were also indicated that determined the percentage of men undergoing the FP procedure who, after completing cancer treatment, returned and used cryopreserved reproductive material (semen) for fertilization under ART (n = 10) ,48,49,50According to the results of individual studies, the return/usage rate did not exceed 22%, and it ranged from 6.5%  to 21.5%Below are the individual results of the studies on the number of men undergoing cancer treatment who underwent cryopreservation as part of FP and the return/usage rate of the material under assisted reproduction after the completion of cancer treatment .On the basis of five studies, the results regarding the decision to transfer or reject unused cryopreserved reproductive material were presented ,45,46,50Depending on the observation period, cancer patients most often continued to store the cryopreserved material  ,46,50. IIn turn, the main reasons for the rejection of the cryopreserved material (semen) were regaining fertility through the return of reproductive functions after cancer treatment ,46 and tThe individual results of the studies concerning the decisions and disposal related to the unused reproductive material of cancer patients are presented below .In the Sankara-Narayana 2019, Muller 2016, and Johnson 2013 studies, the results concerning the disposal of unused cryopreserved material and the usage rate refer to all banked samples (100%). In the Ferrari 2016 study, the discard rate included a meta-analysis of eleven studies (n/N = 691/4291), and the usage rate refers to the number of all patients included in the meta-analysis . In the case of the Moravek 2018 study, the results refer to the material disposal selected at the time of cryopreservation.Based on the results of studies found as part of the systematic search, the usage of cryopreserved reproductive material, collected from patients before cancer treatment as part of fertility preservation, was analysed. The publications included in the review covered various fertility preservation methods .The usage rate varied depending on the study and the type of cryopreserved material. The usage rate of cryopreserved sperm ranged from 2.6% to 21.5% ,48,49,50Considering the above results, attention should be paid to potential organizational problems related to the costs of storage, the space needed, and the usage or disposal of material  . Considep < 0.05). Therefore, there is a visible difference in the return rate in the case of healthy women and women suffering from cancer. The above situation may be related primarily to the death of oncological patients and the lack of loss or recovery of the functions of reproductive organs after recovery from the disease. These situations reduce the level of use of frozen reproductive materials. In the case of women not suffering from cancer, it should be borne in mind that freezing reproductive materials is most often associated with diagnosed infertility and planned in vitro fertilization procedures. The use of frozen eggs/embryos will therefore be more frequent (including due to the sometimes necessary repetition of in vitro fertilization procedures). To complete the analysis, databases and websites of scientific societies were manually searched for clinical practice guidelines on fertility preservation against gonadotoxic therapy. Recommendations from the last 10 years were sought. The main conclusions are presented below.In two studies included in this review, the control groups were non-oncological women  and womeThe vast majority of recommendations indicate the need to inform all people diagnosed with cancer about the potentially harmful impact of the planned treatment on fertility. Before the commencement of treatment, the available fertility preservation methods should be discussed with the patient ,64,65,66Almost all documents found indicate cryopreservation of oocytes and/or embryos as a method of fertility preservation in women of reproductive age, which should be implemented before commencing cancer treatment with gonadotoxic potential ,66,67,68In the population of men and boys after puberty, in whom cancer treatment with gonadotoxic potential is planned, the basic method of preserving fertility is semen cryopreservation ,66,67,68Cryopreservation of testicular tissue is an option for pre-pubertal fertility preservation in boys. However, the societies emphasize that this is an invasive and experimental method, and should only be considered in clinical trials ,62,63,66When analysing the issue of cryopreservation of reproductive materials, the costs associated with this procedure should also be taken into account. In one publication, the authors attempted to estimate the costs of fertility preservation activities among women undergoing cancer treatment. Cost estimates in this case referred to oocyte cryopreservation. The publication did not indicate the general cost-effectiveness threshold for the implementation of the discussed intervention; however, based on the collected data from the systematic search, it was estimated that the total cost of one cycle  ranges between USD 7000 and USD 14,000. In addition, pharmaceuticals used in the collection of oocytes from women also play an important role. In this case, the cost of drugs for one egg cell ranges from USD 2000 to USD 7000. An additional element significantly affecting such high costs of oocyte cryopreservation involves the costs associated with long-term storage of the collected cells. In this case, annual fees for stored cells range from USD 350 to USD 600 .In another publication, the authors attempted to estimate the cost-effectiveness of semen cryopreservation as a method of preserving the fertility of men with testicular cancer. The publication did not indicate the general cost-effectiveness threshold for semen cryopreservation; however, according to estimates, the average cost of the procedure using a single vial was USD 754 in the first year of intervention. In turn, each subsequent year of storage of the collected reproductive material costs an average of USD 343 .Taking into account the high level of non-use of frozen reproductive materials among people with cancer, attention should be paid to the validity of the distinction between gametes and embryos in legal acts, with particular emphasis on the handling of each of these reproductive materials. For example, in the Polish Act on the Treatment of Infertility, a reproductive cell is defined as “a human male reproductive cell (human sperm) or a human female reproductive cell (human egg cell) intended for use in a medically assisted procreation procedure”. In turn, an embryo is defined as “a group of cells resulting from the extracorporeal fusion of female and male reproductive cells, from the completion of the process of fusion of the germ cell nuclei (karyogamy) to the moment of implantation in the uterine mucosa” .Pursuant to the provisions of the Act, reproductive cells may be destroyed or donated for research purposes at the express request of the donor at any time. In turn, embryos created from the collected reproductive cells are transferred for embryo donation  after the expiry of the deadline specified in the agreement concluded between the reproductive cell and embryo bank and the donors. However, this deadline cannot be longer than 20 years from the date on which the embryos were transferred to the bank of reproductive cells and embryos for their storage. Embryo donation may also take place in the event of the death of both embryo donors or—if the embryo was created as a result of non-partner donation—the death of the recipient and her husband or a person in cohabitation with her . HoweverA very important issue is the conscious decision of people with cancer and their partners regarding the method of future fertility protection they choose. In potential legal cases described in the literature, there may be situations where the partner does not consent to the woman’s use of embryos after the couple breaks up ,72. For Another aspect is the need to carry out the in vitro fertilization procedure for people who decide to freeze and later use eggs, embryos, or sperm. For some people, carrying out such a procedure may be unacceptable due to their beliefs. The solution for these people may be collecting and freezing ovarian tissue and its subsequent autotransplantation. In the case of men, the solution of freezing testicular tissue is not a procedure with proven effectiveness, therefore other procedures are performed in their case (mainly sperm freezing), which consequently lead to in vitro fertilization.Fertility preservation in the case of gonadotoxic treatment is widely recommended and increasingly used in cancer patients. The basic methods include cryopreservation of oocytes and embryos in women and semen in men. The usage rate is an important indicator for monitoring the efficacy of these methods. In all of the methods described in the literature, this indicator did not exceed 31%.Unused cryopreserved reproductive material was usually stored further. Some of the material was donated for scientific purposes or to other people in need. Important factors that prevent the usage of cryopreserved material include the patient’s death or regaining fertility through the return of reproductive functions after cancer treatment.It is necessary to create legal and organizational solutions regulating the collection and storage of material and to create clear paths for its usage in the future, including by other recipients.Only publications in English were included in the review. The studies were characterised by high heterogeneity . In the context of the analysed endpoints, individual studies covered a varied follow-up period, which ranged from several months to several decades (in the case of retrospective studies).


Cancer cachexia is a multifactorial condition characterized by body weight loss and skeletal muscle wasting that negatively impacts the quality of life and survival of cancer patients. In this paper, we found that ursolic acid (UA) alleviated cancer cachexia and prevented muscle wasting via activating SIRT1, and thence inhibiting phosphorylation levels of NF-κB and STAT3. Furthermore, UA remained effective in the advanced stages of cancer cachexia. This is significant because no approved drug for the treatment of cancer-related muscle atrophy is available. UA may be a potential drug for cancer cachexia and has the possibility of translational application to clinical treatment.Skeletal muscle wasting is the most remarkable phenotypic feature of cancer cachexia that increases the risk of morbidity and mortality. However, there are currently no effective drugs against cancer cachexia. Ursolic acid (UA) is a lipophilic pentacyclic triterpene that has been reported to alleviate muscle atrophy and reduce muscle decomposition in some disease models. This study aimed to explore the role and mechanisms of UA treatment in cancer cachexia. We found that UA attenuated Lewis lung carcinoma (LLC)-conditioned medium-induced C2C12 myotube atrophy and muscle wasting of LLC tumor-bearing mice. Moreover, UA dose-dependently activated SIRT1 and downregulated MuRF1 and Atrogin-1. Molecular docking results revealed a good binding effect on UA and SIRT1 protein. UA rescued vital features wasting without impacting tumor growth, suppressed the elevated spleen weight, and downregulated serum concentrations of inflammatory cytokines in vivo. The above phenomena can be attenuated by Ex-527, an inhibitor of SIRT1. Furthermore, UA remained protective against cancer cachexia in the advanced stage of tumor growth. The results revealed that UA exerts an anti-cachexia effect via activating SIRT1, thereby downregulating the phosphorylation levels of NF-κB and STAT3. UA might be a potential drug against cancer cachexia. Cancer cachexia is a multifactorial syndrome characterized by persistent loss of skeletal muscle, with or without loss of fat mass, which is not completely reversed by conventional nutritional support and leads to progressive functional impairment . Cancer Systemic inflammation and oxidative stress play important roles in skeletal muscle atrophy ,6. Many SIRT1, an NAD+-dependent protein deacetylase, is the best studied of the seven members of the mammalian sirtuin family and has been shown to regulate a large number of biological processes , such asUrsolic acid UA, A is a peIn this study, we explored the role and mechanisms of UA treatment in cancer cachexia. We found that UA is effective at reversing Lewis lung carcinoma tumor cell (LLC)-conditioned medium-induced myotube atrophy in vitro and ameliorating LLC-induced animal cachexia and muscle atrophy in vivo. Mechanistically, UA inhibited inflammatory responses, decreased the concentration of cytokines in serum, and alleviated symptoms of cancer cachexia via activating SIRT1, thence downregulating the ubiquitin E3 ligase expression levels and the phosphorylation of NF-κB and STAT3. An inhibitor of SIRT1 (Ex-527) could restore the above phenomena triggered by UA, indicating that the therapeutic effect of UA was mediated by SIRT1 activation. Our studies provide insights into potential treatment strategies for cancer cachexia and suggest that UA might be a promising new approach against cancer cachexia.UA and Ex-527 were acquired from MedChemExpress . The primary antibody against myosin heavy chain  was acquired from R&D Systems . The primary antibodies against STAT3 (#9139), p-STAT3 (Yyr705) (#9145), NF-κB (p65) (#8242), p-NF-κB (p-p65) (Ser536) (#3033), and SIRT1 (#9475) were acquired from Cell Signaling Technology ; FOXO3a (#10849-1-Ap), FOXO1 (#18592-1-Ap), NOX4 (#14347-1-Ap), and GAPDH (#10494-1-Ap) were acquired from Proteintech ; and Atrogin-1 (ab168372) and MuRF1 (ab172479) were acquired from Abcam .2 in high-glucose DMEM medium . In DMEM medium, 10% fetal bovine serum  was added, as was 1% penicillin/streptomycin . When C2C12 myoblasts grew to 80–90%, myotube formation could begin to be induced. DMEM medium containing 2% horse serum  was used as a differentiation medium (DM) in place of the proliferation medium. The DM was transposed daily. On days 6–8, myotube differentiation was complete.Murine myoblasts (C2C12 cells) and LLC cells were purchased from Peking Union Medical College Cell Bank . C2C12 and LLC cells were incubated in a 37 °C incubator with 5% COLLC cells were seeded in 10 mm dishes and reached 90% confluence. The proliferation medium was replaced with a serum-free DMEM medium and cultured for 48 h continuously. LLC medium was purified by centrifugation at 4 °C for 20 min at 3000 rpm, and then a sterile filter of 0.22 mm was used to filter the sample. LLC-conditioned medium (LCM) was the filtered medium and kept at −80 °C or used immediately. LLC tumor-conditioned medium (TCM) was a 1:1 dilution of LCM and DMEM containing 2% horse serum containing cachexia factors that caused myotube atrophy.4 cells/well and differentiated into myotubes. The myotubes were cultured with various concentrations of UA  at 37 °C for 24, 48, and 72 h. After incubation, 10 μL of CCK-8 solution was added to each well and incubated for another 2 h at 37 °C. Microplate readers  were used to measure absorbance at a wavelength of 450 nm.To evaluate cell viability, the CCK-8 assay was used. In 96-well culture plates, C2C12 myoblasts were seeded at a density of 1 × 10In a 24-well plate, different concentrations of UA were used to treat C2C12 myotubes for 72 h. After being washed with PBS 3 times, C2C12 myotubes were fixed for 20 min with 4% paraformaldehyde, permeabilized for 30 min with 0.5% Triton X100, and blocked with 1% BSA  at room temperature. After that, the primary antibody against MyHC was incubated overnight at 4 °C, followed by the secondary antibody  at room temperature for 1 h. DAPI was then used to stain myotubes. Following this, a fluorescence microscope  was used to capture myotube images. Over 100 myotube diameters were measured with ImageJ software in at least 10 fields.6 cells/100 μL PBS) were inoculated subcutaneously on the right flanks of the mice. The endpoint for all animal studies was day 21 post-inoculation.All animal experiments complied with the ARRIVE guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. They were approved by the Institutional Animal Care and Use Committee of the Tongji Medical College, Huazhong University of Science and Technology. Six-week-old healthy male C57BL/6 mice were raised in a specific-pathogen-free environment with a constant temperature and humidity with a dark/light cycle of 12:12 h. LLC cells : control group, LLC tumor-bearing group, LLC tumor-bearing + 100 mg/kg UA-treated group, and tumor-bearing + 200 mg/kg UA-treated group. The control group was injected with the same amount of PBS without LLC cells. After tumor inoculation on day 7, mice received different doses of UA treatment by oral gavage daily. The detailed protocol of the experiment is depicted in 2 . Tumor-fThe second study investigated the molecular mechanism of UA alleviating tumor-induced cachexia. All mice were randomly divided into five groups (n = 10 per group): control group, LLC tumor-bearing group, LLC tumor-bearing + 200 mg/kg UA-treated (i.g.) group, LLC tumor-bearing group + 10 mg/kg Ex-527-treated (i.p.) group, LLC tumor-bearing + 200 mg/kg UA-treated + 10 mg/kg Ex-527-treated group. The xenograft tumor models were used to select the doses of Ex-527, which is a SIRT1 inhibitor  . The conThe third study assessed the efficacy of UA in different stages against cancer cachexia. All mice were randomly divided into four groups (n = 10 per group): the control group, which received the same amount of solvent, and the tumor-bearing group which started receiving UA medication  on days 6, 10, and 14 after LLC cell inoculation. All mice were sacrificed on day 21. As explained in the first study, the main characteristics and sample acquisition methods were prepared in the same way.In each group, three gastrocnemius muscles were selected at random, fixed in 4% paraformaldehyde, and embedded in paraffin. Staining with hematoxylin and eosin (H&E) solutions was performed on paraffin-embedded tissues by standard procedures. A Leica microscope was used to capture images of muscle sections. The cross-sectional areas of myofibers were quantified using ImageJ software . In each group, the cross-sectional area of the gastrocnemius muscle per mouse/muscle was approximately 50 fibers.Total RNA was isolated from C2C12 myotubes and muscle samples using RNAiso Plus . The quantity and quality of purified RNA were assessed using a NanoDrop 2000 Spectrophotometer  by measuring the light absorbance at 280 nm, 260 nm, and 230 nm and calculating the 260/280 (A260/280) and 260/230 (A260/230) ratios . Then, 1% agarose gel electrophoresis was applied to check the RNA integrity, followed by visualization using Syngene G . One microgram of total RNA was reverse transcribed into cDNA using Hi Script II QRT SuperMix . ChamQ Universal SYBR qPCR Master Mix  was used to carry out the qRT-PCR according to the instructions. Quantitative detection of mRNA including SIRT1, NOX4, FOXO3a, FOXO1, MuRF1, and Atrogin-1 was performed using a StepOne Real-Time PCR System . These genes, which were normalized to GAPDH and β-actin, were analyzed. The ΔΔCt method was used to analyze the relevant expression levels . The priTotal gastrocnemius muscle and C2C12 myotube protein were extracted on ice for 30 min with RIPA lysis buffer , which contained 1% phenylmethylsulfonyl fluoride (PMSF) and 1% Phosphatase Inhibitor Cocktail . Next, the supernatant proteins were collected after lysis by centrifugation at 12,000 rpm at 4 °C for 15 min. The concentration of protein was measured using BCA Protein Assay Kits . Equal amounts of protein were separated using 10% SDS-PAGE gel and transferred to a 0.45-µm polyvinylidene fluoride membrane . After 1 h of blocking with 5% skim milk at room temperature, the membrane was placed in primary antibodies and incubated at 4 °C overnight. Then, the membranes were incubated with secondary antibodies  at room temperature for 1 h after being washed 3 times each for 10 min with Tris-Buffer saline (TBST) containing 0.1% Tween-20. Finally, A three-time wash was followed by visualization with ECL solution . Syngene G  was used to capture images, and ImageJ software v1.8.0 was used to quantify each blot band.The supernatant was collected by centrifugation of blood collected from mice at 3000 rpm at 4 °C for 15 min. According to the manufacturer’s instructions, levels of IL-6 and IL-1β were quantified using a commercial mouse ELISA kit acquired from Multsciences , and the TNF-α mouse ELISA kit was obtained from Thermo Fisher Scientific ; the serum assay was repeated for each animal.www.rcsb.org (accessed on 13 May 2022)), and we downloaded the structure of UA from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/ (accessed on 13 May 2022)). The exhaustiveness was set to 10, and the rest of the parameters were set by default. Before molecular docking, the protein structures were processed with AutoDocktools1.5.6. The interaction mode of the docking results was analyzed with PyMOL2.3.0. To understand how the SIRT1 protein and UA are bound, molecular docking experiments were conducted in AutoDock Vina 1.1.2. We downloaded the crystal structure of SIRT1 from the RCSB Protein Data Bank  for 24, 48, and 72 h. The results of the CCK8 assay showed that UA was not toxic to C2C12 myotubes at concentrations less than 5.0 μM B, so 1.0Then, we evaluated the anti-cachexia effect of UA in LLC cachexia animals A. The tuTo explore the underlying mechanism of the anti-cachexia effect of UA, we examined the interaction pattern of SIRT1 protein with UA, as well as the influence of SIRT1 activation and muscle atrophy-related signaling pathway changes. Then molecular docking was performed to predict specific binding modes within the active site of SIRT1 by AutoDock Vina 1.1.2. The theoretical binding mode is shown in The mRNA and protein expression of SIRT1 and NOX4 was significantly downregulated in the LLC tumor groups compared to the control group, as well as FOXO1 and FOXO3a in vitro. Meanwhile, UA could dose-dependently increase the expression of the aforementioned proteins B,C. BaseWe examined the alterations of intracellular signaling pathways related to muscle wasting. UA remarkably downregulated the phosphorylation levels of STAT3 and p65 in vitro and in vivo as shown by Western blot A,B. MoreWe used the SIRT1 inhibitor Ex-527  to inveTo delve into the role of SIRT1, we used Ex-527 and assessed its effects on muscle wasting and cancer cachexia in vivo. The detailed protocol of the experiment is depicted in Western blot showed that Ex-527 decreased the enhancive effect of UA on SIRT1 at the protein expression level while reducing the inhibitory effect of UA on NOX4, FOXO1, and FOXO3a protein levels A. MeanwhThe diagnosis of early cancer cachexia has limitations due to the complex mechanism and multiple phenotypes of cachexia, so the early cancer cachexia treatment strategy has poor clinical practicability. In the preceding experiments, UA treatment was initiated at an early stage of the disease when no overt signs of wasting were detected. To investigate whether initiating UA treatment at a later time point could also prevent cancer cachexia, we initiated UA treatment in LLC tumor-bearing mice at 6, 10, and 14 days after tumor cell injection A.On day 6, 10, or 14, LLC tumor/tumor-bearing mice received UA treatment, and body weight loss was limited to 6.0%, 10.1%, and 16.4% C, whilstCancer cachexia is a multifactorial condition characterized by body weight loss and skeletal muscle wasting that negatively impacts the quality of life and survival of cancer patients ,51. ReseUA has shown potential anti-inflammatory , antioxiUA has been demonstrated to increase the sensitivity of cancer cells to chemotherapy drugs by inhibiting NF-κB and enhancing the chemotherapeutic agent-induced cancer cell apoptosis . MoreoveTo explore the molecular mechanism by which UA alleviates cachexia and prevents muscle wasting, we performed molecular docking on UA and SIRT1 protein. The results revealed a good binding effect. The binding energy of UA to SIRT1 protein was −8.5 kcal/mol, and UA formed hydrogen bonds with amino acid residue ASM-147 with lengths of 3.0 Å and 3.1 Å. The regulatory effect of UA on SIRT1 was confirmed in vitro and in vivo. UA upregulated SIRT1 expression and downregulated the MuRF1 and Atrogin-1 expression in both cell and animal experiments, which is consistent with the report that treatment of skeletal muscle satellite cells with UA upregulates the expression of SIRT1 (~35-fold) significantly . MeanwhiSystemic inflammation is one of the important characteristics of cancer cachexia, and inflammatory cytokines IL-1β, IL-6, and TNF-α in the serum of cancer cachexia patients have been confirmed . ElevateIn conclusion, we found that UA alleviates cancer cachexia and prevents muscle wasting via activating SIRT1, and thence inhibiting phosphorylation levels of NF-κB and STAT3. Furthermore, UA remains effective in the advanced stages of cancer cachexia. UA may be a potential drug for cancer cachexia.


Background: Primary care physicians not only coordinate referrals to oncology services but can play a crucial role in successful fertility preservation referrals in cancer-diagnosed patients. Hence, it is important to assess their knowledge and attitudes towards fertility preservation.Methods: An eighteen-item oncofertility survey was administered to primary care physicians between May 2019 to September 2020. Results: A total of forty-six responses were received and analysed. About 60% of primary care physicians did not have adequate knowledge about available fertility preservation options and only 26-32% were aware of international guidelines recommending fertility preservation in cancer patients. Conclusions: Imparting awareness and knowledge of fertility preservation and its options to primary care physicians could enable an integrated cancer care model while also facilitating successful oncofertility referrals in countries like India. However, successful implementation of oncofertility care requires a coordinated referral system among healthcare providers. While it is established that the role of oncologists and reproductive specialists is key for successful oncofertility,, it is also believed that primary care physicians (PCPs) can play an important role in ensuring successful fertility preservation referrals.Oncofertility – integrating cancer care with fertility preservation (FP) – is expected to be an essential part of the standard care for patients, PCPs are the initial contact point for most cancer patients, who are then referred to specialists such as medical oncologists or paediatric hemato-oncologists. However, the role of PCPs in cancer care has expanded further across the cancer care continuum from prevention, diagnosis, treatment to cancer survivorship as it has been reported that cancer patients preferred their PCPs to be involved in their cancer care., A recent study has reported patients discussing their cancer treatment options with their PCPs, in addition to their oncologists, demonstrating the important role they play in decision-making. It has been reported that about two thirds of patients with long-term or complex health issues prefer interpersonal continuity with a PCP whom they know, trust and who also knows their medical history., Hence, PCPs not only coordinate referrals, but are also involved in providing general medical care, emotional support and overall decision making for the patient during cancer care. The American Society of Clinical Oncology breast cancer survivorship guidelines, recommend post-treatment physical and psychosocial surveillance by primary care clinicians and expediting referrals to reproductive endocrinologists in survivors experiencing infertility.PCPs inclusive of general physicians (GPs) and paediatricians (PEDs), are considered as gatekeepers of the healthcare system who provide comprehensive, individual-centric and coordinated continuous care. This also allows for families to gather the information required for taking informed decisions regarding fertility preservation thereby maximizing the chances for preserving their fertility before initiation of cancer therapy. Similar approaches are applicable to patients with non-oncological conditions requiring gonadotoxic treatments. It is therefore important for PCPs dealing with cancer patients or patients with non-malignant conditions requiring gonadotoxic therapies, to be knowledgeable both of the adverse gonadotoxic effects of chemo- and radiotherapy as well as the options available to mitigate them. The awareness of PCPs of international fertility preservation guidelines and on the available fertility preservation options are expected to strengthen the likelihood of fertility preservation referrals. Therefore, this study was aimed to assess the awareness, knowledge and perceptions of PCPs towards fertility preservation, along with exploring the existing barriers to oncofertility establishment in India.Fertility preservation while at times is considered to be a burden for patients and their families, can provide opportunities for maximizing reproductive potential of the patient. A multi-disciplinary team that includes the patient’s primary care physician along with the cancer specialists and reproductive specialists will allow for a comprehensive decision-making by the patients. The earlier work included the use of an eighteen-item survey, which was given to oncologists and gynaecologists to explore their attitudes towards fertility preservation. The survey was designed in collaboration with the Oncofertility Consortium, USA. Content validation for the survey was done by experts in the field and a pilot study was conducted on a small group of healthcare providers to validate the comprehensibility of the survey questions (unpublished). In our new study, the survey was administered randomly to GPs and PEDs of the country at various national conferences and virtual platforms from May 2019 to September 2020. Written informed consent was taken from all the subjects participating in the survey.The study was approved by the Institutional Ethics Committee, Kasturba Medical College & Kasturba Hospital, Manipal, India (IEC No: 880/2017), and is in continuation of a previously published study.In brief, the survey contained a total of eighteen questions, of which ten were aimed at understanding the participants’ knowledge, attitudes, and referral trends in oncofertility, including their familiarity with existing oncofertility barriers in India, and sought the respondent’s suggestions, if any, for effective use of fertility preservation. The remaining eight questions covered the demographics of the survey respondents and contextual details such as the number of new cancer patients treated in a month and the patient age groups. The questions were of different complexities, including those of a dichotomous nature, assessment tools with grading scales, multiple choice items where only one response could be selected, multiple response items where more than one answer could be selected, and even open-ended questions. During the analysis, the survey responses were assigned numerical values , and the data was analysed using descriptive statistics calculated with Microsoft Excel. In questions containing grading scales or multiple choice, options such as ‘not aware’, ‘aware but inadequate knowledge’, ‘knowledgeable’, ‘very knowledgeable’, were clubbed as two categories – ‘inadequate knowledge’ and ‘knowledgeable’. For open-ended questions such as suggestions for oncofertility practice, all the responses were considered and grouped based on similarity of the suggestions made. Questions that showed a high variation in responses, such as more than half of the participants not answering, were not included in the analysis.Out of 105 survey forms distributed, 23 responses each from general physicians (GPs) and pediatricians (PEDs) were received, with an average response rate of 43.8%. The demographics of the PCPs are shown inAbout 32% of GPs (7 of 22) and 26% of PEDs (6 of 23) were aware of the American Society for Clinical Oncology (ASCO) guidelines on fertility preservation . When asWhen asked their opinion on the statement, “offering fertility preservation compromises cancer treatment”, 56% of GPs (13 of 23) and 52% of PEDs (12 of 23) disagreed with the statement, indicating a positive attitude towards oncofertility . FurtherThe majority of PCPs (>60%) in both groups were comfortable discussing most of the available FP options. However, when it came to female fertility preservation options, a minority of GPs were not comfortable discussing oocyte freezing , ovarian tissue freezing  or IVF  options, which could be reflective of inadequate knowledge in this area.Participants were asked to select the most appropriate of the listed barriers for effective oncofertility implementation in India. PCPs from both groups felt ‘financial burden’ , ‘lack of patient awareness’  and ‘lack of physician awareness’  to be the biggest barriers to FP. PCPs felt that ‘lack of FP facilities’  and ‘emotional status of patient’  were other important barriers .One of the major suggestions given, in the open-ended questions, was to create ‘awareness among primary healthcare providers in fertility preservation’ . More than 40% of GPs and PEDs felt there was a need for bringing about social awareness among the general public about oncofertility programs and educating the patient and their family. Other suggestions included, ‘routine medical education’, and ‘affordable costs’, which would help bring about effective implementation of oncofertility programs.This study showed, for the first time, the perceptions and attitudes towards oncofertility among PCPs in India. Lack of knowledge about the current international fertility preservation (FP guidelines) and of the available FP options were key observations made in this study. However, the fertility preservation referral rates are only about 4-12% even in European countries. In India, while there are no studies reporting referral rates, the number of patients receiving fertility preservation counselling is quite low. A study reported that only 15% of childhood cancer survivors received information on infertility risks though they were about to receive high-gonadotoxic risk cancer treatment. About 87% of patients with newly diagnosed cancers reported a desire to understand the long-term risks resultant of their disease and treatment.In India, the established fertility preservation options include sperm, oocyte and embryo banking for post-pubertal and adult cancer patients while prepubertal options that are available include ovarian or immature testicular tissue cryopreservation, which are still considered experimental. Other options such as gonadal shielding, GnRH analogs are also offered to cancer-diagnosed patients, similar to developed countries. There could be several contributing factors to the above finding in our study, one of which could be the lack of knowledge about FP in India, since more than 90% of healthcare workers in other parts of the country reported the need for continued medical education programs or seminars in oncofertility. Secondly, the ASCO-FP guidelines are mainly targeted to oncologists and other cancer-care providers, and not to primary care physicians, though organizations such as the American Academy of Pediatrics have recommended fertility preservation counselling for pediatric and adolescent patients. While ASCO has released primary care focused cancer survivorship guidelines for a few cancers,, there is a need for fertility preservation guidelines before the onset of cancer treatment in pediatric and reproductive-age patients, that can be applicable to primary care settings. Also, PCPs’ depth of knowledge pertaining to long-term effects of cancer treatment could be limited due to their lack of exposure to relevant literature pertaining to the field, therefore, conducting educational programs for PCPs to increase awareness and knowledge of the various fertility preservation options could aid PCPs in their discussions with patients. In an online survey conducted by Nahataet al., it was seen that the majority of pediatricians felt inadequately trained in fertility risks and sexual function, which was reflective in their comfort level when discussing such issues. Though our study reported a higher comfort level among PEDs in discussing most FP options, a majority of them have suggested the need for fertility preservation awareness programs.The majority of PCPs participating in our study reported inadequate knowledge of even the established fertility preservation options along with a reduced awareness of the American Society of Clinical Oncology Fertility Preservation (ASCO-FP) guidelines. This is in line with a cross-sectional study in India on female cancer patients of reproductive age revealing that 68% reported a lack of information received from primary physicians about fertility risks and FP options. Among children of 0-14yrs and children and adolescents of 0-19yrs, approximately 52,366 individuals and 76,805 individuals develop cancer every year, respectively. However, the ratio of new cancer cases per clinical oncologist is 677:1 and it has been reported that medical oncologists from low- and middle-income countries such as India have a substantial workload compared to high-income countries.– While the onus of FP counselling is placed on oncologists and gynecologists, in the given situation, an integrated care model involving PCPs would be more appropriate. As PCPs have a continual relationship with the patients and are more familiar with their wishes, PCPs can be a part of the FP referrals or discussions, to enable patients to think through their decisions, rather than these discussions taking place only with the oncologists or surgeons.– For this to succeed however, knowledge dissemination and awareness building of fertility preservation among PCPs in developing countries like India is essential, as emphasized by the findings of the present study. Our earlier study specifically looked at oncologists and gynaecologists’ attitude towards fertility preservation, who also emphasized the need for oncofertility awareness.In India, the burden of cancer is steadily increasing, with the Globocan project predicting 1.7 million new cases by 2035. The successful establishment of fertility preservation in (non)-oncological conditions therefore requires physicians, both primary care and specialists, to have adequate knowledge of fertility preservation and its related guidelines. A shared care model that facilitates sustained collaborations between healthcare providers, both primary and acute care, will ensure successful execution of fertility preservation goals in patients whose fertility could be impacted. Robust strategies including co-ordinated referral pathways, use of tele-health platforms and decision-aids will promote oncofertility referrals by enabling patients to take informed decisions towards preserving their fertility. Anazodoet al., identified several themes for delivery of oncofertility care such as age-appropriate referral pathways, collaboration between healthcare providers in pediatric or adult hospitals and fertility clinics, and oncofertility training of both cancer and non-cancer HCPs in order to improve the quality of care.The scope of oncofertility has started to include fertility preservation in patients with non-oncological conditions who are at high risk of infertility due to gonadotoxic treatments, or individuals with sexual development or auto-immune disorders, and also, in the gender-diverse population. this is one of the biggest hurdles that need to be overcome. Similar economic barriers have been reported in other developing as well as developed countries., Another important barrier reported in the study were lack of fertility preservation awareness among physicians.Barriers in providing fertility preservation care can lead to their under-implementation. Our study reported financial burden on the patient, as an important barrier to oncofertility by majority of the PCPs. As fertility preservation procedures are not covered under insurance costs in India,,This can be overcome by providing educational resources, for e.g., decision aids, brochures or conducting Continuing Medical Education (CME) programs which is expected to increase the knowledge and confidence in healthcare workers thereby improving the frequency as well as quality of fertility preservation discussions. Development of clinical guidelines with an objective to provide guidance on the gonadotoxicity of (non)-oncological treatments can be focused towards primary care to bridge the knowledge gaps among PCPs in the country.Strategies for effective implementation of FP services across the country can include formation of fertility preservation consortiums that allow for knowledge and resource distribution among healthcare providers would facilitate successful establishment of oncofertility care in India.Our pilot study highlights the dearth of oncofertility awareness among PCPs. With increasing involvement of PCPs in the cancer care continuum, this group could play a crucial role in patient decision-making, thereby helping in the successful establishment of oncofertility as part of standard cancer care. As the scope of fertility preservation is expanding to also include non-oncological patients requiring gonadotoxic therapies the involvement of PCPs whom the patients know and trust and who are also familiar with the patient’s wishes or life goals, in fertility preservation discussions could prove beneficial to the cancer-diagnosed patients as well as increase the frequency of fertility preservation referrals. The present study emphasizes the need for knowledge dissemination and awareness about oncofertility among PCPs. This study should contribute to what is currently only limited literature in this area. Limitations of this study include a small sample size and selection bias due to random recruitment of survey participants at conferences. Due to the small sample size conclusions could not be drawn on the characteristics of PCPs with increased knowledge on fertility preservation.PT: Collected and analyzed the data, wrote paper; SU, RKJ, KSU, GK, NS, TKW: Revised the manuscript critically for important intellectual content; SKA: Conceived and designed the study, wrote paper. PT is the guarantor of this work and as such, had full access to all the data and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors have given final approval for publication. The manuscript entitled "Oncofertility awareness among primary care physicians in India" provides a preliminary survey based analysis highlighting the dearth of oncofertility awareness among PCPs. Specific comments are:Any published estimates of reproductive cancers/prepubertal cancers in India per population/incidences.et al. (2019) described a model to increase the oncofertility implementation - worth mentioning in the current manuscript. A paragraph on the current status of fertility preservation in IVF units across the country and gaps in services. AnazodoThe authors have mentioned about the need for the shared care model for oncofertility referrals. Please explain how this could be achieved in an Indian context tailored to the identified determinants and how the improvement strategies for the guideline-specific barriers should be developed. 1) The discussion section can have the following additional details: Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Assisted Reproductive TechnologiesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I reviewed thoughtfully the manuscript Oncofertility awareness among primary care physicians in India. In this the authors present an exploration of the current knowledge and barriers to fertility preservation in India among PCPs. I congratulate the authors for addressing a topic needing assessment in LMICs. My main concerns are regarding the paper's impact in the current knowledge, as usually young patients diagnosed with a malignancy are under the care of a specialized team (pediatric oncologist/hematologist), and I do not fully understand if the lack of knowledge of PCP in such a specialized topic is something of importance in their care-continuum. I think if the authors could really justify the importance of understanding the knowledge of PCP in their paper, it could strengthen it. Overall, I found the paper well-written and understandable. The data presented was only a description of the obtained answers, were there any correlations made to understand? For example, what are the characteristics of the physicians with the highest knowledge of oncofertility? During the discussion, there have been other publications in LMICs for example, in Mexico, also in other parts of the world outside the US like Italy. I recommend making a more exhaustive review of the currently published literature to correlate your results with the currently available literature. During the discussion I would also recommend a deeper exploration of the reported barriers, are these similar to their context? Or as these unique in the Indian population? In the conclusion, once again I would clearly state why you consider that this is relevant information for the reader.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?NoAre all the source data underlying the results available to ensure full reproducibility?NoIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?NoAre sufficient details of methods and analysis provided to allow replication by others?NoReviewer Expertise:Urologic oncology, disparities in cancer careI confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. It is a well conducted and well written manuscript. It addresses a relevant issue. It is interesting that the authors have gone beyond the specialist and interviewed primary care physicians. It is an important step towards improving awareness about oncofertility. The limitations of the study are the small numbers included.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Ovarian reserveI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.