Nanoparticles based on amphiphilic copolymers with tunable physicochemical properties can be used to encapsulate delicate pharmaceutics while at the same time improving their solubility, stability, pharmacokinetic properties, reducing immune surveillance, or achieving tumor-targeting ability. Those nanocarriers based on biodegradable aliphatic polycarbonates are a particularly promising platform for drug delivery due to flexibility in the design and synthesis of appropriate monomers and copolymers. Current studies in this field focus on the design and the synthesis of new effective carriers of hydrophobic drugs and their release in a controlled manner by exogenous or endogenous factors in tumor-specific regions. Reactive groups present in aliphatic carbonate copolymers, undergo a reaction under the action of a stimulus: e.g., acidic hydrolysis, oxidation, reduction, etc. leading to changes in the morphology of nanoparticles. This allows the release of the drug in a highly controlled manner and induces a desired therapeutic outcome without damaging healthy tissues. The presented review summarizes the current advances in chemistry and methods for designing stimuli-responsive nanocarriers based on aliphatic polycarbonates for controlled drug delivery. Tumors consume an enormous amount of glucose compared to healthy cells, due to a fast rate of glycolysis, to provide the energy needed to increase proliferation . Such a  cancers . Further cancers  develope cancers . PolymerVery recently, Domiński et al.  developevia photoinduced radical thiol-ene addition, and tetrazine via inverse electron demand Diels–Alder reaction. The other advantage of the prepared system was the incorporation of the acetal group for the pH-triggered release of a drug functionalized with norbornene molecule. The ability to accurately control the amount of drug or imaging agents grafted on the polymer backbone provides many possibilities for applications in biomedical fields. The acetal group is commonly used in pH-responsive anticancer drug delivery systems due to its rapid hydrolysis in the acidic tumor environment. However, obtaining stable nanoparticles with acetal groups is not an easy task. The acetal groups hydrolyze with the formation of two hydroxyl groups in the polymer chain, which significantly increases the hydrophilicity of the hydrophobic core of nanoparticles. It disturbs hydrophilic/hydrophobic balance, which is a crucial parameter for the amphiphilic copolymers to self-assemble into nanoparticles. Hence, Zhong et al. [co-acryloyl carbonate) copolymer containing an acetal group for pH-triggered drug release and photo-crosslinkable acryloyl groups was synthesized. The micelles formed using such a copolymer were photo-crosslinked, resulting in extremely stable nanoparticles at physiological pH, while at an acidic pH they were able to hydrolyze resulting in the release of drugs. In vitro drug release studies confirmed that crosslinking does not influence drug release at acidic pH (4.0 and 5.0), while increased stability was observed at pH 7.4. In addition, in vitro cellular studies showed that cross-linked and non-cross-linked micelles exhibited similar anti-tumor activity, which may indicate that the micelles core photo-crosslinking does not affect drug release  trimethylene carbonate) for pH-triggered drug release with norbornene group for versatile post-polymerization modifications . Norborng et al.  develop  release . b-poly(ε-caprolactone). Both amphiphilic copolymers form in aqueous solutions a type of mixed micelles with an average size around 100 nm and then are photo crosslinked utilizing the strategy mentioned above. The in vitro and in vivo studies revealed that galactose-decorated core-crosslinked pH-responsive micelles have excellent stability, biocompatibility, and are actively targeting the hepatoma cells due to receptor-mediated mechanism [co-pyridyl disulfide carbonate) a copolymer that formed approximately 60 nm micelles in aqueous solutions [via core-crosslinking was developed by Huang et al. [co-nucleobase-grafted-APC) (adenine or thymine respectively) were mixed to form micelles with an average size ~100 nm. The base-pairing interactions between nucleobases localized in hydrophobic micelles core caused core-crosslinking due to multiple hydrogen bonds between adenine and thymine, as occurs in the DNA double helix structure. The core-crosslinking significantly reduces the possibility of micelles reorganizing. Therefore, core-crosslinked micelles have greater stability and a lower critical micelle concentration value. The nucleobase core-crosslinked micelles were stable at pH 7.4 leading to a significant decrease in drug release. However, in an acidic environment (pH 5.0) the drug was released much faster. It is caused by the dissociation of hydrogen bonds between nucleobases in the acidic environment. He et al. [p-quinoneimine bond by a mechanism that mimics the Raper-Mason pathway of melanogenesis. Interestingly, the addition of PEGylated N-methylimidazole-functionalized APC to catechol-functionalized APC during DOX conjugation with concomitant micelles formation results in higher drug loading efficiency  with imine linkage between hydrophilic and hydrophobic blocks [via combined ROP and RAFT polymerization techniques which gives a huge amount of possibilities to functionalize such copolymers to achieve superior tumor-targeting efficiency. Such an approach to develop the pH-cleavable bond between hydrophilic and hydrophobic polymeric chains might expand the range of applications of aliphatic polycarbonates as tumor-targeting drug delivery systems. Lately, Kuckling and coworkers developed a versatile platform for designing and synthesizing novel pH-responsive nanocarriers based on diblock copolymer poly(dimethylacrylamide)-c blocks . The copN-substituted 8-membered cyclic carbonates using organocatalyst or through the combination of ROP and post-polymerization modification. Feng et al. [co-ε-caprolactone) blocks [The possibility of using nanocarriers containing ionizable groups in copolymer structure for pH-triggered drug release was also studied. When these ionizable groups become protonated below the acid dissociation constant, the nanoparticles are destabilized by the charge repulsion causing reorganization and consequently, the release of the encapsulated cargo. Venkataraman et al.  developeg et al.  developeg et al. . Further) blocks , or amph) blocks  were alsb- The micelles obtained had a size of ~165 nm. However, at pH 5.8, which is simulating the tumor microenvironment, the tertiary amines are ionized, leading to the micelles swelling and the encapsulated drug release. Extended studies using confocal laser scanning microscopy (CLSM) have shown an enhanced cellular internalization of camptothecin or doxorubicin-loaded micelles by cancer cells [N,N′-dibutylethylenediamine (pKa = 4.0) and 2-pyrrolidin-1-yl-ethyl-amine (pKa = 5.4) to obtain responsivity at different pH. The amines promote the disintegration of micelles in acidic pH and release of the encapsulated anticancer agents gemcitabine and a Hedgehog inhibitor (GDC 0449) in the extracellular tumor microenvironment (pH 6.0–7.0) and intracellular compartments (pH 5.5–4.5) of tumor tissues, respectively. It was also reported that a stoichiometric mixture of micelles formed from two types of pH-sensitive copolymers, enabled to achieve drug release depending on corresponding extra- and intracellular tumor microenvironment. This system was found to inhibit the proliferation of pancreatic cancer cells and showed selective internalization of nanoparticles in pancreatic tumor tissues -er cells . Quadir er cells  prepared tissues .The acidic extracellular and intracellular microenvironment of tumor tissues led to a comprehensive study of pH-triggered nanocarriers for controlled anticancer drug release. The pH-triggered drug administration is considered to be the most basic strategy to target tumor cells. Despite the many advantages of such nanocarriers, limited stability is one of the major issues. Nanocarriers must be stable before entering the cancer cell to prevent drug leakage. Obtaining stable nanoparticles is difficult due to the dynamic process of their formation and reorganization. Therefore, the combination of pH-triggered drug release with cross-linking of the micelle core  seems to be a promising strategy to improve stability against dilution and prolonged circulation. Moreover, to enhance internalization by tumor tissues, targeting ligands that can differentiate tumor tissues from normal ones might be added. In the future, the combination of pH-responsive copolymers with other stimuli and/or the addition of active targeting ligands to nanoparticle surfaces might mitigate some of the challenges in chemotherapy.3-SS-Paclitaxel via azide-alkyne click reaction. The paclitaxel-conjugated amphiphilic copolymer was used to encapsulate doxorubicin to provide combination chemotherapy. To enhance stability and on-demand drug release, the nanoparticles were crosslinked by a redox-sensitive linker. The excess of propargyl groups was crosslinked by azide-alkyne click reaction with bis(azidoethyl)disulfide. Both drugs were released slowly from the nanocarriers at physiological pH, however, were significantly accelerated in the presence of intracellular reducing agent concentration (10 mM dithiothreitol). The in vitro studies conducted on HeLa and MCF-7/ADR cells revealed that the co-delivery of two anticancer drugs exhibited a synergistic effect for inhibiting the proliferation of cancer cells. Chen et al. [vβ3 integrin which overexpression occurs in most tumor tissues. That is why it has been extensively studied for tumor-targeting in the treatment of cancer [co-dithiolane trimethylene carbonate). The micelles with an average size of ~150 nm formed from this amphiphilic copolymer were loaded with doxorubicin. The micelle cores were crosslinked via a disulfide bond to minimize drug leakage and improve stability. The in vivo biodistribution investigation showed better therapeutic outcomes and enhanced accumulation of cRGD peptide decorated nanoparticles in tumor tissues compared to clinically used PEGylated liposomal doxorubicin. Recently, the same group has improved the cRGD-decorated nanoparticles by conjugating the anticancer drug (mertansine) via a disulfide bond to impart redox-sensitivity to drug release. Whereas, excess of thiol groups in the copolymer structure was used to crosslink micelle cores to enhance stability. The confocal microscopy studies have shown active tumor-targeting by cRGD-decorated nanoparticles to αvβ3 integrin overexpressing melanoma cells. In vivo experiments revealed a significant tumor growth inhibition with cRGD-functionalized nanoparticles loaded with mertansine when compared with non-targeting nanoparticles loaded with drug and free mertansine [The differences between intracellular and extracellular levels of reducing agents are broadly exploited as an endogenous stimulus for the controlled release of genes and drugs . The redn et al.  developef cancer . Zhong af cancer  synthesirtansine . Anotherrtansine . This fortansine  to desigThe emerging therapeutic potential of nitric oxide in cancer treatment has made the design of nanoparticles for co-delivering nitric oxide and anticancer drugs a hot research topic in recent years. To overcome multi-drug resistance, Chen and coworkers have designed reduction-triggered micelles for co-delivery of nitric oxide and doxorubicin . The micb-poly micelles for GSH-triggered doxorubicin release [b-poly via click chemistry with propargyl 3,3′-dithiopropionate. Subsequently, the authors reported such prepared nanoparticles loaded with paclitaxel and showed the reduction-responsive intracellular drug release, which was verified by confocal laser scanning microscopy studies [b- trimethylene carbonate)) with a crosslinked core using click chemistry with disulfide containing dialkynyl linker. In this area, Yi et al. [b-) to which 6-bromohexanoic acid and azide-functionalized α-lipoic acid were attached via click reaction. The disulfide core-crosslinked is formed by the addition of a catalytic amount of dithiothreitol [via “click” reaction with bis-(azidoethyl)disulfide were made of diblock copolymer: PEGylated propargyl-functionalized APC. In vitro studies of such GSH-sensitive micelles loaded with doxorubicin were conducted using HeLa cells, 4T1 cells, and doxorubicin-resistant ADR/MCF-7 cells which showed a more efficient inhibition of tumor cell growth than the free drug. More importantly, DOX-loaded micelles possessed significantly higher anti-tumor activity against ADR/MCF-7 cells compared to free doxorubicin. It is caused by the “stealth” endocytosis that overcomes the biological barriers of drug-resistant ADR/MCF-7 cells [2000-b-poly, which was cross-linked by Michael addition reaction between pendant acrylate groups and cystamine. Extended in vivo studies using confocal laser scanning microscopy performed with HeLa tumor-bearing mice revealed that DOX-loaded small-sized micelles (DPP) penetrate deeper into tumor parenchyma compared to micelles with an average size ~50 nm (CDPP)  . FurtherIn summary, it is evident that redox-responsive nanocarriers based on GSH-triggered disulfide/diselenide bond rupture are capable to efficiently improve the cellular uptake of nanoparticles into tumor tissues, demonstrating their high potential for applications in chemotherapy. However, based on the studies outlined above, redox-sensitive bonding has a dual-use: i.e., triggering the drug release and, equally important, cross-linking of micelles core to enhance their stability and preventing drug leakage. Combining the redox-responsive core cross-linked micelles with another stimulus for drug triggering release could provide a promising platform for cancer treatment.2O2), superoxide (O2−), singlet oxygen (1O2), etc.) as the byproducts of aerobic metabolism caused by oncogenic transformation, intensive metabolism related to increased proliferation, or mutations in mitochondrial DNA [b-(carbonate-thioether)) was synthesized by lipase-catalyzed ROP of the cyclic diethylene sulfide carbonate dimer. The thioether groups located in the copolymer main chain caused the nanocarriers to exhibit ROS-sensitivity by oxidizing the thioether groups to hydrophilic polar sulfoxides or sulfones. This causes the size of the micelles to decrease, which was attributed to the reduction in the core of the micelles. Further oxidation increased the hydrophilicity of the copolymer and led to an increase in the size of the micelles, which was caused by the formation of loose aggregates (There are a lot of studies pointing that tumor tissues are characterized by a higher level of reactive oxygen species (ROS) compared to healthy cells. Endogenous cancer cells constantly produce ROS , hydrogen peroxide  caused micelles to form once again. This phenomenon occurred due to the azobenzene cis-trans reverse isomerization  for indomethacin drug delivery system. Likewise, Kuckilng and coworkers developed a series of light-degradable nanocarriers consisting of PEGylated APC with an o-nitrobenzyl ester group as side chains [co-glycolide) in ratio 1:3 . Preliminary in vitro cellular studies of the above-mentioned nanoparticles and degradation products confirmed that systems have the potential to be used for in vivo studies. In this context, the same group reported light-sensitive micelles consisting of PEGylated APC functionalized with spiropyran chromophore group in the side chain . The phoreported . The phog et al.  reportede chains ,150,151.atio 1:3 . The nano-nitrobenzyl one, shows better biocompatibility, does not form toxic degradation products and characterizes a higher photolysis rate. Based on this strategy, a variety of photoactivated nanoparticles can be obtained to expand the family of aliphatic polycarbonates nanocarriers as highly controlled spatially and temporally drug delivery systems. Overall, light-sensitive nanocarriers represent a promising option for controlled drug delivery systems. Although the endogenous stimuli-responsive nanocarrier approach is limited due to the heterogeneity of the tumor tissues, the complexity of the light-triggered drug release mechanism and the potential toxicity of the photosensitive groups and their degradation products are still major disadvantages of photosensitive systems. However, the simultaneous combination of endogenous and exogenous stimulus-responsive groups into a biodegradable nanocarrier may provide more precise cancer treatment with reduced side effects.Very recently, the same group reported light-degradable APCs nanoparticles by introducing bromocoumarin groups into the side chain . Upon liThe rapid and uncontrolled growth of cancer cells is associated with the unique characteristic tumor microenvironment. As cancer develops, tumors become more and more heterogeneous . Therebyb-(disulfide bond)-poly for dually-triggered intracellular release of doxorubicin [b-) obtained by organocatalyzed ROP. The propargyl groups were used to cross-link the core via “click” reaction with 1,6-diazidohexane or bis(azidoethyl) disulfide to obtain insensitive and GSH-sensitive crosslinking, respectively. Yang and coworkers designed and prepared smart pH/redox dual-responsive micelles with high drug loading for anticancer drug delivery [b-(APC-SS-COOH)). Interestingly, the effect of hydrophilic/hydrophobic block lengths and drug loading efficiency was studied. The DOX loading capacity increased with an increasing ratio of the carboxylic acid groups in the copolymer side chain. In vitro drug release showed that at the endolysosomal pH (~5.0) and in the presence of GSH at the cytoplasmic concentration (10 mM GSH), DOX release was significantly accelerated. Moreover, these DOX-loaded dual-sensitive nanocarriers accumulate in tumor tissues, as shown by confocal microscopy studies, and demonstrated better inhibition of tumor growth in nude mice bearing BT-474 xenografts. In this context, Hu et al. [Recently, most research has focused on combining pH sensitivity with various endogenous stimuli, e.g., redox, ROS. The combination of pH and redox-sensitivity is attractive because tumor tissues are always characterized by low pH and elevated level of intracellular GSH. Taking advantage of this, Zhong and coworkers designed and developed a pH/GSH-responsive micelles consisted of diblock copolymer poly(ethylene glycol)-orubicin . The aceorubicin  reporteddelivery  and repou et al.  developeAnother research reported dual-responsive micelles obtained through mixing positively charged pH/reduction-sensitive APC copolymer possessed hydrazone linked doxorubicin and disulfide functional pendant groups, with negatively charged PEGylated APC copolymer containing acid-labile β-carboxylic amide as side groups . The hydb-polycarbonate with incorporated selenide and tertiary amine groups into the APC backbone  PEGylated carboxylic acid-functionalized thioether-containing APC and (ii) PEGylated phenylurea-functionalized thioether-containing APC . The addbackbone  was alsoo-nitrobenzyl ester group and doxorubicin molecules conjugated via acid-sensitive Schiff-base linkage. Interestingly, both triggers can be used individually or together for adjusting the drug release rate  obtained via enzymatic copolymerization of ε-caprolactone and diselenic carbonate macrolide. A detailed study of the copolymerization made it possible to obtain a copolymer with a controlled number of diselenide groups in the main chain to ensure a highly controlled GSH/ROS-responsive drug release from the nanoparticles. Sun et al. [Very recently, Wang et al.  reportedn et al.  designed® as an anticancer agent has shown how powerful a weapon the use of nanoparticles can be in the fight against cancer. Furthermore, it is also worth noting that an analysis of the American Food and Drug Administration and European Medicines Agency databases indicates that more and more novel nanocarriers are approved for the diagnosis and treatment of cancer, including systems using active targeting or stimulus-responsivity mechanisms [The introduction of Doxilchanisms . This prchanisms . In the In the fight against cancer, current advances in research on stimulus-responsive systems are a promising strategy with a bright future. The successful clinical implementation of stimuli-responsive nanocarriers based on aliphatic polycarbonates remains a major challenge, even though recent results are promising. In future studies, there is an expectation of more efficient nanocarriers with a superior balance between safety, tumor-targeting ability, and clinical outcome.


Importance of sperm-derived transcripts and chromatin imprints in organismal development is poorly investigated. Here using an integrative approach, we show that human sperm transcripts are equally important as oocyte. Sperm-specific and sperm-oocyte common transcripts carry distinct chromatin structures at their promoters correlating with corresponding transcript levels in sperm. Interestingly, sperm-specific H3K4me3 patterns at the lincRNA promoters are not maintained in the germ layers and somatic tissues. However, bivalent chromatin at the sperm-specific protein-coding gene promoters is maintained throughout the development. Sperm-specific transcripts reach their peak expression during zygotic genome activation, whereas sperm-oocyte common transcripts are present during early preimplantation development but decline at the onset of zygotic genome activation. Additionally, there is an inverse correlation between sperm-specific and sperm-oocyte lincRNAs throughout the development. Sperm-lincRNAs also show aberrant activation in tumors. Overall, our observations indicate that sperm transcripts carrying chromatin imprints may play an important role in human development and cancer.  •Sp-lincRNAs carry distinct chromatin structures correlating with transcript levels•Sp-lincRNAs are active during ZGA in preimplantation developmental stages•SpOc-lincRNAs are active in pre-ZGA and decline at the onset of ZGA•Sp-lincRNAs are silent post implantation but show aberrant cancer-specific activation Chromosome Organization; Endocrinology; Transcriptomics Maternal or oocyte-derived transcripts are known to regulate maternal to zygotic transition (MZT), which involves zygotic genome activation (ZGA) and maternal transcript degradation. These are the two important events necessary for subsequent preimplantation embryonic development . On the Cancer testis antigens (CTAs) are a group of testis-specific proteins that show predominant expression on cancer cells. This observation suggests a functional connection between testis-specific expression and cancer. Majority of the CTA genes are preferentially expressed in testicular cell types such as spermatogonia or spermatocytes. Hence, we were particularly interested in characterizing whether sperm-derived RNAs show any preferential expression in different tumor types, which would in turn pave the way for understanding the contribution of sperm-derived transcripts in tumor development and progression .At the outset, we analyzed the transcriptome profiles of sperm, oocyte, and preimplantation embryo stages (two cell to late blastocyst) in human and mouse  Figure A. We incOur analysis show that sperm and oocyte, from both mouse and human, harbor comparable number of transcripts, and in particular, mouse harbors nearly twice as many transcripts in sperm compared with oocyte B and 1C.-K27-)  A. In Sp-) (-K27- B. In mou) (-K27- C. In the) (-K27- . Mouse S) (-K27- D. We nexspecific A and 2E.specific  and S3. specific B–S1D. Th-K27-) showed immune defense-related functions such as immune response, neutrophil chemotaxis, and defense response  from sperm-derived chromatin clusters show highly temporal expression patterns with no expression in early preimplantation-stage embryos and specific activation between four- and eight-cell stages of embryos A. Like i-K27- and low-K4 sperm-derived chromatin clusters from four-cell stage onward. More importantly, SpOc PCGs from low-K4 group become super activated during preimplantation development , mesoderm (middle layer), and endoderm (innermost layer) A. Each iromoters A.Figure -K27- clusters maintain the same chromatin structure at their promoters in all the three germ layers and somatic tissues. Interestingly, sperm-derived chromatin clusters from SpOc-PCG promoters showed higher enrichment of H3K4me3 in all the three germ layers and their derived somatic tissues. However, sperm-derived bivalent domains from SpOc-PCG promoters maintained their bivalent chromatin structure in all the three germ layers but in the germ-layer-derived tissues only traces of bivalency were maintained , meiosis , and post-meiosis (round spermatid) stages of spermatogenesis . Sp-lincIn summary, tracing the expression dynamics from different spermatogenic cell types, gametes (matured sperm and oocyte), and preimplantation (zygote to late blastocyst) and post-implantation stages (germ layers and matured tissues) reveal that Sp and SpOc transcripts have opposing expression patterns throughout the human development C.cis- or trans-acting regulatory elements can control the transcription of genes , prostate (PRAD), testis (TCGT), and uterine or ovary -related cancers.Previous studies have shown that RNAs involved in organism development and having testis-specific expression are known to take part in cancer development and progression . To democlusters A; thus, clusters B. One coclusters . A detaiclusters A and S9AP4HA3-AS1, LINC01518, and AP001476.1), representing each of the three sperm-derived chromatin clusters, in human embryonic kidney cell line HEK-293 and cervical carcinoma cell line HeLa. We found that the Sp lincRNAs show higher expression in HeLa cells compared with the HEK293 cells  occurs, even though their expression is restricted to meiotic or pre-meiotic spermatogenic cell types. These observations indicate that low levels of transcription may persist during sperm maturation, and this notion is consistent with the data that sperm transcripts differs in caput and cauda and moreover, as discussed earlier, sperm RNA alterations in response to motility and capacitation  transcripts were identified based on the microarray analysis of RNA from the ejaculated sperm of nine individuals . The precitation . Thus, fThe next important question is whether genes encoding sperm-specific transcripts and/or the transcripts per se have any role in preimplantation development. Multiple lines of evidence suggest that sperm transcripts play an important role in early preimplantation development. For example, defective blastocyst embryos generated from dicer knockout germ cells can be rescued with sperm RNA . SimilarAnother interesting aspect of the current investigation is the temporal expression of genes that encode sperm-specific and sperm-oocyte transcripts during preimplantation development. Genes that encode sperm-specific transcripts carry distinct chromatin structures and show stage-specific expression across preimplantation stages. This exclusive stage-specific expression is not seen with genes that encode sperm-oocyte transcripts, rather the majority of these transcripts show expression during early preimplantation stages, primarily between two- and four-cell stages and start to decline or degrade during the onset of ZGA. Highly temporal expression of genes that encode sperm-specific transcripts in the preimplantation stages signifies their importance during early preimplantation development. Moreover, genes that encode sperm-specific and sperm-oocyte transcripts have crucial non-overlapping roles in preimplantation development. It is reasonable to assume that the sperm-inherited chromatin imprints together with the help of preimplantation development associated transcription factors may bring in the observed stage-specific expression . Indeed,-K27-, and bivalent (H3K4me3/H3K27me3) promoters, transcripts are enriched in sperm, and they express throughout preimplantation development, the germ layers, and in multiple somatic tissues. Thus, sperm-encoded information seems to take part in important developmental decisions.Strikingly, the expression of Sp-lincRNAs as well as their promoter epigenetic profiles were lost in the three germ layers and their derived somatic tissues. This again emphasizes the importance of the genes that encode sperm lincRNAs in fertilization and preimplantation development. On the other hand, genes that encode Sp-PCGs and SpOc transcripts (SpOc-lincRNAs and SpOc-PCGs) continue to express in all the three germ layers and the germ-layer-derived multiple tissues. Comparison of the histone profiles of sperm, germ layers, and mature tissues revealed that Sp-PCGs and SpOc-PCGs retain their sperm-specific bivalent chromatin in the germ layers and somatic tissues; however, the ratio of H3K4me3 and H3K27me3 is different from that in the sperm. These observations indicate that the sperm-derived bivalent chromatin imprints at the Sp-PCGs and SpOc-PCGs promoters are maintained during the germ layers formation and these sperm-inherited chromatin imprints may play an important role in crucial developmental decisions by regulating spatiotemporal expression of Sp-PCGs and SpOc-PCGs. Thus, our study found two sets of genes: one set, sperm-specific genes with high-K4 promoters, whose transcripts are enriched in sperm, activated during ZGA and transcriptionally silenced in the three germ layers and somatic tissues. In the other set of genes, comprising Sp-PCGs, SpOc-lincRNAs, and SpOc-PCGs with high-K4, low-K4, K4-K27-). Interestingly, the level of their deregulation in cancer matches with the sperm H3K4me3 levels at the promoters. For example, lincRNAs with high-K4 levels at their promoters showed more deregulation in cancer compared with the lincRNAs with low-K4, indicating that mechanisms that establish sperm-specific chromatin imprints are recapitulated in cancer. Their higher expression in tumors compared with the corresponding normal tissues indicate that they may behave as oncogenes. We have also tested the oncogenic properties of selected lncRNAs from high-K4, low-K4, and K4-K27- clusters in HeLa cell line by measuring their effect on crucial cancer cell hallmarks that define the oncogenic drivers such as cell proliferation, cell cycle progression, and apoptosis. Loss of function of these lncRNAs in HeLa cells using siRNAs resulted in decrease in cell proliferation, cell cycle progression, and apoptosis, indicating that these sperm lincRNAs harbor oncogenic properties. Thus, Sp-lincRNAs may serve as comprehensive resource for diagnostic/prognostic markers and potential therapeutic targets. SpOc-lincRNAs also showed deregulation in multiple cancers but to a lesser extent compared with the Sp-lincRNAs. Surprisingly, Sp-PCGs and SpOc-PCGs were mostly downregulated in cancers, and this in particular is consistent with their role in immune response regulation as immune response genes are mostly suppressed in many cancers.Previous studies have proposed a functional link between early embryonic gene expression program and cancer development and progression . In partIn sum, the abundance of RNA, in particular lincRNAs, in sperm necessitates further investigation on their importance in sperm maturation, fertilization, and preimplantation development and cancer. More importantly, our study laid a strong basis for further investigation on the functional role of sperm-inherited chromatin imprints in organismal development.Lack of chromatin data (ChIP-seq) for different stages of human preimplantation embryos limited our possibility to explore whether sperm-derived chromatin imprints are preserved during preimplantation development. Exploring this would have added an in-depth resolution to our analysis.kanduri.chandrasekhar@gu.se.Correspondence to lead contact Chandrasekhar Kanduri, E-mail: This study did not generate any new sequencing data. Analysis was performed using public datasets.https://dx.doi.org/10.17632/695c4zvr6d.1.The processed datasets generated during this study are available publicly at Mendeley Data repository with the following DOI All methods can be found in the accompanying


Idiopathic inflammatory myopathies (IIMs) are rare, heterogeneous, autoimmune musculoskeletal diseases, characterised clinically by muscle weakness. Extramuscular involvement includes the skin, respiratory and cardiovascular systems. Genetic and environmental factors contribute to IIM susceptibility, and viral or bacterial infection may contribute to disease pathogenesis.Both the innate and adaptive immune systems are important in IIM pathology. Two-thirds of affected individuals have known myositis-specific or associated autoantibodies, often linked to particular clinical features,We recently used a high-throughput approach combining disease-specific immunoglobulin epitope signature enrichment and antigen identification from the total microbial ‘exposome’  and human proteins.Human coronaviruses are associated with the common cold, but can lead to fatal inflammatory responses and acute lung injury. Emergence of a novel coronavirus has caused a recent global pandemic of severe acute respiratory syndrome (SARS) in humans (COVID-19).Due to the current coronavirus pandemic, here, we focused our analysis on epitopes mapping to the coronaviridae family. In dermatomyositis patientsHLA-A*01:01 restriction.We subsequently investigated whether these epitopes have been experimentally identified as B cell and T cell immunogenic epitopes from studies of epidemic-causing virus SARS-CoV, or computationally predicted from SARS-CoV-2. Epitope ‘DDAVVC’ in the RNA-dependent RNA polymerase protein is a highly ranked CD8 T cell predicted epitope identified from the Immune Epitope Database and Analysis Resource, showing The coronavirus genome encodes four structural proteins; spike, nucleocapsid, membrane and envelope proteins. SARS-CoV-2 spike glycoproteins promote cell entry through attachment to the host ACE 2 receptor, and subsequent fusion between viral and host cell membranes to facilitate viral entry,


The effects of DHU001, a mixed herbal formula consisted of 7 types aqueous extracts for treating respiratory disorders were observed on xylene-induced acute inflammation. The xylene was topically applied 60 min after administration of 500, 250 and 125 mg/kg of DHU001, and all animals were sacrificed 2 hrs after xylene application. The changes on ear weights, histolopathological analyses of ear were evaluated and compared to those of indomethacin and dexamethasone (15 mg/kg treated) - Both of drugs are well-known by anti-inflammatory agents. Xylene application resulted in marked increases in induced ear weights as compared with intact control ear. Severe vasodilation, edematous changes of ear skin and increase in the thickness of the ear tissues, neutrophil infiltration as acute inflammation were detected in xylene-treated control ears at histopathological observation. However, these xylene-induced acute inflammatory changes were dose-dependently decreased by oral treatment of DHU001. Therefore, it is concluded that DHU001 has favorable anti-inflammatory effects on xylene-applicated acute ear inflamed mice.


In recent decades, a new method of cellular immunotherapy was introduced based on engineering and empowering the immune effector cells. In this type of immunotherapy, the immune effector cells are equipped with chimeric antigen receptor (CAR) to specifically target cancer cells. In much of the trials and experiments, CAR-modified T cell immunotherapy has achieved very promising therapeutic results in the treatment of some types of cancers and infectious diseases. However, there are also some considerable drawbacks in the clinical application of CAR-T cells although much effort is in progress to rectify the issues. In some conditions, CAR-T cells initiate over-activated and strong immune responses, therefore, causing unexpected side-effects such as systemic cytokine toxicity , neurotoxicity, on-target, off-tumor toxicity, and graft-versus-host disease (GvHD). To overcome these limitations in CAR-T cell immunotherapy, NK cells as an alternative source of immune effector cells have been utilized for CAR-engineering. Natural killer cells are key players of the innate immune system that can destroy virus-infected cells, tumor cells, or other aberrant cells with their efficient recognizing capability. Compared to T cells, CAR-transduced NK cells (CAR-NK) have several advantages, such as safety in clinical use, non-MHC-restricted recognition of tumor cells, and renewable and easy cell sources for their preparation. In this review, we will discuss the recent preclinical and clinical studies, different sources of NK cells, transduction methods, possible limitations and challenges, and clinical considerations. As we expand our knowledge and understanding of how the immune system acts against cancer, we are more likely to believe in the immune system’s power. Therefore, in recent decades, we relied more on novel immune-based therapies than the classic chemical drugs (chemotherapies) to treat cancer. Various novel immunotherapy methods are in development to achieve effective and durable therapies for patients with hardly treatable or refractory cancers . Cancer ICIs block the checkpoint receptors/ligands on immune cells and cancer cells then prompt the killing mechanisms in immune effector cells. This type of immunotherapy was found to be advantageous in various malignancies, e.g., Hodgkin’s lymphoma , 6. ICIsIn addition, Bi- and tri-specific antibodies as the novel immunotherapy strategy can engage the immune effector cells toward tumor cells by binding simultaneously to both cells. Then, the forced proximity between effector cells and tumor cells causes the release of cytotoxic molecules and apoptosis of tumor cells. This method showed good anticancer activity against several types of non-Hodgkin lymphomas (NHL) such as diffused large B cell lymphoma (DLBCL) and follicular lymphoma (FL) –15.Also, another branch of immunotherapy is comprised of genetic modification of immune cells as living drugs. The method is currently under investigation for understanding its usefulness in the treatment of different cancers particularly hematological malignancies. The first successful forms of transduced cells with CAR were autologous T cells capable of targeting CD19-specific antigen expressed on B cell malignant tumor cells . CAR is CARs have been successfully utilized in many research and clinical trials to redirect autologous T cells against lymphoid leukemia and lymphoma . CAR-expTherefore, the clinical application of CAR T cells has been mainly constrained to CD19-expressing B cell blood cancers . Many reCD19-CAR T cell adoptive immunotherapy has achieved successful results in clinical trials in the treatment of ALL and diffuse large B cell lymphoma , but theNK cells are a key member of the innate immune system, and while they have some similarities to cytotoxic T lymphocytes (CTLs) in term of killing features, they have an extra intrinsic capability to detect and kill transformed/mutant cells independent of specific antigen recognition processes indicating MHC-unrestricted cytotoxicity done with the help of a wide range of receptors such as the natural cytotoxicity receptors (NCRs) , 35. IntIn this article, we tend to discuss in the field of recent advances in CAR-engineered NK cell immunotherapy, the advantages of using CAR-NK cells toward CAR-T cells, and different sources of NK cells used for CAR manipulations. Also, various transduction methods to introduce CARs into both primary NK cells and NK cell lines regarding increase efficacy have been discussed. Besides, the results of pre-clinical and clinical studies and the strategies for overcoming challenges and improving safety have been summarized.Natural killer (NK) cells were first identified in 1975 as a subpopulation of cells that were different from T and B lymphocytes , 39. For+CD16+ CD3− phenotype. By considering the expression levels of CD56, NK cells are subdivided into two separate subsets; CD56bright and CD56dim [dim NK cells are almost 90–95% of NK cells in the peripheral blood (PB) and inflammatory sites and are capable of mediating high cytotoxicity responses [dim cells also express Fcγ receptor CD16, they can detect and destroy tumor cells or virus-infected cells covered by antibodies and therefore can initiate antibody-dependent cell-mediated cytotoxicity (ADCC) [bright NK cells are immature and comprise only about 5–15% of NK cells in PB [bright NK cells are present in secondary lymphoid tissues and are predominantly powerful in cytokine secretion but with limited cytotoxic properties. CD56bright cells have immunoregulatory functions by producing cytokines or chemokines [NK cells are innate immune cells that reside in the blood, spleen, liver, lung, bone marrow, and to a lesser extent in lymph nodes . They pl CD56dim . CD56dimesponses . Since Cy (ADCC) . In contls in PB . CD56briemokines , 51 suchemokines . Given temokines , 54.Unlike T and B lymphocytes, NK cells do not express antigen-specific recognizing receptors . Abnormal cell recognition by NK cells mainly relies on a balance between inhibitory and activating signals. NK cells have a complex collection of receptors capable of signaling either inhibiting or activating messages. The receptor repository of NK cells contains mainly the non-specific primary receptors that make NK cells capable of exerting more general and not antigen- and TCR-restricted recognition (unlike the T cells). The receptors can be considered as a kind of pattern recognition receptors, not antigen-specific receptors . In otheThe inhibitory receptors interacting with MHC molecules contain a large repertoire of receptors. The repertoire includes Ig-like receptor (KIR) on human NK cells while Ly49, a family of polymorphic type II proteins with C-type lectin domains, on murine NK cells , 59 and Members of the KIR family normally recognize a subset of classic MHC-I molecules, including human leukocyte antigen  . In a siBased on mentioned points above, NK cells recognize target cells through one of these models: “missing-self recognition,” “stress induced-self recognition,” and “non-self-recognition.” Since MHC molecules expression on host cells downregulated or cell transformation occurred, NK cells activated in a process called “missing-self recognition” . Under cIt is more than a decade that NK cells have been reported as efficient effector cells in adoptive immunotherapy , 67. UnlIn recent years, genetic manipulation of NK cells has been considered to improve cytotoxicity, tumor-targeting capability, and persistence in the tumor microenvironment (TME) . One of Similar to CAR-T cells, CAR-engineered NK cells have three parts; an extracellular region comprised of a single-chain variable fragment (scFv) to target a tumor-specific antigen, transmembrane, and intracellular domains . First-gThere are different sources that functional NK cells can be extracted and generated from . One of However, working with blood NK-cells is challenging because of some difficulties in the collection, in vitro expansion, and transduction of these cells . AutologThe second source of NK cells is derived from immortal NK cell clones. These cell lines have good cytotoxic potential. Some of these cell lines are NKG, NKL, KHYG-1, HANK-1, YT, NK-YS, YTS, SNK-6, IMC-1, and NK-92 cells . They arHowever, the strengths and weaknesses usually come together, and the NK cell lines suffer from some disadvantages such as the lack of CD16 expression in most cell lines and non-permanent expression or lack of important activating receptors like natural cytotoxicity receptors (NCRs) , 93. FurAlthough in experimental studies NK-92 cells succeed in restraining tumor burden, due to the short lifespan, they lack sufficient long-term therapeutic efficacy in xenograft mouse models , 95. FurNK-92MI and NK-92CI cells were derived from NK-92 cells and have biological characteristics similar to their progenitors, but are not dependent on IL-2 stimulation . In receFurthermore, stem cell progenitors have been introduced as a unique source to be differentiated into NK cells. Additionally, NK cells can be directly isolated from the umbilical cord blood (CB). The low immunogenicity of cord blood cells significantly reduces the risk of relapse and GvHD . RecentlNaturally, NK cells have a short lifespan. Primary NK cells or CAR-NK cells can be induced to proliferate and last longer within the body. Some endogenous or exogenous stimulating mediators  can be used to provide stimulus. Using growth/proliferation promoters in vitro requires time-consuming and sometimes high-cost techniques and procedures , 106. AlFor more than a decade, scientists have followed different methods to genetically modify NK cells to utilize them as immunotherapeutic agents in cancer. There are various modification strategies to improve effector functions, persistence, safety, and efficacy of NK cells.Increased persistence was achieved by genetically manipulating the cytokine-expressing NK cells which as a result, enhanced their stability within TME via self-expression of IL2 and IL-15. Therapeutic efficacy was achieved by the expression of CAR on NK cells to target specific tumor antigens. Better safety can also be obtained by the incorporation of suicide genes into NK cells . In summOne of the advantages of lentiviral vectors over retroviral vectors is their capability to infect both dividing and non-dividing cells. Retroviral vectors can only infect dividing cells . AnotherThe efficacy of retroviral transduction is very high in expanded primary NK cells with a median of 69% (43–93%) , but thiThere are also much safer non-viral methods to deliver genetic material into the target cells such as electroporation, sonoporation, microinjection, magnetofection, and patented nucleofection technique. Electroporation-based methods are safer compared to viral methods due to the transient nature of delivered foreign genetic materials , applicable in both dividing and non-dividing cells, and no need for direct manipulation of the cell’s genome –126. ThiIn the last two decades, an interesting biologic phenomenon was discovered referring to the transfer of intact membrane patches during immune-cell contacts through an immunological synapse (IS) which was later named trogocytosis. It was also found that this mechanism of exchanging cellular components   can alsoPiggyBac transposon system is another effective non-viral method for genetic element delivery. The system is based on an enzyme  that can target-specific sequences on DNA. The enzyme called transposase recognizes the transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of a transposon vector and then simply cut the sequence and paste it somewhere on the target DNA. Therefore, upon recognition, the transposase easily integrates genetic elements from the original sites into chromosomal sites. This creates stable/permanent genomic integration of genomic material . This teCAR-based immunotherapy in hematological malignancies such as leukemia and lymphoma has been probably the most efficient therapeutic method to date because of its impressive power to elicit an anti-tumor response. CAR-NK cells and CAR-T cells both have been successful to open new clinically relevant opportunities for the treatment of these malignancies.Many ex vivo studies have investigated the efficacy of CAR-NK cells, and some of them are still in progress to improve the efficacy of CAR constructs. Many of the earlier preclinical studies of CAR-NK cells focused on targeting B cell malignancies through utilizing the anti-CD19 and CD20-CARs , 138. InAccording to some evidence, acute and chronic B cell leukemias tend to develop resistance against normal/unmodified NK cells, especially after repeated and inappropriate treatment. To overcome the resistance, first-generation CD19-specific CAR NK cells were designed to target CD19-positive leukemic cell lines and primary leukemia cells . It was In vivo studies using CD19-CAR-NK92 cells have shown the efficient distribution of the cells throughout the circulation and also good killing activity in leukemia murine models , 138. Innull mice to exert high antileukemic activity and inhibit cancer progression [Adoptive immunotherapy by (CAR)-engineered NK cells has been also examined for anti-cancer activity against Pre-B cell acute lymphoblastic leukemia (Pre-B-ALL). Exposure of Pre-B-ALL cell lines and primary blasts to FLT3-specific CAR NK-92 cells resulted in notable selective cytotoxicity of NK cells against them. Besides, FLT3-specific CAR NK-92 was effective in an SEM Pre-B-ALL xenograft model in NOD-SCID IL2R γgression .Furthermore, the anti-cancer efficiency of CAR-NK has also been studied in non-Hodgkin’s lymphomas (NHLs) with poor prognosis such as aggressive T cell malignancies. It was found that CD5 or CD3 specific CAR-NK92 cells can maintain their stable expansion , 143 andMM as another type of malignant hematological cancer is characterized by abnormal proliferation of plasma cells in the bone borrow. These abnormal plasma cells then produce and secrete abnormal antibodies which can be detected in the serum or urine . ApplicaCAR NK cell-based immunotherapies are also a promising therapeutic option for solid tumors. Glioblastoma (GB) is one of the most aggressive primary malignancies that can affect the central nervous system (CNS) . SeveralIt has been shown that NK cell line YTS transduced with EGFRvIII-specific CAR construct can significantly display higher anti-cancer activity than the non-transduced cells which have no CAR. In this study, a CAR structure was designed and produced, consisting two parts, an antibody named MR1-1 with a scFv fragment capable of targeting EGFRvIII, and an intracellular part called DNAX-activating protein-12 (DAP12) to transfer the received signals into the cell . DAP12 cIn another study, KHYG-1 NK cells with EGFRvIII-specific CAR showed good cytolytic activity against cancer cells . In anotThe receptor tyrosine kinase ErbB2 belongs to the same receptor family that EGFRvIII and EGFR are present, i.e., EGFR-related family of receptor tyrosine kinases. ErbB2 has also been incorporated in CAR-engineered NK-92 cells to be evaluated in cancer experiments . It was In some preclinical studies, NK-92 cells were transduced with second-generation CARs containing a composite CD28-CD3ζ signaling domain to target ErbB2-positive cancer cells. These new CARs showed the strong and selective cytotoxicity against ErbB2-bearing target cells originated from different types of solid tumors such as the GB-related cell lines and primary GB stem cell cultures , 159. Re125I has been examined in a murine model of orthotopic pancreatic tumor. The results illustrated that those groups treated with 125I seed +CAR-NK had higher tumor repression than 125I seed treatment alone. Consequently, this study indicated that 125I seed brachytherapy accompanied with Robo1-specific CAR-NK immunotherapy leads to better anti-cancer efficacy in a murine model of orthotopic pancreatic cancer [2-specific CAR NK cells in primary donor-drive NK cells and NK-92 cell line which carrying first- or second-generation CARs have been utilized in preclinical in vitro and in vivo models of neuroblastoma [Another group, Qing Zhang et al. constructed specific second-generation CAR-NK92 cells against an epithelial cell adhesion molecule- (EpCAM-) in colorectal cancer cells (CRC) . In vitrc cancer . A novelc cancer . Reprogrc cancer . Contactc cancer . Besidesblastoma , 166 andblastoma  which haAlthough CAR T cell therapy has shown promising results in the treatment of acute lymphoblastic leukemia the method has not been so good at the treatment of acute myeloid leukemia. As an alternative approach, CD33-directed CAR-NK-92 cells were evaluated in phase 1 clinical trial in patients with refractory acute myeloid leukemia without major adverse effects, indicating that CAR-NK cells may be a safe alternative for CAR-T cells . HoweverFurthermore, to evaluate the safety and feasibility of CAR-NK cell treatment in patients with metastatic solid tumors phase 1 of a clinical trial are now in progress. For this aim, CAR-NK cells targeting NKG2D ligands are being utilized and following infusion of CAR-NK cells interleukin-2 (IL-2) will be injected subcutaneously into some patients to support the survival of CAR-NK cells in vivo (NCT03415100).125I seed brachytherapy in vitro and in vivo in pancreatic cancer models [Treatment with Robo1 BiCAR-NK could improve r models . It has Despite the many advantages of using NK cells, some challenges limit these effector cells to generate CAR-modified NK cells for clinical application . To begiAnother concern about immunotherapy with NK cells goes back to using allogeneic NK cells, which has the possibility of infusion contamination with T or B cells in the expanded NK cell preparation, which can initiate undesirable immune responses in receiver’s body such as the GVHD or post-transplant lymphoproliferative disease .Furthermore, one more potential challenge to use NK cell-based immunotherapy is keeping NK cell expansion in the host’s body after infusion. Because these cells are short-lived, it is necessary to use a new strategy for maintaining their numbers and activity , 190. ReAnother new approach is the incorporation of both IL-2 and IL-15 genes into the CAR constructs to be expressed and secreted within the TME. This approach has been conducted in a mouse model of Raji lymphoma. The results showed that a single infusion of CAR.19/IL15+ CB-NK cells can control tumor progression and increase survival through improving their cancer recognition and cytotoxic activity in comparison to CAR.19/IL15-negative NK cells. These results indicate the pivotal impact of IL-15 on the antitumor activity of NK cells by improving their expansion and persistence capacity .The CAR-engineered immune effector cells (CAR-T and CAR-NK) are developed over the last two decades as the adoptive cell therapy of cancers. However, their therapeutic efficacy against solid tumors remained restricted. Consequently, some new solutions and approaches are being evaluated to improve the effectiveness of these cells.One of the recently developed methods for enhancing the efficacy of CAR-NK cells in a solid tumor is utilizing chemotherapy drugs in combination with CAR NK cells. Qing Zhang and his colleagues for the first time have determined the synergistic effects of Cabozantinib and EGFR-specific CAR-NK-92 cells in vitro and in vivo. Cabozantinib is a multikinase inhibitor that has repressing effects on myeloid-derived suppressor cells and Tregs and also is synergistic in combination with immunotherapies in the treatment of cancers , 198. ThGenerally, mature NK cells have limited persistence in the tumor site, so permanent side effects are not observed. Nonetheless, there are types of NK cells with higher long-lived, such as those derived from cord blood or hematopoietic stem cells as that may cause longstanding toxicity . To addrCAR-engineered NK cells can be successfully employed to improve the therapeutic outcome of cell-based cancer immunotherapy. They have been investigated in many research and also clinical trials with acceptable therapeutic results while keeping the side-effect at the minimum level in comparison to other cell-based immunotherapies. However, technical and biological challenges are still associated with gene delivery into NK cells and therefore have hindered this approach to become a routine anti-cancer treatment at the moment. There are still some difficulties in CAR delivery into NK cells and in NK cells’ persistence and expansion within the body after infusion. Despite all these problems, CAR NK cells have gained much attention due to their lower safety concerns and the possibility of triggering negative immune reactions such as GvHD. NK cells can provide a homogenous, off-the-shelf, standardized cell-based medicine for the treatment of patients with resistant/hardly treatable cancers. CAR NK cells have come to light as top anti-cancer agents with great effectiveness; however, for attaining this goal, improved CRA-NK cells with fewer side effects are required. Besides, we need better transfection methods, better than routine NK cell sources that are much more safer and efficient for CAR NK cells. Many advanced strategies including further optimization of transfer methods, novel CAR construction and delivery methods such as novel gene-editing techniques based on CRISPR/Cas9, and finally combination of CAR NK cells with other treatments such as the checkpoint inhibitors, mAbs, drugs targeting TME, and chemotherapies. These strategies may strengthen the immune system overcome cancer and open a new chapter in cancer treatment. With the improvement of CAR NK cells, it is not surprising that in the near future, CAR-NK cell-based immunotherapy will revolutionize the treatment of cancer.


MRI gradient-fields may induce extrinsic voltage between electrodes and conductive neurostimulator enclosure of implanted deep brain stimulation (DBS) systems, and may cause unintended stimulation and/or malfunction. Electromagnetic (EM) simulations using detailed anatomical human models, therapy implant trajectories, and gradient coil models can be used to calculate clinically relevant induced voltage levels. Incorporating additional anatomical human models into the EM simulation library can help to achieve more clinically relevant and accurate induced voltage levels, however, adding new anatomical human models and developing implant trajectories is time-consuming, expensive and not always feasible.MRI gradient-field induced voltage levels are simulated in six adult human anatomical models, along clinically relevant DBS implant trajectories to generate the dataset. Predictive artificial neural network (ANN) regression models are trained on the simulated dataset. Leave-one-out cross validation is performed to assess the performance of ANN regressors and quantify model prediction errors.More than 180,000 unique gradient-induced voltage levels are simulated. ANN algorithm with two fully connected layers is selected due to its superior generalizability compared to support vector machine and tree-based algorithms in this particular application. The ANN regression model is capable of producing thousands of gradient-induced voltage predictions in less than a second with mean-squared-error less than 200 mV.We have integrated machine learning (ML) with computational modeling and simulations and developed an accurate predictive model to determine MRI gradient-field induced voltage levels on implanted DBS systems. More than 60% of patients implanted with a deep brain stimulator (DBS) need an MRI within 10 years after implantation . StructuDeep brain stimulation systems should also be designed for time-varying MRI gradient-fields, which can induce electric fields (E-fields) and current flow in the human body that may cause unintended tissue stimulation . MRI graElectromagnetic simulation methods described in Annex A.3.4 for ISO/TS 10974  are commin vivo MRI gradient-field induced extrinsic voltage levels on implanted DBS systems.Incorporating additional human body models and therapy implant routing pathways into the EM simulation library can help to achieve more clinically relevant and accurate test exposure levels, however, adding new anatomical human models into the EM simulation library is time-consuming, expensive and not always feasible. In this work, we integrated machine learning (ML) with computational EM modeling and developed a predictive algorithm using artificial neural networks (ANN) to predict E-fields are induced inside the human body due to time-varying MRI gradient magnetic fields. In the presence of AIMDs with long conductive components such as DBS leads and extensions, MRI gradient-field induced E-fields can generate extrinsic electric potential between spatially separated electrodes of an AIMD. Gradient-field induced extrinsic electric potential can be calculated by integrating the tangential component of the E-field along trajectory of the implanted system:L is the length of the AIMD trajectory and Vgradient is the MRI gradient-field induced extrinsic potential.where In this work, MRI gradient-field induced extrinsic electric potential between DBS therapy electrodes and the implanted conductive neurostimulator are evaluated. Terms induced voltage and voltage levels are used interchangeably to refer to MRI gradient-field induced extrinsic potential between DBS electrodes and neurostimulator.in vivo gradient-field induced voltage levels on implanted DBS systems , mean-squared-error (MSE) and median-absolute-error (MAE) metrics are calculated to evaluate the performance of these ML algorithms.Performance of five ML regression algorithms are evaluated on the Condensed-Dataset: support vector regression with linear kernel (SVMLIN), support vector regression with radial basis function kernel (SVMRBF), random forest regressor (RF), gradient boosting regressor (GB), and ANN with two fully connected hidden layers (NN). For all five algorithms, model parameter optimization is performed using “GridSearchCV” from scikit-learn package . To evalNeural network model training is performed using the Condensed-Dataset and performance metrics are evaluated on the Extended-Dataset. Leave-one-out cross validation is performed by training the NN model on five body-models and evaluating the performance on the body-model that is not included in training to assess model generalizability. Model training history with training and validation losses are evaluated to avoid overfitting. Final NN model with optimized parameters is then trained using the entire Extended-Dataset including all six body-models. Since a separate validation dataset was not available to evaluate the final NN model trained on the entire dataset, most conservative NN model prediction uncertainty levels from the leave-one-out cross validation analysis are used to estimate gradient-induced voltage prediction uncertainty .A list of software scripting and analysis tools used in this work are provided in the Three axis sum-of-magnitude gradient induced voltage levels at each principal axis slewing at 200 T/m/s are calculated. Example boxplots shown in As described in the Feature Selection section, selected features cannot uniquely identify DBS implant routing trajectories that are in the Extended-Dataset. For example, replicate routing pathways shown in R2 of RF and GB models are higher than 0.8, both models are biased and consistently underestimate “Fats” induced voltage levels. The NN model has the lowest MSE, highest R2, does not have a consistent bias, and therefore NN algorithm is selected for this study.To evaluate generalizability of ML algorithms, training is performed on five anatomical-models excluding “Fats” and performance are evaluated on the “Fats” induced voltage values. Performance of tuned SVMLIN, SVMRBF, RF, GB, and NN predictive models are shown in Neural network models with two to five fully connected hidden layers are evaluated and no significant performance boost is observed beyond two. Optimal model parameters are found to be a fully connected two layer NN with 25 and 120 units in first and second hidden layers, with no dropout, a batch size of 32 and 300 epochs; using GridSearchCV hyperparameter optimization method. Leaky rectified linear unit (ReLU) activation functions are used in all layers. As described before, all model training has been performed using the Condensed-Dataset. The two-layer ANN predictive models are capable of producing thousands of gradient-induced voltage predictions in less than a second.Leave-one-out training history plots are shown in Leave-one-out cross validation results on the Extended-Dataset are shown in R2 = 0.86, MSE = 124 mV) compared to other five body models . Final NN model prediction uncertainty is derived from the leave-one-out analysis, and most conservative MSE and MAE values of 124 and 191 mV, respectively, are used.Neural network models on Extended-Dataset exhibited a similar pattern, with induced voltage predictions on “Fats” body model having worse performance metrics  therefore takes at least 9 min. After gradient-induced E-field simulations are completed, extracting tangential component of the E-field along hundreds of DBS implant trajectories and computing the induced voltage take less than a second. In the most conservative scenario, ANN predictive model offers a speed-up of 500-fold (1 s vs. 9 min). On the other hand, Tier 3 EM simulation approach requires availability of anatomical body models and clinically relevant DBS routing pathways. Obtaining, creating, or morphing an anatomical body model can be time-consuming, expensive and may not always be feasible. Implementing a clinically relevant DBS implant trajectory in anatomical body model using an in-house software take between 10 to 100 s depending on the implanted system length, complexity of the DBS trajectory and quality of the anatomical body model tissue segmentation; or several hours if done manually by an experienced design technician. In cases where additional anatomical body models and/or DBS routing pathways are not available, Tier 3 EM simulation approach would require days/weeks to complete, whereas the predictive ANN model doesn’t require availability of additional anatomical body models or DBS routing pathways.Two-layer ANN predictive model developed in this study can perform thousands of gradient-induced voltage predictions in less than a second. In comparison, Tier 3 EM MRI gradient induced E-field simulations take between 3 and 5 min on a workstation with Intel Xeon E3-1535M CPU (2.9 GHz). Simulating E-field distributions inside a human body model using a set of gradient coils (max) and peak slew rate are desired to improve image quality and speed for echo planar imaging, diffusion-weighted imaging and several other advanced imaging protocols  with mean-squared-error less than 200 mV. The predictive model developed in this study can be used to determine clinically relevant MRI gradient-induced voltage exposure levels on implanted DBS systems.arcan.a.erturk@medtronic.com.The datasets for this article are not publicly available because datasets are Medtronic confidential. Requests to access the datasets should be directed to ME, ME conceived the study, performed the computational modeling and simulations, trained the machine learning models, and wrote the manuscript. EP helped with machine learning model development and analysis. MC and JE helped with gradient-induced voltage modeling and simulations. EP, MC, JC, and RB helped with the study design. All authors reviewed and edited the manuscript.The authors declare that this study received funding from Medtronic. The funder had the following involvement with the study: study design, collection, analysis, interpretation of data, the writing of this article, and the decision to submit it for publication.


N-arylation reactions to benchtop analysis for the efficient generation of fragment-based libraries. Technological advances in photoreactor design facilitated reliable and reproducible experimentation. Knowledge on the reactivity under previously reported reaction conditions of spirocyclic and strained heterocyclic building blocks, viewed as chemistry informers, could thus be rapidly accessed, identifying privileged or challenging scaffolds and paving the way for further exploration.Herein we report a workflow coupling photoredox-nickel dual-catalyzed  The higher reactivity of azaspirooctane example 3d highlights the distinct reactivity of azaspiroheptanes. On one hand, a rationalization of this observation could be traced back to the first step of the catalytic cycle. Reduction of the nickel(II) pre-catalyst to nickel(0) is believed to occur by β-hydride elimination on a sacrificial amount of amine [4c, 4d, 5a and 5b, provided that steric hindrance was not too high (see 5c). Surprisingly, oxetanes appear to be incompatible with the reaction conditions , despite a successful report on their decarboxylative arylation under nickel-photoredox-catalyzed conditions [Much information could be obtained from this screening. Electron-deficient aryl bromides led to better yields than neutral and electron-rich partners, as observed in previous reports on photochemically- or electrochemically-mediated nickel-catalyzed cross-couplings. Electron-deficient aniline products are less prone to oxidative decomposition. BCP-amines were viable coupling partners but gave the corresponding products of amine . The strof amine –18. Thisnditions . As thisnditions –21.2–sp3 fragments.In summary, we have developed a new workflow allowing to efficiently perform photoredox-nickel dual-catalyzed reactions in parallel for fragment-based library generation. We have screened 29 combinations of complex spirocyclic amines and (hetero)aryl halides to get an insight into the specific reactivity of these building blocks. The workflow combined a powerful photoreactor with precise and reproducible conditions control with benchtop analytical tools such as TLC–MS and low-field NMR. This enabled the identification of privileged spirocyclic scaffolds compatible with this chemistry, and challenging substrates requiring additional investigations. The knowledge obtained within this four-day experimentation period could be built-upon in-house and allowed the efficient synthesis of original heterocyclic spFile 1Experimental part.


Cytokine storm is an acute hyperinflammatory response that may be responsible for critical illness in many conditions including viral infections, cancer, sepsis, and multi-organ failure. The phenomenon has been implicated in critically ill patients infected with SARS-CoV-2, the novel coronavirus implicated in COVID-19. Critically ill COVID-19 patients experiencing cytokine storm are believed to have a worse prognosis and increased fatality rate. In SARS-CoV-2 infected patients, cytokine storm appears important to the pathogenesis of several severe manifestations of COVID-19: acute respiratory distress syndrome, thromboembolic diseases such as acute ischemic strokes caused by large vessel occlusion and myocardial infarction, encephalitis, acute kidney injury, and vasculitis . Understanding the pathogenesis of cytokine storm will help unravel not only risk factors for the condition but also therapeutic strategies to modulate the immune response and deliver improved outcomes in COVID-19 patients at high risk for severe disease. In this article, we present an overview of the cytokine storm and its implications in COVID-19 settings and identify potential pathways or biomarkers that could be targeted for therapy. Leveraging expert opinion, emerging evidence, and a case-based approach, this position paper provides critical insights on cytokine storm from both a prognostic and therapeutic standpoint. The coronavirus disease 2019 (COVID-19) pandemic has caused a public health crisis with profound long-term socioeconomic fallout. COVID-19 results from infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus . Much ofObservations from the first cohort of 41 COVID-19 patients in Wuhan, which led to the discovery of the novel SARS-CoV-2 virus, revealed a cytokine profile similar to that of secondary hemophagocytic lymphohistiocytosis (sHLH), a hyperinflammatory condition triggered by viral infection . A combiPatients who die from severe COVID-19 disease experience endothelial cell infection and an endotheliitis affecting many organs , 8. The per se can generate little inflammation  and chemotactic cytokines (IL-8 and MCP-1) rise early in hypercytokinemia, facilitating a sustained increase in IL-6. IL-6 binds to either membrane bound IL-6 receptor (mIL-6R) or soluble IL-6 receptor (sIL-6R), forming a complex that acts on gp130, regulates levels of IL-6, MCP-1 and GM-CSF via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, and thereby perpetuates the inflammatory processes . IL-6, aIn reaction to SARS-CoV-2 infection, macrophages  and dendWe propose that the immune system cytokine network may also communicate with the central nervous system (CNS) cytokine network, especially when the blood-brain-barrier (BBB) is compromised. Microglia and IL-1 activation can cause increased reactive oxygen species (ROS) production, phagocytosis, apoptosis, and increased cytokine expression see  within tSome of the severe complications associated with COVID-19 are acute respiratory distress syndrome (ARDS) , acute cardiac injury including myocarditis, myocardial infarction and cardiac arrest, sepsis, multi-organ failure, ischemic stroke, Kawasaki-like syndrome in children, acute pulmonary embolism, sHLH, and secondary infections such as bacterial pneumonia  3). The. The3). Although the association between cytokine storm and the radiological manifestations of COVID-19 pneumonia infection require further investigation, computer assisted tomography (CT), the lungs of patient with COVID-19 pneumonia typically demonstrate findings typical of underlying hyperinflammatory pathway , 54. On n = 81), 25% of the patients not receiving pharmacologic VTE prophylaxis developed lower extremity DVT, and 40% of those patients died. A D-dimer level of >1.5μg/mL predicted VTE with high sensitivity and specificity , elevated D-dimer and prothrombin time were correlated with a higher mortality rate  in all hospitalized COVID-19 patients unless the risk of bleeding outweighs thrombosis risk . FondapaCertain cancer patients, especially those with hematopoietic or lymphoid malignancies, are at higher risk of severe COVID-19 disease because they are immunocompromised , 73. GloHematopoietic stem cell transplantation recipients should practice self-isolation prior to transplantation. If such a patient becomes infected with SARS-CoV-2, the procedure should be delayed . CompletHIV-infected patients should be provided sufficient supply of medications to avoid treatment gaps and to allow them to maintain a viral load below the level of detectability. HIV-infected patients who display symptoms of COVID-19 should be prioritized for diagnostic testing because they are at risk for severe complications .Patients with systemic autoimmune conditions, including systemic lupus erythematosus (SLE), vasculitis, multiple sclerosis, progressive systemic sclerosis, or rheumatic disease affecting the lungs are at a greater risk of developing complications secondary to respiratory viruses . This inVarious stages of the cytokine storm pathway can be targeted for therapeutic effects  110). C. C110). Corticosteroids and NSAIDs can effectively suppress hyperinflammatory responses; however, delayed viral clearance could lead to further complications and also increase the risk of transmission. Although corticosteroids could be used acutely to target cytokine storm, their use in respiratory viral infection is associated with increased mortality, increased risk of secondary bacterial or fungal infections, and prolonged ICU admission. Furthermore, corticosteroids may mask COVID-19 related fever. As such, corticosteroids and NSAIDs are not recommended for routine management of COVID-19 patients , despiteInterleukin-6 (IL-6) plays a key role in cytokine storm . BlockinSARS-CoV-2 enters host cells via receptor-mediated endocytosis, which is regulated by numb-associated kinases (NKA) such as adaptor complex protein 2 (AP2)-associated protein kinase (AAK1) and G-associated kinase (GAK). The high affinity AAK1 blocker ruxolitinib is under investigation for treating COVID-19 (ChiCTR2000029580). To achieve NAK inhibition, toxic doses of AAK1 blockers are required. Baricitinib can inhibit both AAK1 and GAK (approved dosage of 2–4 mg daily) and can selectively inhibit JAK 1 and 2, thus reducing the inflammatory effects of Il-6 via the JAK-STAT signaling pathway. Furthermore, baricitinib can be considered in combination antiviral and anti-inflammatory therapies due to its minimal interaction with cytochrome P450 (CYP) enzymes and low plasma protein binding , 114. EaBecause of the association between cytokine storm and severe COVID-19 complications, we propose a cytokine storm-based diagnosis and management workflow for patients with or suspected of COVID-19  120). O. O120). The diagnosis and management of cytokine storm are clinically challenging and controversial due to lack of proven treatment. Our proposed algorithm may be used as a possible approach  120–122122; howeThe original contributions presented in the study are included in the article/SB devised the project, the main conceptual ideas, including the workflow targeting cytokine storm, proof outline, and coordinated the writing and editing of the manuscript. SB and AS wrote the first draft of the manuscript. SB encouraged AS to investigate and supervised the findings of this work. All authors discussed the results and recommendations and contributed to the final manuscript. The opinions expressed in this article are those of the authors and do not necessarily represent the decisions, official policy, or opinions of the affiliated institutions.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.


Granulosa cells (GCs) are somatic cells surrounding oocytes within follicles and are essential for folliculogenesis. Pathological changes in GCs are found in several ovarian disorders. Recent reports have indicated that long non-coding RNAs (lncRNAs), which modulate gene expression via multiple mechanisms, are key regulators of the normal development of GCs, follicles, and ovaries. In addition, accumulating evidence has suggested that lncRNAs can be utilized as biomarkers for the diagnosis and prognosis of GC-related diseases, such as polycystic ovary syndrome (PCOS) and premature ovarian insufficiency (POI). Therefore, lncRNAs not only play a role in GCs that are involved in normal folliculogenesis, but they may also be considered as potential candidate biomarkers and therapeutic targets in GCs under pathological conditions. In the future, a detailed investigation of the in vivo delivery or targeting of lncRNAs and large-cohort-validation of the clinical applicability of lncRNAs is required. Granulosa cells (GCs) are somatic cells of the sex cord , which aDevelopment of sequencing technology has led to in-depth genome and transcriptome analysis, which showed that over 85% of the human genome is transcribed . HoweverNcRNAs can be roughly divided into two groups: a group of short RNAs with a length less than 200 nucleotides long, such as microRNAs (miRNAs), small interfering RNA (siRNA), and piwi RNA (piRNA). The other category is long ncRNAs (lncRNAs) with a length longer than 200 nucleotides. Due to the lack of protein-coding capability, non-coding transcripts have been known as “junk DNA” or “transcriptional noise” for the past few decades . HoweverMoreover, emerging studies have demonstrated that some lncRNAs affect the function of ovarian GCs and are thereby involved in both physiological conditions and pathological processes, such as human oocyte maturation, fertilization, embryo development, tumorigenesis,  and ovarLong non-coding RNAs are extensively transcribed and play a role in a variety of biological functions in the human genome. Communication between GCs and oocytes is essential for oogenesis . StudyinYerushalmi et al. reported that compact GCs (CGCs) from germinal vesicle cumulus oocyte complexes (COCs) were isolated from patients undergoing in vitro maturation (IVM). Expanded GCs (EGCs) from metaphase 2 COC were isolated from patients undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). Global transcriptome profiles of CGCs and EGCs were compared, and 89 lncRNAs with significantly altered expressions were identified. Among them, 12 lncRNAs were encoded within introns of genes that were associated with GC development, suggesting a potential involvement of lncRNAs in cumulus expansion and oocyte maturation. Yerushalmi et al. also generated a library of genes regulated during cumulus expansion and oocyte maturation processes using RNA sequencing. Analysis of these genes led to the identification of novel lncRNAs that are potentially involved in COC maturation and cumulus expansion, which might boost our understanding of the process of oocyte maturation and ultimately improve the IVM efficiency .Using microarray analysis, another study compared the expression profiles of lncRNAs in GCs from three pairs of mature oocytes that gave rise to high-quality embryos and poor-quality embryos. A total of 20,563 lncRNAs were identified in GCs. Among them, one hundred and twenty-four lncRNAs were upregulated and five hundred and nine lncRNAs were downregulated in GCs from poor-quality embryos when compared to GCs from high-quality embryos. In addition, Gene ontology (GO) was used to analyze these significantly differential expressed lncRNAs. It is believed that GC-lncRNAs may exert their functions via interactions with coding transcripts and proteins in the development of oocyte and early embryos. Therefore, lncRNAs in GCs may contribute to the process of oogenesis .Another interesting study by Macaulay et al.  showed tPublished sequencing data , 33 wereThe endogenous expression of LncRNA-LET in KGN cells is quite low. Overexpression of this lncRNA in KGN cells repressed proliferation and migration and induced apoptosis. LncRNA-LET also repressed KGN cell epithelial-mesenchymal transition (EMT) by inducing E-cadherin and reducing N-cadherin and vimentin expression. An essential EMT-associated factor, tissue inhibitor of metalloproteinases 2 (TIMP2), was directly induced by lncRNA-LET. In addition, TIMP2 overexpression generally mimicked the lncRNA-LET function in KGN cells, validating the idea of TIMP2 being downstream target of lncRNA-LET. Moreover, lncRNA-LET and TIMP2 activated the Wnt/β-catenin and Notch pathways in KGN cells. Overall, lncRNA-LET repressed proliferation, migration and EMT, and promoted apoptosis of KGN cells by activating TIMP2 and inducing the Wnt/β-catenin/Notch pathways .Anti-Müllerian hormone (AMH) is mainly produced and secreted from GCs and is essential for repressing Mullerian duct development . The lonProlyl oligopeptidase (POP) is a serine endopeptidase that is involved in progesterone secretion . To furtLncRNA-AK124742 was identified as an antisense of proteasome 26S subunit, non-ATPase, 6 (PSMD6). A positive association was found between AK124742 and PSMD6 in cumulus GCs. Up-regulations of AK124742 and PSMD6 were observed in high-quality embryos (HCCs) compared to poor-quality embryos (PCCs). The relative expressions of AK124742 and PSMD6 were up-regulated in the pregnancy group compared to those in the non-pregnancy group. AK124742-PSMD6 is a potential lncRNA-mRNA biomarker in cumulus GCs involved in embryo selection [Cumulus expansion is a luteinizing hormone (LH)-mediated ovulatory process. Hyaluronan synthase 2 (HAS2) affects the synthesis of hyaluronic acid (HA), a major component of cumulus expansion. LncRNA HAS2 antisense RNA 1 (HAS2-AS1) expression was low in immature GCs and up-regulated in mature GCs. HAS2-AS1 inhibition caused the suppression of HAS2 and repressed the migration of cumulus GCs, suggesting that HAS2-AS1 is an LH-targeted gene that modulates cumulus expansion and migration by increasing HAS2 expression .To summarize, these studies in the above section focused on the roles of five individual lncRNAs in normal GCs, and their findings suggested that lncRNAs could affect the GCs and ovaries via different mechanisms. Although the role of lncRNAs in GCs have not been well-established under physiological conditions, the aforementioned reports show that they play an essential role in GCs. Specifically, lncRNAs  regulate host or adjacent gene function in GCs. In addition, lncRNAs (such as AK124742) could be potential biomarkers of embryo selection. Individual lncRNA (LncRNA-LET) also affects the functions of GCs  by affecting traditional signaling pathways . The role of individual lncRNA in normal GCs was summarized in Fig. Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility in women, mainly manifesting polycystic ovary and hyperandrogenism. The potential role of lncRNAs in the pathogenesis of PCOS was studied in human primary GCs and a GCs cell line, human granulosa-like tumor cell line (KGN). Microarrays were performed to compare “lncRNAome” in primary GCs obtained from seven PCOS patients and seven control women. The primary human GCs were mainly cumulus GCs. In total, 862 lncRNAs and 998 mRNAs were differentially expressed in the PCOS GCs. Real-time reverse transcription PCR (qRT-PCR) was used to validate lncRNA HCG26 expression in GCs obtained from 53 PCOS patients and 50 controls. HCG26 expression was up-regulated in PCOS patients and positively correlated with follicle number at a mature stage. HCG26 knockdown in KGN cells inhibited cell proliferation and cell-cycle progression. In addition, aromatase gene (CYP19A1) expression and estradiol production were also increased in HCG26-knockdown KGN cells. This study was the first to report the lncRNA profiles in GCs obtained from PCOS patients, and suggested that dysregulated lncRNAs might play vital roles in GCs proliferation and steroidogenesis .Another high-throughput study showed that 623 lncRNAs and 260 mRNAs were significantly altered and these differences could be used to discriminate GCs from PCOS patients and normal controls. Interestingly, some differentially expressed lncRNAs that are transcribed from chromosome 2 could act as enhancers, regulating neighboring coding genes. Therefore, we compared all differentially expressed lncRNAs that are transcribed from chromosome 2 and enhancer-like lncRNAs that were differently expressed in PCOS GCs. Fourteen overlapping lncRNAs were found among these two lncRNA sets  had increased in the peripheral blood of PCOS patients. SRA overexpression promoted GCs  growth and regulated the cell cycle by elevating several cell-cycle associated proteins, including Cyclin B, Cyclin E, and Cyclin D1. SRA also inhibited cell apoptosis by increasing the ratio of bcl-2:bax and repressing the levels of cleaved-caspase 3 and cleaved-Poly (ADP-Ribose) polymerase (PARP). In addition, SRA elevated the production of estradiol and progesterone, and that of two key enzymes, CYP19A1 and CYP11A1, in GCs, suggesting that SRA is a potential risk factor for PCOS . A folloPrader-Willi region non-protein coding RNA 2 (PWRN2) expression was associated with oocyte nuclear maturation in PCOS patients. In cumulus GCs, PWRN2 regulates 176 lncRNAs and 131 mRNAs. Based on these microarray data, a PWRN2-miR-92b-3p-transmembrane protein 120B (TMEM120B) competing endogenous RNA (ceRNA) network was established. This network was further validated by a luciferase report assay that revealed that miR-92b-3p directly binds to PWRN2 and TMEM120B. Therefore, PWRN2 is involved in oocyte nuclear maturation in PCOS patients by acting as a ceRNA . InteresBRAF-activated non-protein coding RNA (BANCR) is a well-studied lncRNA that plays pivotal roles in various malignancies, such as melanoma  and endoIn human primary cumulus GCs obtained from PCOS patients a negative association was found between LncRNA LINC-01572:28/p27 protein and PCNA. Also, there was a positive correlation between LINC-01572:28 expression and testosterone secretion from primary cumulus GCs. Furthermore, LINC-01572:28 inhibited cumulus GCs proliferation and prevented G1/S transition, which were partially reversed by p27 knockdown. This indicated that LINC-01572:28 suppressed cumulus GCs proliferation and cell cycle progression by reducing the degradation of p27 protein via competitive binding to S-phase kinase associated protein 2 (SKP2)-a key component of the SCF-SKP2 ubiquitin-ligase complex, which mediates cyclin E/CDK2-dependent ubiquitination and degradation of p27 .Androgen overdose is a key feature of PCOS. Researchers used microarrays to measure the profile of the abnormally expressed lncRNAs in GCs from 8 PCOS patients. A very highly expressed lncRNA in PCOS GCs was named as HUPCOS, which was positively correlated with follicle testosterone in PCOS patients. HUPCOS knockdown increased aromatase expression, which also promoted the conversion of androgens to estrogen. In addition, RNA-binding protein with multiple splicing RBPMS was identified as a direct binding protein of HUPCOS. This study showed that a novel lncRNA, HUPCOS, mediated androgen excess in the follicular fluid of PCOS patients by inhibiting aromatase expression via interaction with RBPMS .Liu et al. studied the role of lncRNA-PVT1/miR-17-5p/PTEN axis in PCOS ovarian GCs. Binding between PVT1 and miR-17-5p and the targeting between miR-17-5p and PTEN are determined by bioinformatics analysis, luciferase activity assay, RNA-induced silencing complex assay, and RNA pull-assay. PVT1 and PTEN were both highly expressed in PCOS ovarian GCs and follicular fluid, whereas miR-17-5p was not. MiR-17-5p overexpression and PVT1 knockdown could slow apoptosis and promote the cloning formation and proliferation of ovarian GCs in PCOS. In addition, PVT1 overexpression and miR-17-5p inhibition reversed these results. This study showed that both PVT1 down-regulation and miR-17-5p up-regulation lead to PTEN inhibition, which promoting proliferation and repressing apoptosis of ovarian GCs in PCOS .LncRNA TUG1 was significantly high in PCOS GCs and was associated with the antral follicle count. TUG1 was mainly expressed in GC nucleus. TUG1 knockdown in KGN cells inhibited proliferation and promoted apoptosis of GCs. TUG1 knockdown led to increased expression of bax, bak, cleaved caspase-3, caspase-9, cleaved caspase-9, LC3B and p-ERK proteins and decreased expression of bcl-2 and p62 proteins. In addition, using the ERK/MAPK pathway inhibitor U0126, the TUG1 knockdown-induced change in KGN cells was partially restored. Therefore, TUG1 was significantly higher in the PCOS group than that in the control group, TUG1 may inhibit cell apoptosis and autophagy in GCs through ERK/MAPK pathway inhibition and contribute to excess antral follicles. TUG1 has potential diagnostic value in PCOS .Taken together, these studies suggested that GC-specific lncRNAs are involved in PCOS, which paves a novel pathway for better understanding of the molecular mechanisms underlying this reproductive disease. However, these high-throughput experiments of lncRNA only identified GC-specific lncRNAs at the transcriptome level Table . To idenPremature ovarian insufficiency  is a common cause of female infertility. GC apoptosis is one important mechanism underlying this decline in ovarian function . CyclophWang et al. found lncRNA HCP5 decreased in GCs that isolated from patients with biochemical POI . HCP5 stabilized the interaction between YB1 and ILF2, mediating the YB1 transfer into GC nucleus. HCP5 knockdown affected this transfer and reduced its binding to to the promoter region of MSH5 gene and repressing its transcription. Reduced HCP5 expression in GCs of biochemical POI patients contributed to the generation of dysfunctional GCs by modulating MSH5 transcription via the interaction with YB1. This study showed a novel epigenetic mechanism for POI pathogenesis .To investigate whether lncRNA-FMR6 is involved in the development of fragile X-associated premature ovarian insufficiency (FXPOI), 22 consecutive fragile X mental retardation 1 (FMR1) premutation carriers undergoing IVF and pre-implantation genetic diagnosis (IVF-PGD) were studied. Eleven patients undergoing IVF- intracytoplasmic sperm injection (ICSI) were recruited as a control group and their cumulus GCs were isolated after oocyte retrieval. In FMR1 premutation carriers, there was a nonlinear association between the number of CGG repeats and the transcriptional level of lncRNA-FMR6. A negative linear correlation was observed between the number of oocytes retrieved and lncRNA-FMR6 expression in cumulus GCs, indicating that the RNA toxic gain-of-function is a potential pathophysiological mechanism of FXPOI .Ovarian reserves depend primarily on the number and quality of oocytes. Initially, insufficient or accelerated depletion of the primordial follicle pool will result in ovary insufficiency. It has been determined that mammalian GCs significantly contribute to the development of follicles 39, 40). In addition, reduced GCs induced follicle atresia, which eventually lead to POI . So far, the main research field of POI pathogenesis was protein-coding genes (4–6). However, the causing-gene mutations can only explain ~ 15% of POI cases . There is currently a lack of research on the role of individual lncRNA in POI GCs , which is characterized by enlarged ovaries and up-regulated vascular permeability, GCs were obtained from women with varying OHSS risks, for high-throughput sequencing. A total of 23,815 lncRNAs were detected and 482 of them showed differential expression: 205 lncRNAs were up-regulated and 277 lncRNAs were downregulated. Several ovarian biological processes were significantly involved in this phenomenon as demonstrated by KEGG pathway and GO analyses. Meanwhile, an lncRNA/miRNA interacting network was also established according to ceRNA regulatory mechanisms, a recent finding that adds to the complexities of miRNA-mediated gene modulation. ceRNAs are RNAs that mutually regulate other miRNAs via competitively binding the same miRNA recognition elements (MREs). In addition, expression screening identified eight novel lncRNAs  and aryl hydrocarbon receptor (AhR), were identified among the genes that were trans-regulated by the differentially expressed lncRNAs. The mRNA-lncRNA co-expression analysis showed that the TCDD-regulated lncRNAs might be involved in numerous cellular activities in GCs, such as cellular response to xenobiotics, proliferation, and dioxin metabolism. Therefore, these GC-specific lncRNAs may be involved in TCDD-induced reproductive defects [The environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes reproductive defects, such as anovulation and disorder of follicular steroidogenesis, in several mammals, including human, mouse, rat and pig , 61. In  defects .Endometriosis and its surgical treatment have an adverse effect on the ovarian reserve and on oocyte development. Metastasis associated lung adenocarcinoma transcript 1 (MALAT1), also known as nuclear-enriched abundant transcript 2 (NEAT2), is a well-studied lncRNA that is highly evolutionarily conserved  and is eTaken together, these data reflect the important role of GC-specific lncRNAs in a series of GC-related reproductive diseases, suggesting that GC-specific lncRNAs may be used as diagnostic markers or potential therapeutic targets for these diseases. However, the underlying mechanisms have not been systematically illuminated and these studies used in vitro assays. Therefore, to elucidate the specific function and mechanism of these lncRNAs in GCs obtained from patients suffering from ovarian diseases, advanced animal models and a large number of clinical samples will be required in subsequent studies.To investigate the potential application of lncRNAs in clinical diagnosis and therapy of GC-related diseases, researchers currently focus on the function and underlying mechanisms of lncRNAs in GCs. High-throughput technologies, such as lncRNA sequencing, have been applied to screen lncRNAs in GCs in several pathological conditions. These differentially expressed lncRNAs may be useful for the diagnosis or prognosis of GC-associated diseases. Moreover, studies of individual lncRNAs in GCs highlight the potential use of lncRNAs as therapeutic targets for GC-related diseases. For example, encapsulated siRNA may be delivered to target specific lncRNAs for the treatment of GC-related diseases. Due to the complex spatial structure and unclear molecular regulatory mechanisms, studies of lncRNAs are still at a very early stage. Therefore, a deeper understanding of lncRNA functions in GCs will provide a promising foundation for a potential future use of lncRNAs in the diagnosis or treatment of GC-related diseases.Granulosa cells are widely known for playing an essential role in both normal folliculogenesis and various ovarian disorders. LncRNAs exert their effects in GCs via multiple mechanisms. Until now, all regulatory mechanisms of lncRNA in GCs could be divided into five categories: 1) lncRNAs that regulate classical signaling pathways, such as lncRNA-LET (which induces the Wnt/β-catenin/Notch pathways), or lncRNA-SRA (which activates the NF-κB pathway) and lncRNA-MALAT1 and lncRNA-TUG1(which represses the ERK/MARK pathway) in GCs; 2) lncRNAs that activate transcription via activating their host genes or adjacent coding genes in GCs, including lncRNA-Amhr2, which activates the transcription of its neighboring gene Amhr2, or lncRNA-lncPrepþ96kb, which promotes its adjacent coding gene POP and HAS2, which was activated by its antisense lncRNA-HAS2-AS1; 3) ceRNA mechanism in GCs: PWRN2-miR-92b-3p-TMEM120B ceRNA network, LncRNA-LINC-01572:28/p27-SKP2-p27 ceRNA network and PVT1-miR-17-5p-PTEN ceRNA network in GCs; 4) P53-associated lncRNAs: lncRNA-BANCR and LncRNA-Meg3; 5) transcriptional factors-associated lncRNAs: lncRNA-HUPCOS interacts with RBPMS, lncRNA-CHBRP interacts with YY1 and SIX5 and lncRNA-HCP5 interacts with YB1(Table Additional file 1. The fourteen overlapping lncRNAs that are transcribed from chromosome 2 and enhancer-like lncRNAs that were differently expressed in PCOS GCs. Additional file 2. The eight novel lncRNAs in GCs that were associated with risk factors for OHSS.