Innumerable studies associated with cellular differentiation, tissue response and disease modeling have been conducted in two-dimensional (2D) culture systems or animal models. This has been invaluable in deciphering the normal and disease states in cell biology; the key shortcomings of it being suitability for translational or clinical correlations. The past decade has seen several major advances in organoid culture technologies and this has enhanced our understanding of mimicking organ reconstruction. The term organoid has generally been used to describe cellular aggregates derived from primary tissues or stem cells that can self-organize into organotypic structures. Organoids mimic the cellular microenvironment of tissues better than 2D cell culture systems and represent the tissue physiology. Human organoids of brain, thyroid, gastrointestinal, lung, cardiac, liver, pancreatic and kidney have been established from various diseases, healthy tissues and from pluripotent stem cells (PSCs). Advances in patient-derived organoid culture further provides a unique perspective from which treatment modalities can be personalized. In this review article, we have discussed the current strategies for establishing various types of organoids of ectodermal, endodermal and mesodermal origin. We have also discussed their applications in modeling human health and diseases , applications in regenerative medicine and evolutionary studies. Cell culture has been among the essential articulations used to study the mechanisms by which cells assemble into tissues and organs. Although two-dimensional (2D) cell culture is one of the major techniques through which researchers have elucidated a variety of aspects of cellular interactions, the uniform distribution of cells in the petri dish and the absence of nutrient gradients tend to diminish its physiological relevance  cell culture involves an artificially created growth environment where cells can interact in all three dimensions. 3D cell culture systems have special importance in fields such as drug discovery, as 2D culture models may not accurately reflect the response of a tissue to a particular compound , which can be embryonic (ESCs), induced (iPSCs), and adult stem cells (ASCs). Knowledge generated through the innumerable studies performed in the field of developmental biology have paved way for the establishment of protocols to culture various differentiated cell types from stem cell progenitors. Figure ETV2). These vascularized organoids acquired several blood–brain barrier characteristics and supported the formation of perfused blood vessels in vivo  of vertebrates is derived from the ectoderm after inductive signaling from the mesoderm. Su et al.  pioneereThe human brain is sub-divided into three distinct regions: forebrain, midbrain and hindbrain. Forebrain, the largest of the three regions, comprises of epithalmus, hypothalamus, subthalamus, thalamus and cerebrum. Cerebral aqueduct, cerebral peduncle, tectum and tegmentum form the midbrain whereas the hindbrain comprises cerebellum, medulla oblongata and pons.Paşca and colleagues have also generated region-specific organoids that resembled the dorsal and ventral forebrain, called cortical and subpallial spheroids respectively, which showed a transition from fetal to postnatal phenotype at around the 280th day in vitro , LDN193189 (BMP type I receptor inhibitor) and insulin for 8 days followed by thalamic patterning media containing BMP7 and PD325901 (MEK inhibitor) for another 8 days and subsequently grown in neural differentiation media , FGF1, FGF2, FGF9, PAX2 and Lim homeobox 2 (LHX2)  with hippocampal identity. Embryoid bodies were treated with antagonists of sonic hedgehog pathway and various factors that mimic the patterning of the forebrain, such as transforming growth factor- β (TGF-β) inhibitor SB431542, Dickkopf WNT signaling pathway inhibitor 1 (DKK-1) and Noggin (NOG). Hippocampal dentate gyrus-like granular neurons expressing PROX1 can be produced by treating the NPCs with high concentrations of WNT3a . TROY+ cells of human origin can be cultured in vitro to generate organoids resembling tissue from the corpus of the stomach . McCracken et al.  showed t+ intestinal mesenchyme  cells and type II (surfactant secreting) cells, but the culture conditions for these cells have not been completely defined, as co-culture with mouse lung fibroblasts is required for growth , each of which has certain drawbacks that make it suboptimal for disease modeling . The study used 5 PDAC tumoroid samples and treated them with an epigenetic drug, UNC1999, an inhibitor of histone methyltransferase, enhancer of zeste homolog 1 (EZH2) (H3 lysine 27 trimethylation writer). Out of the 5 tumoroids, the drug was effective against only 3 samples. Genetic analysis of the tumoroids revealed that only those 3 tumoroids retained the H3 lysine 27 trimethylation mark. This is indicative of tumoroids property of retaining the epigenetic signatures of the original tumor  live cell imaging of organoids have been utilized to study the tissue dynamics and also lineage tracing. Intravital imaging involves stable fluorescent labeled organoid culture followed by its transplantation in vivo. This is followed by tracing and imaging the primary and metastasized cells of the labeled organoids in vivo up to one year  gene , the level of which is increased by forskolin, an activator of adenylyl cyclase. They developed a forskolin-induced swelling (FIS) assay that could be used to screen drugs against specific mutations of the CFTR gene to create personalized treatment modalities for CF patients. CF patient biobanks are now being developed with organoids generated from intestine, lung, kidney and pancreas . CF is an autosomal recessive condition that is caused by a mutation in the cystic fibrosis transmembrane conductance regulator  mutant in RP  caused due to either X-linked or autosomal mutations. Retinal organoids generated from patient-derived iPSCs have been used to study the tRNA nucleotidyl transferase, CCA-adding 1 (TTC7A gene (tetratricopeptide repeat domain 7A). Intestinal organoids derived from two HMIA patients showed the pathogenicity of TTC7A mutations causing gain of function of differentiation and loss of function of epithelial proliferation  is an autosomal recessive genetic disease caused by mutation in JAG1 gene that encode Notch receptor ligand associated with ALGS. The organoid model study helped in the rescue of impaired ducts on reversal of JAG1 mutation in vitro  is an autosomal genetic disease affecting multiorgan systems including liver, heart, skeleton and others. The liver organoid model generated from patients was used to study the pathogenicity of the mutant An organoid’s property of retaining the genetic signatures of the original tissue makes it an excellent model to study host-microbe interactions Bartfeld . Many ofHelicobacter pylori, for example, infects about 50% of the human population, but only a small subset of individuals develops gastric cancer and gastric ulcers  infection  and could provide insight into its neurobiology  is caused due to accumulation of Lewy bodies that lead to selective degeneration of dopaminergic neurons. PD can be studied by modeling of midbrain organoids  can be addressed. TOCs fail to recapitulate the precise microenvironment and its cues for organ development in vivo. One such area where organ-on-chip can help improvise the organoids is in the management of biochemical microenvironment. In TOCs controlling the morphogen gradients at different time points becomes a challenging task. The organ-on-chip model uses a pair of microchannels that can act as a source and sink to precisely control the biochemical environment  which was lost in humans during evolution. The hPSCs used for the generation of cortical organoids were CRISPR-Cas9 genome edited and the human NOVA1Hu/Hu gene replaced with the archaic NOVA1Ar/Ar. There was an observed increase in the rigid surface complexity, slower growth, and altered electrophysiological properties in cortical organoids of NOVA1Ar/Ar archaic variant as compared to NOVA1Hu/Hu human cortical organoids. This study revealed the mutations were exclusively found in the modern day humans. The human-specific mutations in NOVA1Hu/Hu not observed in archaic NOVA1Ar/Ar may be due to functional role during the course of evolution. Various researchers, although commended this work, have been skeptical due to two reasons: a) there is a limitation to validate these evolutionary claims in the not well-preserved soft tissues of Neanderthal brains and b) the organoid may not represent the actual Neanderthal brain, moreover, the observed difference in organoid morphology by this single protein change to archaic one would be due to compounding effect (Remmel Brain organoids have been used to understand the changes that occurred during the evolution of man from Denisovan and Neanderthals who are the extant closest relatives to modern humans. One method described by Trujillo et al.  uses intt Remmel .Despite the potential of organoids in understanding disease and development, like any other system, there are limitations that need to be addressed. First, although organoids have been demonstrated to mimic the cellular microenvironment of tissues more faithfully than 2D culture systems, there are minute differences in gene expression patterns between organoids and the corresponding in vivo tissues (Pollen et al. Growth of organoids in Matrigel, a tumor-derived ECM matrix, also impedes their potential clinical application due to the risk of transfer of pathogens and immunogens (Clarke et al. As organoids are found to have more and more relevance and applications, large scale manufacture of organoids becomes inevitable. Due to heterogeneity of cell types, generation of viable organoids with higher yield has been a major challenge. For instance, while generating the intestinal organoids, not all the aggregate of cells bud into organoids. However, by selectively culturing the aggregate of cells that has the potential to develop into organoids, would increase the yield in cultures. Arora et al.  developeEthical challenges in organoid culture are related to the use and derivation of PSCs. Informed consent of the donor in case of patient-derived organoids for further use and limitations of their applications have been considered in the organoid culture research similar to those in developmental biology and clinical research (Munsie et al. . Organoids derived from patients do not deviate in their phenotype, and thus are good models to study disease. Furthermore, organoid biobanks hold the promise of helping us understand the contributions of genetic variations to drug response, helping improve existing therapies and discover new ones. Thus, organoids hold the promise of bridging the gap between animal model systems and human subjects. In the future, this technology may help reduce the use of animals in in vivo drug testing, provide an ethical alternative to animal testing. Use of organoids in evolutionary biology, may additionally serve along with the conventional methods of fossil studies and computational biology.This review has aimed to collect information on the various strategies employed in organoid culture and to address the potential of this form of cell culture in modeling diseases, as a tool for drug testing, and to better understand the developmental processes involved in the formation of various organs in the human body. The ability of organoids to recapitulate the genetic and epigenetic signatures of the original tissue is of relevance in the field of personalized medicine and can help find therapies against various diseases and conditions. Methods to co-culture tumoroids with tumor environmental cells have allowed the study of tumor-environment interactions, which could possibly lead to a better understanding of the progression of cancer and its potential treatment. The combination of organoid culture with CRISPR/Cas technology and organ-on-chip devices to incorporate and study vasculature are methods that may help better recapitulate in vivo tissue biology


Diabetoporosis, diabetic-related decreased bone quality and quantity, is one of the leading causes of osteoporotic fractures in subjects with type 2 diabetes (T2D). This is associated with lower trabecular and cortical bone quality, lower bone turnover rates, lower rates of bone healing, and abnormal posttranslational modifications of collagen. Decreased nitric oxide (NO) bioavailability has been reported within the bones of T2D patients and can be considered as one of the primary mechanisms by which diabetoporosis is manifested. NO donors increase trabecular and cortical bone quality, increase the rate of bone formation, accelerate the bone healing process, delay osteoporosis, and decrease osteoporotic fractures in T2D patients, suggesting the potential therapeutic implication of NO-based interventions. NO is produced in the osteoblast and osteoclast cells by three isoforms of NO synthase (NOS) enzymes. In this review, the roles of NO in bone remodeling in the normal and diabetic states are discussed. Also, the favorable effects of low physiological levels of NO produced by endothelial NOS (eNOS) versus detrimental effects of high pathological levels of NO produced by inducible NOS (iNOS) in diabetoporosis are summarized. Available data indicates decreased bone NO bioavailability in T2D and decreased expression of eNOS, and increased expression and activity of iNOS. NO donors can be considered novel therapeutic agents in diabetoporosis.  The prevalence of type 2 diabetes (T2D) is increasing worldwide and is estimated to reach 693 million by 2045  bioavailability can contribute to diabetoporosis. NO is produced in the osteoblast and osteoclast cells by the three isoforms of NO synthase (NOS) enzymes , and iNOS . eNOS aThe eNOS gene is constitutively expressed in osteoblasts and osteocytes in both the fetus and adults . HoweveSome studies have failed to detect nNOS expression in the bone cells . In addAs shown in Figure 1Decreased NO bioavailability has been reported in bones of humans and animals with T2D and can be considered as one of the main mechanisms in diabetoporosis. As shown in Figure 2eNOS uncoupling in the bones of T2D patients is at least in part due to increased production of bone morphogenetic protein 4 (BMP4)  that leBone remodeling is a life-long process and is achieved within anatomical structures that are known as a basic multicellular unit (BMU). These provide a unique microenvironment to facilitate coupled bone resorption and formation , the osteocytes sense signals for initiating remodeling; these include mechanical forces, changes in calcium homeostasis, or changes in hormone levels that translate into biological signals , the decreased TGF-β in osteocytes recruits hematopoietic stem cells (HSC) from the bone marrow or the circulation; these HCSs are then differentiated to osteoclasts in the presence of monocyte/macrophage colony-stimulating factor (M-CSF) and the RANKL . Low leMononuclear macrophage-like cells in step 3  engulf and remove demineralized undigested collagen and generate transition signals that stop bone resorption and start bone formation . Step 4 , in response to a decrease in SOST within osteocytes, mesenchymal stem cells (MSC) are recruited and differentiated into osteoblasts that start the bone formation and mineralization process. When an equal quantity of resorbed bone has been replaced, the remodeling cycle is terminated . Some oIn T2D and the bone remodeling process, there is a decrease in eNOS-derived NO and an increase in iNOS-derived NO; this leads to inhibition of steps 1 and 4, activation and bone formation, respectively; and at the same time, step 2, bone formation is stimulated. As shown in Figure 3eNOS-derived NO directly activates and facilitates osteoblastic differentiation from MSC  throughAvailable treatments for osteoporosis are limited by cost, side effects, and efficacy, with limited impact on the cortical bone  contributed equally as corresponding author.This study was supported by Shahid Beheshti University of Medical Sciences [grant No. 27443-1], Tehran, Iran.The authors declare that they have no competing interests.


Transplanting stem cells with the abilities of self-renewal and differentiation is one of the most effective ways to treat many diseases. In order to optimize the therapeutic effect of stem cell transplantation, it is necessary to intervene in stem cell differentiation. Inorganic nanomaterials (NMs), due to their unique physical and chemical properties, can affect the adhesion, migration, proliferation and differentiation of stem cells. In addition, inorganic NMs have huge specific surface area and modifiability that can be used as vectors to transport plasmids, proteins or small molecules to further interfere with the fate of stem cells. In this mini review, we summarized the recent advances of common inorganic NMs in regulating stem cells differentiation, and the effects of the stiffness, size and shape of inorganic NMs on stem cell behavior were discussed. In addition, we further analyzed the existing obstacles and corresponding perspectives of the application of inorganic NMs in the field of stem cells. Stem cells refer to cells with self-renewal and differentiation capacity, which can be roughly divided into embryonic stem cells and somatic stem cells. Embryonic stem cells are derived from blastocysts . Typical2) nanoparticles (2) nanoparticles  and graphene have good physical properties, stability and biocompatibility, which can maintain the adhesion and proliferation of stem cells and can influence the differentiation fate of stem cells . But dif2+-doped CDs and Zn2+-doped CDs, which were internalized by cells as a biomarker and simultaneously promoted the osteogenic differentiation of mouse embryo osteoblast precursor cells (MC3T3-E1) by increasing their ALP activity.Being able to simultaneously monitor and direct stem cell differentiation is important for the application of stem cells. In vivo experiments have shown that AuNPs can promote bone regeneration at bone defect sites and play a positive role in bone healing  to promote osteogenic differentiation of MSCs . Patel e+ -based alloys are a common type of bone implants. Inert Ti alloy implants can quickly form TiOtibility .TiO2 filmaterial . Ti-baseoporosis . At the oporosis . TiO2 nain BMSCs . Apart fntiation .2 nanotubes to obtain Crystalline SrTiO3, which can realize the slow release of Sr. It further enhances the ALP activity and matrix mineralization ability of MSCs. SrTiO3 nanotube arrays have good biocompatibility and are ideal implants for osteoporotic bone (Strontium (Sr) has biological effects to promote osteogenesis, and moderate supplementation of Sr enhances calcium absorption . Zhang etic bone . Meanwhitic bone . In additic bone .3O4 nanoparticles (Fe3O4 NPs) are typical magnetic materials for promoting bone tissue regeneration  in the skin injury model.Feneration . Fe3O4 Nof hMSCs . Magneti . Moreover, the ππ stacking interaction affects the charge-carrier density between catecholamines and MnO2 nanoellipsoids through which the catecholamines secreted by PC12 cells can be monitored in real time (One of the most important applications of MnO in vivo . MnO2 nang (MRI) . Mn2+ caeal time .2 as an antioxidant can alleviate the oxidative environment of injured tissues. Modification of MnO2 on the surface of MSCs can improve cell survival in oxidative disease tissue while secreting more proangiogenic factors , with excellent adsorption properties, stability and biocompatibility, are often used as vehicles for various active molecules to direct the fate of stem cells . BMP2 dein vivo to form calcium salts, thus accelerating the biomineralization of bone defects and enhancing bone regeneration  determine the differentiation fate of stem cells . ModulatIn vivo transplantation can also promote new bone formation and osseointegration at the femoral defect   . In contorted by  to be thTaken together, a variety of inorganic NMs are capable of intervening in the differentiation of stem cells and are used for the treatment of specific diseases. Meanwhile, the physicochemical characteristics including stiffness, shape and size of inorganic NMs greatly influence their differentiation guidance see .ClinicalTrials.gov database yielded only four results and none of the clinical studies were related to stem cells, so we can see the extremely low clinical translation of nanomaterials. Combined with the analysis of the current published papers on the effects of inorganic NMs on stem cell differentiation (see in vitro and in vivo. Articles usually emphasize that inorganic NMs possess good biocompatibility. However, these experiments only carry out short-term tests in vivo and in vitro, and even do not detect the distribution of nanoparticles in various organs in the body, which is far from the actual clinical application. In addition, due to the profound differences in anatomy, physiology and genetics between humans and animals (We searched the literatures through the PubMed website using “stem cells and nanomaterials” as keywords in the past 5 years. A total of 3,526 results were displayed, and only two remained after adjusting article type to clinical trial, both about nanocurcumin. In addition, our keywords search for “nanomaterials and nanoparticles” in the tion see , it is n animals , success animals . In the  animals , put for2 nanoparticles increase the secretion level of pro-angiogenic factors in MSCs (3O4 NPs-treated hMSCs significantly promoted the angiogenesis of hUVECs. In addition, cell-derived nanoparticles have emerged as a promising alternative to synthetic nanocarriers for safer clinical outcomes (Previous studies have suggested that the efficacy of stem cell transplantation depends on the differentiation into specific cell types, but there is growing evidence that the efficacy depends on paracrine behavior, which produces neuroprotective, angiogenesis, and immunomodulatory effects through the secretion of a large number of cytokines and proteins. MnO in MSCs . Li et aoutcomes . Exosom-Stem cell transplantation has enabled the cure of many diseases. Based on previous studies, we know that the unique physicochemical characteristics of inorganic NMs greatly influence stem cell fate , and thein vivo distribution, visceral toxicity, as well as metabolic pathways of the NMs. Next, HSCs are known to play an important role in the field of regenerative medicine. But there are few publications related to inorganic NMs’ role in HSCs (Biosafety issues are one of the main reasons for the low clinical translation efficiency of inorganic NMs in the field of regenerative medicine, so we should also perform more comprehensive and systematic studies on the biosafety of inorganic NMs, which are not limited to superficial cytotoxicity tests, but should pay more attention to the  in HSCs . Based o in HSCs . The limIn conclusion, inorganic NMs enrich the applications of stem cells, and there are still many problems to be solved, but nanomaterials combined with stem cell therapy is promising and will lead to major breakthroughs in the near future.


Prevention through early screening remains one of the most optimal strategies against breast cancer. The primary objective of this analysis was to determine the prevalence of breast cancer screening using the clinical breast examination (CBE) and breast self-examination (BSE) methods among women aged 15-49 years, and the secondary objective was to explore demographic and socio-economic factors associated with clinical breast examination (CBE) and breast self-examination (BSE) breast cancer screening methods.the study used Demographic Health Survey data collected in 2014. The study participants were Basotho women aged 15-49 years. STATA 17 was employed for developing logistic regressions and weighting for sampling probabilities and non-response. Complex sampling procedures were also considered during testing of statistical significance.variables that were associated with significantly increased odds of having you had a breast cancer either self-examination or clinical test in last 12 months were: i) visiting a health centre in the past 12 months ; ii) completion of primary level education ; iii) being aware of breast cancer ; and iv) age ; while district of origin  was significantly associated with decreased odds of the outcome.our findings suggest that raising awareness about breast cancer is the most effective method of improving breast cancer screening among women in Lesotho. Breast cancer is associated with serious morbidity, low quality of life and mortality . TreatmeSeveral studies have investigated the factors associated with breast cancer screening. The first strand of studies investigated the factors associated with undertaking a mammography -14. The The primary objective of this study is to investigate the prevalence of breast screening among women aged 15-49 using CBE and BSE methods in Lesotho. The secondary aim, is to analyse the socio-economic and demographic factors which are associated with breast cancer screening, using CBE and BSE, among women aged 15-49 in Lesotho. Currently the prevalence of breast cancer screening is unknown for Lesotho due to absence of National Cancer Registry. Secondly, confirmed cases of breast cancer are not treated locally, they are treated in South Africa and India. In most cases, referrals are made where breast cancer is detection at an advanced stage. As a result, policy interventions which promote breast cancer screening are essential for developing countries such as Lesotho.The study participants were Basotho women aged 15-49 years from the 10 districts of Lesotho in Southern Africa.This study is an exploratory analysis of secondary data derived from the 2014 national demographic health survey (DHS) database. DHS is designed to provide estimates of national and regional health indicators for the 10 districts of Lesotho. The sampling frame used for the 2014 LDHS is an updated frame from the 2006 Lesotho Population and Housing Census (PHC) provided by the Lesotho Bureau of Statistics (BOS). The sampling frame excluded nomadic and institutional populations such as persons in hotels, barracks, and prisons.The 2014 LDHS followed a two-stage sample design and was intended to allow estimates of key indicators at the national level as well as in urban and rural areas, four ecological zones, and each of Lesotho´s 10 districts . The firWe included all women aged 15-49 years for whom we had complete data on all the variables-which included the outcome and independent variables described below. As a result, out of 9,942 participants, there were 6621 women and 2931 were men. From a sample of 6,621 women, only 6187 were eligible for this study because they had knowledge of undertaking either a CBE or BSE in the last 12 months as shown in The outcome variable was: have you had a BSE or CBE in last 12 months? (yes/no)Age (in years), wealth index, district of origin, living in a rural or urban area, owning a radio or TV set, having a health insurance policy, awareness of breast cancer, visiting a health centre in the last 12 months, were among the independent variables.We used descriptive statistics - reported as count (percentage) - to describe the characteristics of the respondents included in the analysis. We used a multivariable logistic regression to explore whether the independent factors where associated with the outcome. This analysis appropriately adjusted for the complex sampling design by using the design weights . We alsoIt is estimated that 3,887/6,187 (62.8%) of the study participants were form rural areas as shown in Prevalence of breast cancer screening: it is estimated that 38.5%   of the respondents had undertaken either a BSE or CBE in the past 12 months.Factors associated with breast cancer screening: as shown in Women from Butha Buthe district  were significantly associated with decreased odds of the outcome relative to Maseru which is capital district of Lesotho.There was no significant association between having either a BSE or CBE in last 12 months and owning a medical insurance. Similarly, the outcome variable was not significantly associated with being from a rural area, owning a radio or television set, household wealth and originating from other districts excluding the capital district which was used as a reference district.The study also investigated the relationship between district of origin, breast cancer awareness and the outcome variable. The results of the main and interaction effects shown in Key findings: based on the multivariable logistic regression estimated above, it is evident that having visited a health centre in the past 12 months, completing primary level education, awareness of breast cancer and age group 35-39 years were significantly associated with having you had either a BSE or CBE in last 12 months.The results from the interaction effects reveal that lack of knowledge about breast cancer is the main challenge for women from Butha Buthe, Mokhotlong, Thaba Tseka, Mafeteng, Mohale´s Hoek and Quthing districts to undertake either a CBE or BSE.How results compare with similar studies: these results are congruent with existing literature which indicates that breast cancer screening is highly associated with: (i) higher ager [her ager , 27-29; her ager , 28-30; her ager , 31; TheThe results further indicate that having visited a health centre in the past 12 months, having completed primary level education, and being aware of the breast cancer, were associated with significantly higher the odds of having either a BSE or CBE in last 12 months.The interaction results indicate the importance of breast cancer awareness on undertaking either a CBE or BSE. Firstly, the odds of women undertaking either a CBE or BSE are significantly increased amongst women with knowledge of breast cancer as compared to with women without knowledge of breast cancer. Secondly, women from certain districts of the country, namely Butha Buthe, Mokhotlong, Thaba Tseka, Mafeteng, Mohale´s Hoek and Quthing, are not undertaking either a CBE or BSE because they do not have knowledge of breast cancer. Thirdly, the results reveal that younger women can undertake breast cancer screening if they were aware of breast cancer. Lastly, the results reveal that awareness raising yields significant outcomes when undertaken at health centres as compared to radio or TV.Limitations: this is an exploratory analysis based on cross-sectional data and therefore we caution against inferring causal links in the results. Secondly, the study prevalence is based on self-reported data as opposed to clinical surveillance and we therefore caution its adoption as the national prevalence of breast cancer screening. Thirdly, the study focused on BSE and CBE as methods of breast cancer screening due to limited data on mammography.The prevalence rate of breast cancer screening, measured using BSE and CBE, among women aged 15-49 in Lesotho is estimated at 38.5%. In order to improve it, policy interventions should seek to improve knowledge of women on breast cancer. The interaction results indicate awareness campaigns are most effective when conducted at health centres as opposed to radios or television.Breast cancer screening in developing countries can be measured using BSE and CBE;There are socio-economic factors affecting breast cancer screening, which are country and location specific;Media advocacy is one of the most effect tool for promoting breast cancer screening.The study estimated the prevalence of breast cancer screening in Lesotho, measured using BSE and CBE, which was not known;The study presents new socio-economic variables which are associated with breast cancer screening; having visited a health centre in the last 12 months;The study proves that health education at health facilities on breast cancer is more effective than media advocacy in promoting breast cancer screening amongst women.


Genetic variation databases provide invaluable information on the presence and frequency of genetic variants in the ‘untargeted’ human population, aggregated with the primary goal to facilitate the interpretation of clinically important variants. The presence of somatic variants in such databases can affect variant assessment in undiagnosed rare disease (RD) patients. Previously, the impact of somatic mosaicism was only considered in relation to two Mendelian disease-associated genes. Here, we expand the analyses to identify additional mosaicism-prone genes in blood-derived reference population databases.ASXL1 and DNMT3A, we identified 7 additional autosomal dominant RD-associated genes with known pathogenic single-nucleotide variants present in the reference population databases and good evidence of allelic imbalance: BRAF, CBL, FGFR3, IDH2, KRAS, PTPN11 and SETBP1. From this group of 9 genes, the majority (n = 7) was important for hematopoiesis. In addition, 4 of these genes were involved in cell proliferation. Further assessment of the known 156 hematopoietic genes led to identification of 48 genes (21 not yet associated with RDs) with at least some evidence of mosaicism detectable in reference population databases.To identify additional mosaicism-prone genes relevant to RDs, we focused on known/previously established ClinVar pathogenic and likely pathogenic single-nucleotide variants, residing in genes associated with early onset, severe autosomal dominant diseases. We asked whether any of these variants are present in a higher-than-expected frequency in the reference population databases and whether there is evidence of somatic origin  rather than germline heterozygosity . The mosaicism-prone genes identified were further categorized according to the processes they are involved in. Beyond the previously reported These results stress the importance of considering genes involved in hematopoiesis and cell proliferation when interpreting the presence and frequency of genetic variants in blood-derived reference population databases, both public and private. This is especially important when considering new variants of uncertain significance in known hematopoietic/cell proliferation RD genes and future novel gene–disease associations involving this class of genes.The online version contains supplementary material available at 10.1186/s40246-021-00371-y. The advent of high-throughput sequencing created a revolution in the discovery and diagnostics of Mendelian diseases , 2. LargA powerful way to deprioritize potentially non-contributing variants in patients with undiagnosed Mendelian diseases is to assess their presence, frequency and corresponding genotypes in such reference population databases , 3, 7. Ahttps://www.bcchr.ca/silent-genomes-project/ibvl), the Greater Middle East Variome project (GME) (http://igm.ucsd.edu/gme/) and the Iranome project (http://www.iranome.ir/). Currently, the reference population databases are based on blood-derived DNA, due to the ease of data collection and quality. Here, we explored the blood-derived reference population data for the presence of somatic variants and their potential impact on diagnostics of rare genetic diseases.While reference population databases offer great power for variant interpretation, the information these databases contain and their limitations need to be well understood to ensure appropriate use. For example, public reference population databases may contain data on some individuals who are not healthy despite the efforts to exclude all individuals with severe pediatric disease . Moreover, both public and private reference population databases may contain variants of variable quality due to sequencing errors, and despite attempts to apply various filtering strategies for quality control, they may persist and in some instances complicate analyses. Importantly, all of the currently available public reference population databases also suffer from ancestry and diversity biases and hence the efforts to increase the diversity. Some examples of such efforts include the Silent Genomes Indigenous Background Variant Library  nonsense variants in ExAC. This was unexpected as ASXL1 haploinsufficiency has been implicated in severe, pediatric, autosomal dominant (AD) disease, Bohring–Opitz syndrome (BOPS [MIM: 605039]). The presence of such ASXL1 variants in non-BOPS individuals has not been described  , we first assembled a list of 1388 genes implicated in 2010 AD diseases , Cbl proto-oncogene (CBL), isocitrate dehydrogenase (NADP(+)) 2 (IDH2), KRAS proto-oncogene, GTPase (KRAS) and protein tyrosine phosphatase non-receptor type 11 (PTPN11), genes known to have a role in hematopoiesis, the formation of cellular components of blood  and set-binding protein 1 (SETBP1) were involved in other blood-related functions . This variant can be found in COSMIC in relation to cancer; it has been confirmed to be somatic and is predominantly observed in hematopoietic and lymphoid samples . These results support our decision to use the ExAC database as our primary source of information, which allowed us to get a better overview of the mosaicism phenomenon in blood-derived reference populations in general. Furthermore, our findings emphasize the importance for better ways to flag, rather than filter, potentially mosaic variants based on knowledge on the mosaicism-prone genes in reference population databases.Next, we wanted to compare our findings in nine AD genes with good evidence of somatic mosaicism in ExAC  that may be affected by CHIP driver variants, the list of 156 hematopoietic genes compiled by Jaiswal et al.  was analIn addition to the already known somatic ClinVar pathogenic and likely pathogenic SNVs in ExAC, we found 125 SNVs of the same variant types (nonsense or missense) which have not yet been associated with a Mendelian disease but have at least one allele with read ratio imbalance, residing in 40 of 156 hematopoietic genes. Some of the hematopoietic genes (besides those nine AD genes we described earlier) have two or more of such SNVs, and 84 of these 125 SNVs have been reported in COSMIC  and DNMT3A (for missense SNVs) genes. As mentioned earlier, the severe, early onset AD disease BOPS [MIM: 605039] is caused by nonsense ASXL1 variants. By searching the ExAC dataset, we found two such ASXL1 SNVs which have been reported in ClinVar, and show allelic imbalance in ExAC , which are currently not associated with BOPS, but may be in future. The majority of these SNVs (16 of 20 variants) can be found in COSMIC where they were described as somatic and in relation to malignancies  have been found in COSMIC where they were reported as confirmed somatic and in relation to different malignancies. In addition to ASXL1 and DNMT3A, we found similar examples in genes such as CBL, IDH2 and KRAS  having at least some evidence of somatic mosaicism .ASXL1 and DNMT3A). Given the mechanism by which these variants arise in blood tissue, we expect that our findings are applicable to other public and private blood-derived reference population databases. The list of genes prone to acquiring somatic variants will enable variant interpretation computational pipelines to flag variants residing in those genes as ‘potentially somatic’ and thus select them for closer inspection.Here, we revealed the presence of somatic ClinVar pathogenic and likely pathogenic SNVs associated with early onset severe AD diseases in the reference population database ExAC for seven additional genes   in the lgnancies , 29. ThiThere are several study limitations associated with our work that we would like to highlight to aid in the interpretation of our findings. First, this work was based on the analysis of aggregated data in publicly available reference population databases. Therefore, we did not have access to DNA samples to allow for a direct comparison of sequencing results from different tissues and thus confirm the true genotypes of the individuals. Instead, several important hallmarks of somatic mosaicism were used for characterization of alleles and SNVs. Second, several SNVs with detectable imbalance in ExAC and/or gnomAD were excluded due to low quality  which was done to reduce the number of false positives. Nevertheless, some of the potential false positives might in fact be true positives meaning that our study was not able to capture all genes prone to acquiring somatic SNVs. Third, the SNVs without read support available in the IGV were excluded from this study. Those SNVs could potentially have evidence of somatic mosaicism that is not accounted for here. Also, we only focused on the ClinVar pathogenic and likely pathogenic SNVs associated with early onset severe AD conditions in the reference population databases that are due to mosaicism. Other SNVs that do not show signs of mosaicism could be present because (i) they are not pathogenic even though reported as being so, (ii) it was not possible to detect mosaicism using the chosen methodology for those SNVs, (iii) they may not be contributing to AD conditions, (iv) sequencing errors could have occurred that we do not know of and/or (v) occurrence of incomplete penetrance and/or variable expressivity that affects the likelihood of whether a condition develops or not . FinallyConsideration of the identified genes prone to acquiring somatic variants during the interpretation process will be helpful to reduce the risk of errors due to variant misclassification. We showed that the somatic mosaicism is present in large blood-derived publicly available reference population databases, such as ExAC and gnomAD, to a higher extent than it was previously reported , 15. OurASXL1 and DNMT3A, the list of mosaicism-prone genes was expanded to include BRAF, CBL, FGFR3, IDH2, KRAS, PTPN11 and SETBP1. All of these genes showed the presence of somatic ClinVar pathogenic or likely pathogenic SNVs in a reference population. Focusing on some of the most widely used blood-derived reference population databases, we showed that the current filtering strategies employed to mitigate the problem of somatic mosaicism were only partially successful. In addition to the variants previously described in ClinVar as pathogenic and likely pathogenic, we revealed other SNVs that show allelic imbalance. Some of these variants are associated with non-AD diseases, while the others are currently not associated with any Mendelian disease, but found in the COSMIC database in relation to cancer and are residing in hematopoietic genes. We expect that at least some of these genes will be associated with Mendelian diseases in the future. Overall, our results underline the importance of considering CHIP and genes involved in hematopoiesis and cell proliferation when interpreting the presence and frequency of genetic variants in both public and private blood-derived reference population databases.We identified 7 additional AD disease-associated genes with recurrent appearance of somatic SNVs in blood-derived reference population databases. In addition to the previously reported Genes with at least one phenotype inherited in an AD pattern were identified using gene–disease information stored in the genemap2.txt file . The foUsing the AD disease lists  and severe (scores 1 and 2) AD diseases using the Integrative Genomics Viewer (IGV) . Read su and 2 anThe ExAC data can now be found as a part of the gnomAD dataset, in addition to other exome and genome data. In contrast to the ExAC database, gnomAD applies more stringent filters that remove some variants with allelic imbalance from the allele count (< 0.2) . To inspHomo Sapiens. The GO database was released on 2020-10-09 and the comparisons were made using the statistical software R 4.0.2.To categorize the genes according to the processes they are involved in, we used selected gene ontology (GO) terms. AD genes associated with early onset severe Mendelian diseases were classified according to their involvement in regulation of hematopoiesis GO1903706) (Additional file 903706 (AThe total of 156 previously reported hematopoietic genes  (AdditioAdditional file 1. Supplementary tables S1–S7. Table S1. Summary of the 1388 genes with the associated autosomal dominant conditions. Table S2. Summary of AD genes and the associated conditions with their corresponding ClinVar likely pathogenic and pathogenic SNVs in the ExAC catalog. Table S3. Summary of AD genes and the associated early onset, severe conditions with their corresponding ClinVar likely pathogenic and pathogenic SNVs in the ExAC catalog. Table S4. gnomAD variant assessment for the genes that show evidence of mosaicism in ExAC. Table 5. Mosaicim in hematopoietic genes in ExAC. Table S6. Column names and corresponding descriptions for the tables S1-S5. Table S7. Phenotype assessment criteria.


Memory is vital to human functioning and controls future behavioral responses. Many neurological, neurodegenerative, and psychiatric disorders are associated with memory impairments or the presence of maladaptive memories. Thus, understanding the molecular mechanisms that control both normal and abnormal memory formation and modification is critical for treating a variety of disorders characterized by altered memory function.This Special Issue is a compilation of 11 research papers and reviews, all covering various “Molecular Mechanisms of Memory Formation and Modification”. The topics covered in this Special Issue can be divided into two categories, (1) memory consolidation, and 2) memory reconsolidation. For memory consolidation, included in this Special Issue are three research papers and three review articles. For the former, Smith and colleagues show that proteasome-dependent degradation of the activating transcription factor 4 (ATF4), a potent inhibitor of the cAMP-response element binding protein (CREB), occurs during the induction of long-term potentiation (LTP), the proposed cellular analog of memory, via its phosphorylation by cAMP-dependent protein kinase  memory r. They foIn addition to these research papers, three review articles were included in this Special Issue that all discussed epigenetic mechanisms of memory consolidation. Leonetti and colleagues reviewed the role of methylation of the adenosine base m6A) of RNA in neuronal development and memory formation, emphasizing the overlap in functionality of m6A in these two processes . ImportaA of RNA This Special Issue also includes five articles focused on memory retrieval and reconsolidation: four research papers and one review. All of these articles focus on what happens to memory after the initial consolidation period is over and the memory is placed into long-term storage. Traditionally, memories were thought to be resistant to disruption once consolidation was complete, but more recent evidence has demonstrated that stored memories become labile again when the animal is presented with a reminder cue or retrieval event. This period of transient susceptibility and restabilization of memory, termed “reconsolidation” has generated intense interest as a potential intervention to weaken, update, or even erase maladaptive associations. First, Trask and colleagues demonstrate that both middle-aged (15-month-old) and aged (22-month-old) rats show deficits in retrieving memory for trace fear conditioning, a form of aversive learning that is particularly susceptible to the effects of aging . Then, cNpas4 gene (but not at cFos) shortly after retrieval, suggesting these two markers of transcriptional activity may function in concert to support reconsolidation. Impressively, knockdown of the enzyme responsible for DSBs (topoisomerase IIβ) before retrieval reverses these effects and impairs memory reconsolidation, demonstrating that DSB-induced transcriptional increases are critical for successful reconsolidation. Elahi and colleagues take a different approach to disrupting reconsolidation, attempting to use electroconvulsive shock (ECS) as a non-surgical intervention to interfere with reconsolidation [1 muscarinic agonists, identified by Wideman and colleagues as a mechanism capable of driving memory destabilization even in the face of boundary conditions that prevent reconsolidation, like the age of the original memory [1 muscarinic receptors can override the effects of glutamate (mediated via GluN2A- and GluN2B-containing NMDARs), with an M1 agonist driving destabilization even in the presence of a GluN2B inhibitor that normally blocks destabilization. The M1 mAChR may therefore be a novel target capable of forcing an otherwise resistant memory to become labile and susceptible to modification. Finally, Bellfy and Kwapis review the evidence that the reconsolidation process may allow existing memories to update in response to new, relevant information [The remaining articles focus on understanding the mechanisms that underlie the destabilization and restabilization of memory following retrieval. Navabpour and colleagues  investiglidation . Despitel memory . In a seormation . In theiIn conclusion, this Special Issue covered a range of molecular mechanisms that support both the consolidation and the reconsolidation of memory. Understanding how each of these phases of memory work at a mechanistic level is a key step toward treating the myriad disorders characterized by alterations in memory.


The exploitation of new methods to control material structure has historically been dominating the material science. The bottom-up self-assembly strategy by taking atom/molecule/ensembles in nanoscale as building blocks and crystallization as a driving force bring hope for material fabrication. DNA-grafted nanoparticle has emerged as a “programmable atom equivalent” and was employed for the assembly of hierarchically ordered three-dimensional superlattice with novel properties and studying the unknown assembly mechanism due to its programmability and versatility in the binding capabilities. In this review, we highlight the assembly strategies and rules of DNA-grafted three-dimensional superlattice, dynamic assembly by different driving factors, and discuss their future applications. Self-assembly could arrange individual components into ordered structure, this phenomenon exists widely in nature, such as the double helix structure of DNA, protein aggregation and folding, cell, or some living life, and so on, they are considered to be the result of molecular self-assembly . InspireColloid is often used as a model of atomic system to reveal the crystallization rules and the role of nanoparticle in assembly process . ColloidIn order to realize the concept of designing novel nanomaterials by bottom-up self-assembly, some challenges must be solved. For instance, clearing the relationships between interparticle interaction, assembly environment, and superlattice products. This review aims to highlight the critical factors for DNA-grafted 3D superlattice self-assembly, covering the species of building blocks , assembly methods , dynamic manipulability for 3D superlattice , and their potential application in fabricating functional materials as well as current challenges in this field. The design paradigms in this review call for a move toward the realm of intelligent nanofabrication.Assembly of DNA-grafted 3D superlattice is composed of two pivotal elements: nanoparticles (NPs) core and DNA shell. The progress of chemical synthesis technique enables the chemical composition of NP core unrestricted; it can be metals, oxides, polymers, even biological molecule, which also allow for the integration of multiple functions like luminescent, catalytic, magnetic properties, and so on.Metal NPs are commonly employed because it is facile to modify dense DNA on the surface of NPs via thiol chemistry and their plasmon resonances could monitor the assembly process more easily. Majority of PAEs in superlattice assembly have spherical and isotropic NPs core. As early as in 1996, Mirkin’s group tried to assemble 3D superlattice using spherical gold nanoparticles (AuNPs) as core, and single-strand DNA as shell. Although the random binding of DNA shell on the surface of AuNPs increases the difficulty in assembly of ordered 3D superlattice. Until 2008, Mirkin and Gang realized the assembly of ordered 3D superlattice by accurately controlling the programmed DNA hybridization . After pOn the basis of spherical NPs core, researchers attempted to increase the complexity of PAE core in order to manipulate the superlattice assembly. Endowing the valency and specific directional bonding for NPs is effective in increasing the complexity of PAE core, and main three kinds of strategies of PAE construction were developed. First, patchy  or asymmTo further enrich the lattice library, hollow spherical nucleic acid  NPs compThough assembly strategies and rules of NPs were deeply explored and developed, it is more challenging to create superlattice when the size of colloid increased to micrometer range. The relative thin DNA coating is difficult to rearrange further, which results in disordered aggregation. Crocker et al. introduced a block polymer spacer poly(ethylene glycol) to increase the length and flexibility of thin DNA to mediate the order crystallization of 1-μm diameter polystyrene particles . Meulen 3O4 NPs [Besides the wide range of superlattice structures assembled by the co-crystallization of homogeneous building blocks, researchers also tried to explore the assembly of different building blocks in order to integrate the functions of multiple materials. For instance, Park investigated the co-crystallization of a binary system including two same AuNPs with different sizes . On the 3O4 NPs   DNA structures on the long-range order of NP lattice . The DNADNA tile, derived from Holliday junction like the four-arm junction  in FigurThough the position of NPs could be controlled precisely by DNA array or 3D DNA architecture assembled by DNA tile, there still are some obstacles to form a 3D crystal on the linear, planar template or even polyhedral frame. A groundbreaking work was published by Seeman that a single crystal was obtained by designing a tensegrity triangle . This riDNA origami was first termed by Rothemund in 2006 , in whicInitially, NPs could be arranged to prescribed nanoclusters and low-dimensional arrays via the connection of the DNA frame origami structure . A DNA oApart from connecting the NPs at the vertices of DNA frame, arbitrary positions within the frame could be encoded to anchor homogeneous or heterogeneous NPs. In 2020, Tian et al. connected NPs into the cavity of DNA frame structure to control the valence and coordination of NPs . CompareAdditionally, Liedl’s group assembled 3D crystal merely relying on DNA origami structure which have something in common with aforementioned tensegrity triangle designed by DNA tile . This DNAbove works give a good example for crystallization guiding by single DNA origami structure, but the co-crystallization for multiple anisotropic NPs is still challenging but attractive in engineering intricate materials. Tian’s group employed a couple of anisotropic DNA origami frames to alternatively encage NPs, creating a series of superlattice derivatives . In thisDNA is regarded as an excellent candidate for engineering crystallization due to its high predictability, programmability by tuning the length, binding strength, and flexibility. Most obviously, the parameter of lattice could be modulated by the number of base pairs. Many factors in design influence the binding strength and flexibility and ultimately affect the crystallization ability and quality such as the sticky end sequences, number of the linkers, the complementary shape for blunt end, and so on. Some have been described in the previous section like the integrating ssDNA base or PEG “flexors” to tune the flexibility  and “age+ with duplex in lattice [+-stabilized crystal did not dissociate when moving from water to organic media or air. Similarly, ruthenium coordination complexes [2+, Mn2+, Co2+, Ca2+, Na+, NH4+ actuated the NPs superlattice in varying degrees by screening the negative charges on the DNA backbones. Taking Ni2+ as a typical example, the volume change could reach as high as 65% by optimizing the concentration.The sensitive responsiveness of DNA for external chemical stimulus  to broaden the crystal library. The successful design and assembly of 3D superlattice mentioned above will arguably bring innovative opportunities, the integration of structural DNA nanotechnology and dynamic assemblies of 3D lattice push applications further. Material fabrication with custom high-performance properties has been a long-term goal for NPs self-assembly and also there are enormous possibilities and challenges for such application. The lattice properties are inextricably linked with the lattice structure , while decoupling the interaction and distance between particles from composition and size of particles are important advantages for fabricating materials with novel plasmonic, photonic, electric, magnetic, mechanical, or catalytic properties.DNA-mediated superlattice composed of noble-metal NPs exhibits potential to fabricate plasmonic metamaterials. Mirkin’s group first used electrodynamic simulation and experiment to prove that the superlattice is composed of spherical silver NPs (AgNPs) showing plasmonic properties . ComparePhotonic crystal material could prohibit spontaneous emission of light , showingThe electric property of superlattice also attracts numerous attentions, but the DNA scaffold in DNA-grafted superlattice possesses low conductivity. How to integrate nanomaterial with high conductivity is an effective strategy. But the intrinsically susceptible DNA lattice ascribed to DNA is stable in a narrow range of conditions, which limits the organization of diverse nanomaterials. Mineralization for DNA superlattice with silica of excellent chemical and temperature stability could perfectly break through the above limit while maintaining the original 3D topology structure of lattice though exposing to extreme temperature, pressure, and harsh radiation condition . Silica 3O4 NPs superlattice showed synergistically enhanced magnetic performance [Most advances of magnetic NPs-based superlattice depend on hydrophobic surface ligands, typically the stability agents introduced in colloid synthesis. For instance, Zeng et al. exemplified that magnetic binary FePt and Feformance . Only a formance . With thThe mechanical property of superlattice is closely correlated with phonon/molecular/ion transport function, which possesses potential to be applied to chemoselective separation like ultrafiltration. Elasticity and hardness reflected the mechanical performance, which depended on the surface ligand of superlattice. Macfarlane’s group used DNA-grafted superlattice assembly to investigate the material mechanical behavior . Due to 3O4 superlattice after postprocessing could catalyze the reduction of oxygen to water [Catalytic properties of superlattice could promote the development of another application direction. For example, based on the catalytic property of enzyme, Tian et al. filled two kinds of enzymes glucose oxidase and horseradish peroxide into octahedron voxels, then co-assembled in a 3D lattice. The enzyme lattice displayed increased catalytic activity by the enzymatic cascade  in lattito water  and oxidto water  at a fasTo sum up, a significant amount of assembly experiences including NP cores, connection methods, and preliminary verification of 3D lattice functions in this review will offer new strategies for overcoming the difficulty in material synthesis. A full understanding of the 3D superlattice assembly by further investigating the assembly kinetics and arbitrarily controlling the crystal parameters like symmetry, the size, and shape of crystal habit are still required. Combining the fresh ideas of bottom-up DNA-grated 3D superlattice assembly with traditional postprocessing will be a promising direction of 3D lattice-based nanofabrication and further promote the application in real world.


Pro-protein convertase subtilisin/kexin type 9 (PCSK9) is secreted mostly by hepatocytes and to a lesser extent by the intestine, pancreas, kidney, adipose tissue, and vascular cells. PCSK9 has been known to interact with the low-density lipoprotein receptor (LDLR) and chaperones the receptor to its degradation. In this manner, targeting PCSK9 is a novel attractive approach to reduce hyperlipidaemia and the risk for cardiovascular diseases. Recently, it has been recognised that the effects of PCSK9 in relation to cardiovascular complications are not only LDLR related, but that various LDLR-independent pathways and processes are also influenced. In this review, the various LDLR dependent and especially independent effects of PCSK9 on the cardiovascular system are discussed, followed by an overview of related PCSK9-polymorphisms and currently available and future therapeutic approaches to manipulate PCSK9 expression. Pro-protein convertase subtilisin/kexin type 9 (PCSK9) is a soluble protease that has been widely studied in the field of cholesterol homeostasis and in cardiovascular biology after its discovery in 2003 . OriginaTherefore, in this review the biological and physiological characterisation of PCSK9 will first be outlined in the context of cardiovascular biology, followed by an overview of the LDLR dependent and especially the LDLR independent effects of PCSK9 in this context. Furthermore, we will focus on state-of-the-art PCSK9 inhibitory techniques and their clinical potential.The 22kb human PCSK9 gene is located on the chromosome 1p32 . The C-tPCSK9 also exists in a furin-cleaved form that is 55 kDa in size  Figure . This foVarious transcription factors and cofactors regulate the expression of the PCSK9 gene. The PCSK9 promoter contains an important region that is essential for transcription, called the Sterol regulatory Element (SRE) . Sterol-Although a lot of progress has been made over the last years to fully understand the regulation of PCSK9, it is still not completely understood as this regulation turns out to be highly dynamic. The following sections will discuss how PCSK9 not just interacts with LDLR, but also with several other receptors to influence cardiovascular biology.In 2007, PCSK9 emerged as a novel target to treat dyslipidaemia. Numerous studies have identified a clear link between low density lipoprotein cholesterol (LDL-C) and PCSK9. Normally, LDL-C binds and gets internalised by binding to the LDLR, thereby mediating its clearance by endocytosis. In the presence of PCSK9, the LDLR undergoes lysosomal degradation, leading to an inhibition of the recycling of the receptor and therefore an increase in the plasma levels of LDL-C . This deOn the other hand, PCSK9 also interacts with LDLR in non-destructive ways . The catThe binding of PCSK9 to LDLR resulting in its degradation plays an important role in the cholesterol homeostasis, as it could be shown that a full-body knockout of PCSK9 in mice leads to an extreme reduction of cholesterol levels in the serum. More particularly, liver-specific PCSK9 knockout also leads to a considerable reduction in circulating cholesterol levels concluding that the hepatic PCSK9 contributes most to the cholesterol phenotype . The desThe function of PCSK9 is not just limited to the regulation of the LDLR, it also influences the lysosomal degradation of various receptors that structurally relate to the LDLR. Receptors such as very low density lipoprotein receptor (VLDLR), LDLR-related protein 1 (LRP1)  and apolAtherosclerosis is characterised by chronic inflammation of the vasculature, accompanied by lipid retention in arteries and the formation of plaques. Activation of the endothelial cell layer by atherogenic and pro-inflammatory stimuli initiates atherogenesis, which is followed by the infiltration of monocytes, lymphocytes, and various other immune cells . The rec−/−Apoe mice [PCSK9 is expressed on aortic ECs, SMCs, macrophages, dendritic cells and epithelial cells , suggest−/− mice . Further−/− mice . Additio−/− mice . The inc−/− mice , clearlyBesides ECs, vascular SMCs are also affected by shear stress when the EC layer is disrupted, as it could be demonstrated that low shear stress upregulates their proliferation and migration capability while increasing the secretion of PCSK9 by the SMCs. Several studies have demonstrated that SMCs secrete functional PCSK9 into the atheroma that exerts effects on monocytes migration in the intima. The overexpression of PCSK9 by SMCs in atherosclerotic plaques also reduces the ability of macrophages to ingest aggregated LDL (agLDL) and oxLDL molecules through scavenger receptors and LDLR related proteins ,65. PCSKhi monocytes into the atherosclerotic plaques [−/− mice [Besides its influence on vascular cells, PCSK9 has also been shown to exert pro-inflammatory and pro-atherogenic effects on macrophages in vitro even in the absence of LDLR . For exa plaques . Inhibit plaques ,62. In l plaques . These p−/− mice . Macroph−/− mice . PCSK9 s−/− mice . Similar−/− mice ,75. Ther−/− mice . Althoug−/− mice .Considering all the above-mentioned studies, it can be summarised that PCSK9 has a direct effect on both vascular and immune cells to directly influence atherogenesis not only by modulating the LDLR but also by LDLR-independent mechanisms.Complete cessation or lowering of blood flow to parts of the heart, due to atherosclerosis formation, causes the myocardium to be deprived of oxygen, resulting in a myocardial infarction (MI) which is associated with myocardial cell death and necrosis . PCSK9 lMI is very closely associated with coronary artery disease (CAD), as MI causes occlusion of coronary arteries . A studyTo summarise, in situations of MI, the expression of PCSK9 is elevated to not just alter the lipid metabolism but also to promote the cytotoxic capacity of oxLDL and increase the expression of apoptotic markers and promotes autophagy . InhibitObesity being a major health problem at the moment, leads to the development of metabolic comorbidities in the form of cardiovascular complications. Obesity is characterised by the enlargement of adipocytes and adipose tissue, and secretion of inflammatory cytokines to instigate pathological complications to the cardiovascular system . At firs−/− mice show lower markers for calcification compared to mice with functional PCSK9 [−/− mice overexpressing PCSK9 using an adenovirus containing PCSK9 develop more vascular calcification in response to being fed with Western diet compared to mice with normal PCSK9 levels [Ectopic calcification is associated with old age and is a very relevant comorbidity of CVDs. Calcific aortic valve stenosis (CAVS) is a form of ectopic calcification. Studies have indicated that there is an association between the levels of plasma PCSK9 and the severity of aortic valve stenosis . The expal PCSK9 . Histoloal PCSK9 . As a va9 levels . All theSeveral polymorphisms occur in the PCSK9 gene that tend to cause an effect on CVDs ,103,104.The PCSK9 gene also undergoes gain-of-function mutations (GOF), that usually result in hypercholesterolaemia  causing Cholesterol lowering therapies are the gold standard to reduce the risks of cardiovascular mortality and morbidity. One of the most effective ways to lower circulating cholesterol in blood is the usage of statins. Statins increase the activity of LDLRs, thus increasing the catabolism of VLDL, LDL and intermediate-density lipoprotein (IDL). Furthermore, statins also decrease the hepatic and endogenous cholesterol production by inhibiting HMG-CoA reductase , but theBerberis and other flowering plants such as Coptis rhizomes and Hydratis Canadensis [Studies have identified numerous naturally occurring inhibitors and small molecule inhibitors to rescue PCSK9 induced CVDs. In the context of naturally occurring substances, berberine has the ability to exert inhibitory effects on the transcription and translation of PCSK9 . Berberinadensis . Berberinadensis . Similarnadensis . Furthernadensis , are alsnadensis . In contnadensis . Furthernadensis . Other tnadensis .The administration of monoclonal antibodies (mAbs) against PCSK9 is a novel lipid-lowering therapeutic approach, that inhibits the attachment of PCSK9 with LDLR and LDLR-like receptors . They ac−/−Apoe mice that eventually reduces the atherosclerotic lesion sizes and makes the plaques more stable [+) was tested in mice with dyslipidaemia, where it inhibits the interaction between PCSK9 and LDLR and reduces the progression of atherosclerosis with long term immunity effects and efficacy [As an alternative to mAbs, creating an immune response against PCSK9 through vaccination can be used to provide protection against hyperlipidaemia. In fact, several groups have already developed extremely potent vaccines against PCSK9. For instance, PCSK9Qβ-003 is made of Qβ viral particles to decrease total cholesterol and LDL-C levels in e stable . AT04A ie stable . Peptidee stable . Additioefficacy .Most small molecule inhibitors, mABs and vaccines target extracellular PCSK9, while there are also ways to inhibit PCSK9 intracellularly via modulation of the gene expression . InitialA myriad of studies have discovered synthetic and natural compounds to inhibit the gene expression of PCSK9 aside from hindering the activity of this protein. Despite the extensive research available on monoclonal antibodies to inhibit PCSK9, the end-cost makes it difficult to put this into large-scale clinical use. The development of novel approaches to silence the mRNA of PCSK9 as well as developing immunity against PCSK9 using vaccines looks extremely promising, though large clinical trials are still needed to confirm its efficacy. Further investigation on these novel therapeutic approaches might unveil a big leap in the field of cardiovascular biology.PCSK9 exerts its effect on the cardiovascular system via the degradation of LDLR and by multiple mechanisms that are independent of LDLR. We have already a rather good understanding of the influence of PCSK9 on vascular biology, especially via its LDLR dependent effect, although it is clear that there are still several effects that are yet to be discovered. It has already been shown that PCSK9 inhibitors can be used to reduce cardiovascular complications in patients with well controlled LDL-C plasma levels. Therapeutically, PCSK9 antibodies inhibit the interaction between the EGF-A domain of the LDLR and PCSK9 and reduce LDL-C levels in plasma and have appeared to be used safely in clinical context. However, it remains rather unclear what the effects of PCSK9 manipulation are on LDLR-independent processes, which should therefore be an important focus point of future research. Additionally, based on the effects of the LOF mutations of PCSK9, manipulation of its gene expression might also be an additional approach to treat CVDs.


Meiosis generates genetic diversity in natural populations and during breeding. We review meiosis research in major crop species, including an in-depth resource of cloned crop meiotic mutants and an overview of genome-wide recombination maps. Brassica species). Generally, the functional roles of meiotic proteins are conserved between plant species, but we highlight notable differences in mutant phenotypes. The physical lengths of plant chromosomes vary greatly; for instance, wheat chromosomes are roughly one order of magnitude longer than those of rice. We explore how chromosomal distribution for crossover recombination can vary between species. We conclude that research on meiosis in crops will continue to complement that in Arabidopsis, and alongside possible applications in plant breeding will facilitate a better understanding of how the different stages of meiosis are controlled in plant species.Meiosis is a key feature of sexual reproduction. During meiosis homologous chromosomes replicate, recombine, and randomly segregate, followed by the segregation of sister chromatids to produce haploid cells. The unique genotypes of recombinant gametes are an essential substrate for the selection of superior genotypes in natural populations and in plant breeding. In this review we summarize current knowledge on meiosis in diverse monocot and dicot crop species and provide a comprehensive resource of cloned meiotic mutants in six crop species (rice, maize, wheat, barley, tomato, and  Meiosis is a specialized cell division that takes place during sexual reproduction and leads to the production of genetically unique haploid spores. Meiosis consists of one round of chromosome replication followed by two rounds of segregation, thereby halving the chromosome number . In the Meiosis can be broadly split into five key stages: meiotic entry, recombination initiation, chromosome synapsis, resolution of recombination intermediates, and the second meiotic division. Unlike in animals, the specification of both male and female plant germlines occurs late in development during flowering. The transition from the sporophyte phase to the gametophyte state is initiated by meiotic entry. Upon meiotic entry the replication of chromosomes and establishment of sister chromatid cohesion is followed by the association of telomeres with the nuclear envelope during the leptotene stage. Subsequently the initiation of meiotic recombination occurs via the formation of programmed meiotic DNA double strand breaks (DSBs) by a topoisomerase-like complex containing conserved SPO11 proteins. Recombination between homologous chromosomes is tightly interlinked with the lengthwise alignment of homologous chromosomes (pairing) and the formation of the synaptonemal complex (synapsis). The synaptonemal complex (SC), a protein-rich structure, connects the paired homologous chromosomes during zygotene and pachytene (the stages where recombination occurs). Meiotic DSBs are repaired by homologous recombination, which can be resolved as either a crossover (CO), which is a full reciprocal exchange, or a non-crossover (NCO), which is either a gene conversion (from the homologous chromosome) or sister chromatid-based repair. In plant species studied to date, approximately 1 in 20 meiotic DSB sites are finally repaired as a CO, suggesting selection acts to restrict CO number , rice (Oryza sativa), wheat (Triticum aestivum), barley (Hordeum vulgare), tomato (Solanum lycopersicum) and Brassica species . Rice and maize are by far the most studied crop species with 64 and 22 mutants cloned, respectively  in both Arabidopsis and rice  complexes , rice (OsAM1) and Arabidopsis (AtSWI1/AtDYAD). In four strong maize meiosis arrested at leptotene mutants (mel1 and mel2) have been isolated in rice and provide important insights into the transition to meiosis and the early stages of meiotic prophase I  but defects are found in pre-meiotic divisions of germ cells, chromosome condensation, and meiotic progression, with arrested meiocytes having uncondensed chromosomes like those usually found at leptotene or zygotene  protein that appears to control the translation of meiotic RNAs, and strikingly the pre-meiotic S-phase is asynchronous in mel2 mutants, indicating MEL2 likely functions in a separate pathway from MEL1  that are constitutively expressed in mitotic stages are down-regulated upon the transition to meiosis  could be used in hybrid seed production. Currently, many hybrid seed production systems are based on cytoplasmic male sterility, which is genetically complex and not available in many important crops . ArtificMeiotic recombination is initiated during the leptotene stage of meiotic prophase I by the programmed formation of meiotic DSBs, which is followed by DSB processing and repair by homologous recombination. Meiotic DSBs are produced in a huge excess compared with the final number of COs. In allotetraploid wheat, 1400 DSBs (RAD51 foci) result in 29 class I COs (HEI10 foci) across 14 homologous chromosomes; in maize, 500 DSBs (RAD51 foci) result in 16 COs (chiasma number) across 10 homologous chromosomes; and in Arabidopsis, 200 DSBs (RAD51 foci) result in 10 class I COs (MLH1 foci) across five homologous chromosomes , while RNAi lines of Osspo11-4 do display meiotic defects and have a more severe reduction in fertility , are essential for DSB formation; however, the Arabidopsis homologues AtPCH2 and AtCOMET are not required for DSB formation  are meiotic DSB hot spots while retrotransposons were depleted of meiotic DSBs  it appears that a specific DMC1 gene located on the D genome is required for the stabilization of chromosome pairing at low and high temperatures  and COM1 to release SPO11 proteins, facilitating exonuclease end resection to produce the 3′ ssDNA overhangs . DMC1 ismtopvib mutants  occurs in tandem with meiotic DSB formation and strand invasion. The SC is a proteinaceous connection between the homologous chromosomes and is made up of three main elements: axial/lateral elements (AE/LE), transverse filaments, and the central element . The twoorm a SC .B. oleracea as an AE  have significantly reduced COs compared with wild-type controls (17 chiasmata per meiocyte)  recombinant products. In Arabidopsis, maize, and wheat, only 5% of repair events are through meiotic CO .There are two types of meiotic CO pathways—the class I and class II CO pathways. In Arabidopsis and rice, the majority of CO events occur via the class I pathway, which accounts for 80–90% of total CO events  and chiasmata frequency  (msh5 mutants are similarly sterile but exhibit fewer chiasmata (2.10) per meiocyte than mer3 mutants (msh4 (1.71) and msh4/msh5 (1.76) double mutants compared with other ZMM mutants, suggesting the MutSγ complex may play an earlier role to stabilize progenitor Holliday junctions in rice , the MutSy complex maintains ~85% of all COs and is required for the obligate chiasma. Intriguingly, much like in B. napus, in allopolyploid wheat the loss of function of MutSy genes  has been described, suggesting a possible adaptive role in the control of CO  and Osmlh1 (15.2 chiasmata per cell) mutants have relatively modest reductions in chiasmata frequency (19.5 chiasmata in wild-type)  proteins play a crucial role in the DNA mismatch repair (MMR) pathway, and in eukaryotes the MLH1–MLH3 complex has been co-opted for the resolution of double Holliday junctions as meiotic COs. In tomato, MLH1 foci mark 70% of late recombination nodules and their distribution suggests they mark strongly interfering COs  locus, led to increased homoeologous CO in crosses with related wild species, suggesting ZIP4 gene dosage may influence the choice between homologous and homoeologous CO (Pairing homoeologous 2 (Ph2) mutant phenotype  locus on chromosome 9 (C genome) was identified as a controlling factor of homoeologous chromosome pairing in haploid B. napus  can further increase CO rate in inbred and hybrid Arabidopsis compared with the single pathway mutants  mutants for FANCM and RECQ4 genes have increased CO rates  with varying genome sizes  and compArabidopsis subtelomeres and pericentromeric regions are areas with higher recombination than the genome average . The cenindica and japonica rice subspecies, respectively, demonstrating the existence of CO hot spots  and also simple sequence repeats were over-represented within CO hot spots, while retrotransposon classes are depleted  correlate with CO rate at the whole genome level   .Like mitosis, progression through the meiotic stages is driven by the activity of cyclins–CDKs, which phosphorylate universal cell cycle proteins and meiosis specific factors (OMISSION OF SECOND DIVISION1 (OSD1), encoding an APC/C inhibitor, is required for meiosis II entry in both Arabidopsis and rice  and female (85% penetrance in Arabidopsis and 91% penetrance in rice) gametes  and female (∼30% penetrance) lineages (THREE DIVISION MUTANT1 (TDM1), which is close to a conserved CDK phosphorylation site, terminate at the end of meiosis I and produce diploid gametes, whereas tdm1 loss of function mutants fail to exit meiosis and enter a third aberrant division (Arabidopsis mutants of genes that play a role in the cyclin–CDK–APC/C module have been shown to skip the second meiotic division and give rise to unreduced gametes (spo11-1) or establish sister chromatid cohesion (e.g. rec8) leads to the ‘mitosis instead of meiosis’ (MiMe) genotype  in different rice genetic backgrounds, suggesting possible modification by genetic or environmental factors  and haploid induction (mutation of OsMTL) (osd1 mutants in rice, mutants that modify the meiotic cell cycle are largely underexplored outside of Arabidopsis, and therefore reverse genetic approaches will likely help resolve the function of known genes in various crop species. Forward genetic screens for genes involved in unreduced gamete formation in crops, alongside the isolation of known genetic factors in Brassica and potato (Hybrid vigor contributes to the high yield of commercial seeds and could be fixed through seeds by synthetic apomixis (HEI10 gene control meiotic CO rate in trans (RECQ7 gene was shown to be important for meiotic gene conversion despite its having been lost in many plant species including Arabidopsis (rmi1 mutants cannot progress in meiosis due to chromosome fragmentation and formation of chromatin bridges (rmi1 mutants have no meiotic defects (The core function of meiosis is to recombine chromosomes and reduce the chromosome number by half, and it is therefore essential to the process of sexual reproduction because it derives haploid cells from diploid cells . UnsurprIn the coming years various topics and approaches are likely to play a role in the better understanding of plant meiosis. The interaction between meiosis and the environment appears to be an emerging topic, especially in a time of global climatic variation. Plants are sessile organisms that cannot determine their environment, and meiosis is an important, and potentially vulnerable, aspect of plant reproduction. Already in Arabidopsis and barley, temperature has been demonstrated to alter meiotic CO rates (MiMe system and the application of synthetic apomixis in more crop species could represent a route to fixing hybrid vigor in commercial seed production.Meiosis research in crop species is intrinsically linked with crop improvement and breeding, as meiotic recombination is a key substrate of breeding. The application of approaches that can increase CO rate and change CO distribution can be useful in crop breeding for increasing genetic gain per breeding cycle. The approaches discovered so far have been tested in a limited number of plant species and genetic contexts. Future research will no doubt unravel whether anti-CO and epigenetic mutants, which respectively modify the absolute number and distribution of COs, can have similar or different effects in genetic backgrounds with varying levels of genetic polymorphism. This will help identify if there are particular levels, or types, of genetic polymorphism where modulation of CO level and/or position can lead to negative effects including chromosomal rearrangements or reduced fertility. On the other hand, harnessing the Above and beyond the useful applications of meiosis in plant breeding, using crops as model systems has become much more accessible to academic researchers due to the developments in genome editing and genomics. Using crop species strengthens our understanding of meiosis and should not be obligatorily categorized as applied research. Indeed, we expect that rapid cycling crop varieties that are amenable to generate mutants by CRISPR/Cas9 may be useful not only for reverse genetic studies but as a means for generating mutants for suppressor and/or enhancer screens. As outlined, we propose that meiosis research in Arabidopsis and other plant model systems will continue to complement one another and lead to the discovery of novel genes, processes, and mechanisms that are involved in meiosis.