Despite widespread influenza vaccination programs, influenza remains a major cause of morbidity and mortality in older adults. Age-related changes in multiple aspects of the adaptive immune response to influenza have been well-documented including a decline in antibody responses to influenza vaccination and changes in the cell-mediated response associated with immune senescence. This review will focus on T cell responses to influenza and influenza vaccination in older adults, and how increasing frailty or coexistence of multiple (≥2) chronic conditions contributes to the loss of vaccine effectiveness for the prevention of hospitalization. Further, dysregulation of the production of pro- and anti-inflammatory mediators contributes to a decline in the generation of an effective CD8 T cell response needed to clear influenza virus from the lungs. Current influenza vaccines provide only a weak stimulus to this arm of the adaptive immune response and rely on re-stimulation of CD8 T cell memory related to prior exposure to influenza virus. Efforts to improve vaccine effectiveness in older adults will be fruitless until CD8 responses take center stage. Despite widespread influenza vaccination programs and > 60% vaccination rates in the population age 65 years and older in many countries, influenza remains a serious threat to the health of older people. In the US, the over 65 population accounts for two-thirds of the 200,000 influenza-related hospitalizations independent of whether they are at high or low risk for serious complications of influenza , and oldOlder adults are particularly susceptible to severe outcomes of influenza. This is especially true during seasons when A/H3N2 is the predominant circulating strain where dramatic increases in hospitalization rates occur in the population age 65 and older –9. DysreDespite recent advances in the development of high-dose, adjuvanted, and subunit vaccines, there remain significant challenges to the development of more effective influenza vaccines for older adults, particularly for preventing the most serious complications. Influenza vaccines are annually formulated to stimulate strain-specific neutralizing antibody responses to the predicted circulating strains of influenza. These changes in the circulating strains are the result of antigenic drift wherein new strains emerge with human transmission due to the high mutation rates of the influenza A virus and the selection of strains with epitopes on the hemagglutinin (HA) head that are resistant to antibody binding. Vaccine strain mismatch may also result from the loss of glycosylation sites on the HA head in the process of adaptation of the vaccine strains for optimal growth in eggs. These neutralizing antibodies are stimulated in the response to influenza vaccination and thus prevent infection by binding to the HA head and neutralizing the virus to prevent virus entry into the host cell, so-called “sterilizing immunity”. Vaccine strain mismatch leads to a loss of strain-specific antibody binding to the critical protective epitopes surrounding the receptor-binding domain of the globular head of hemagglutinin. The hemagglutination inhibition (HAI) assay is the standard measure of strain-specific antibody titers and the rise in HAI antibody titers with vaccination is usually used to estimate vaccine efficacy in clinical trials. Much of the decline in vaccine efficacy with aging has been attributed to “immune senescence”, wherein the decline in the antibody response to influenza vaccination is attributed to age-related changes in B cell and T cell development and function (reviewed in –35). TheFrailty is a geriatric syndrome which is multifactorial in etiology, involves complex and dynamic interactions with the elements of biopsychosocial health, and reflects the loss of the adaptive capacity to respond to acute health challenges –39. It hInfluenza illness is associated with increases in frailty, measured using a 40-item Frailty Index, among hospitalized older adults. Importantly, frailty is also an important confounder of vaccine effectiveness, and vaccine effectiveness declines with increasing frailty . Our hosIn our community studies of the response to influenza vaccination in older adults, using the same 40-item Frailty Index, older adults on average have a Frailty Index of 0.1 (Non-frail are < 0.1) (unpublished observations). A systematic review of high-dose relative to standard-dose influenza vaccine trials, showed that the better antibody responses to high-dose influenza vaccine was independent of sex, age > 75 years old, frailty, and chronic conditions . This isTest-negative case control design studies are another way to investigate the effect of frailty on laboratory-confirmed influenza-related hospitalizations, an outcome of vaccination that cannot be captured in sufficient numbers in clinical trials to demonstrate the enhanced benefit of newer vaccines. All patients admitted with an acute respiratory illness are included in these studies where those patients with laboratory-confirmed influenza are compared to those patients who are test-negative for influenza . These sWhen antibodies fail to protect against influenza infection by providing sterilizing immunity, “clinical protection” relies on cell-mediated immune mechanisms to the clear the virus from infected host cells and prevent the pulmonary and extrapulmonary complications of influenza. Previous studies have shown that heterosubtypic immunity  induced in the mucosal tissue of the respiratory tract by influenza infection is mediated by CD8 cytotoxic T lymphocyte (CTL) responses  and by bComparisons of young and old mice have yielded conflicting results, possibly arising from discrepancies in experimental design, macrophage type studied, and exactly how “old” is defined , but at Natural killer (NK) cells have also been shown in mouse models to contribute to the control of influenza during the early stage of infection through granule-mediated (granzyme B and perforin) killing of virus-infected cells –87, but Appropriate selection of TLR agonists which stimulate dendritic cells (DC) is needed to improve the CD8 T cell response to influenza challenge in older adults. The primary targets for TLR agonists are the TLR on myeloid (mDC) and plasmacytoid (pDC) dendritic cells but studies have shown that the age-related decline in the engagement of TLR1/2, TLR2/6, TLR3, TLR5, and TLR8 in mDCs and TLR7 and TLR9 in pDCs, may be mediated by dysregulated cytokine production . These rVaccine adjuvants including TLR agonists have been used to enhance Th and CTL responses to influenza vaccination. Different vaccine adjuvants can stimulate Th1  vs. Th2  responses to influenza vaccination according to the relative degree of Th1 production of IFNγ vs. Th2 production of IL-5; these actions contribute to antibody and cell-mediated protection against influenza . HoweverTo the best of our knowledge, we have published the only prospective studies of T cell correlates of protection against influenza in vaccinated older adults. In multiple studies, we have shown in PBMC that the ratio of IFNγ to interleukin (IL)-10 (IFNγ:IL10) in supernatants of stimulated PBMC and biological activity of the cytolytic mediator, granzyme B (GrB), in lysates of ex vivo influenza challenged PBMC correlate with protection against influenza illness , 71 and Novel strategies for stimulating CD8 CTL responses have long been a priority for new vaccine development to provide heterosubtypic immunity across serologically distinct strains of virus , 103–105The importance of including influenza internal viral proteins such as M1 and NP in vaccines has been highlighted in experiments in the mouse model showing that sequential immunization with whole-inactivated influenza vaccine , but NOT a subunit vaccine , could alleviate the severity of infection with antigenically drifted viruses from the vaccine strains and potentially improve protection to unpredictable seasonal infection . ImportaImportantly, we have shown in in vitro experiments that there was an increase in the IFNγ:IL-10 ratio and GrB activity in response to influenza challenge when the TLR4 agonist GLA-SE was combined with SVV. This response appears to be mediated by a 10-fold reduction in IL-10 levels when older adult PBMC are stimulated with GLA-SE plus SVV compared to SVV alone prior to influenza challenge . Our unpCurrent research efforts to support the development of new influenza vaccines have largely focused on hemagglutination inhibition antibody responses. Defining those changes that occur with immune senescence requires studies of relatively healthy older adults who are not generally representative of the over 65 population with multiple chronic conditions, particularly those age 75 years and older. While antibody responses to influenza vaccination may predict protection against influenza infection in community-dwelling older adults, it does not explain the significant increases in their hospitalization rates that occur during influenza seasons where A/H3N2 (relative to A/H1N1 and B strains) is the predominant circulating strain. Immunologic priming with the first exposure to influenza in childhood (i.e. A/H1N1 for the current older adult cohort) may explain the low hospitalization and death rates during A/H1N1 years, with only the most frail being susceptible to the serious complications of influenza. Further, dysregulated immune responses characterized as ‘inflammaging’ in the context of multiple chronic conditions and frailty appear to be the mechanistic link to these complications, and go beyond those changes in lymphocyte development and function attributed to immunosenescence. More studies are needed in older adults with multiple chronic conditions to determine how varying levels of frailty lead to the loss of adaptive capacity or resilience and are reflected in multiple measures of the interaction of innate and adaptive immune function. Current influenza vaccines are poor stimulators of the heterosubtypic cell-mediated immunity needed to prevent the serious complications of influenza. We propose that there is a requirement to translate new insights into how cell-mediated immune responses to influenza A/H3N2 strains can be enhanced in the design of new influenza vaccines. To address this unmet need, we must first understand the interaction between circulating proteins associated with inflammatory processes, the phenotype and function of different immune cells subsets responding to influenza challenge, and prove that cell-mediated immune correlates of protection are valid in older adults. It will also be necessary to better understand how broadly-neutralizing antibodies contribute to heterosubtypic immunity. Finally, our in vitro system using older adult PBMC to simulate the effect of different vaccine/adjuvants on the response to influenza challenge using our established correlates of protection, and our choice of adjuvants currently in clinical development, serves as a model for pre-clinical testing and an accelerated pathway through the clinical development pipeline.


Enhancer of zeste homolog 2 (EZH2) is a histone-lysine N-methyltransferase enzyme that catalyzes the addition of methyl groups to histone H3 at lysine 27, leading to gene silencing. Mutation or over-expression of EZH2 has been linked to many cancers including renal carcinoma. Recent studies have shown that EZH2 expression and activity are also increased in several animal models of kidney injury, such as acute kidney injury (AKI), renal fibrosis, diabetic nephropathy, lupus nephritis (LN), and renal transplantation rejection. The pharmacological and/or genetic inhibition of EZH2 can alleviate AKI, renal fibrosis, and LN, but potentiate podocyte injury in animal models, suggesting that the functional role of EZH2 varies with renal cell type and disease model. In this article, we summarize the role of EZH2 in the pathology of renal injury and relevant mechanisms and highlight EZH2 as a potential therapeutic target for kidney diseases. Epigenetics refers to the heritable change of gene function and phenotype without alteration in a genes DNA sequence . The corH3K27 trimethylation is mainly carried out by Polycomb Repressor Complex PRC2; . PRC2 isEnhancer of zeste homolog 2 was discovered in 1996 using yeast two-hybrid experiments  and its Increasing evidence shows that EZH2 is associated with a variety of kidney diseases and pathology. In addition to the abnormal expression and activation of EZH2 in renal tumors, its expression levels and activity were also increased in acute kidney injury AKI; , renal fRenal cell carcinoma (RCC), also known as renal adenocarcinoma. originates from renal tubular epithelial cells and accounts for over 90% of adult renal malignancies. The worldwide incidence RCC has increased significantly in recent years . At presIn vitro and in vivo studies confirmed that the abnormal increase of EZH2 can inhibit the expression level of E-cadherin, induce the epithelial stromal transformation of renal cancer cells, and promote the occurrence, development and recurrence of renal cancer. Inhibition of EZH2 with 3-DZNep can reverse these pathological responses . Renal interstitial fibrosis is considered an example of poor self-healing after injury . It is mIn vitro, pharmacological inhibition or siRNA mediated silencing of EZH2 reduced the activation of renal interstitial fibroblasts; in vivo, treatment with the EZH2 inhibitor 3-DZNep attenuated UUO-induced fibrosis in an animal model. The anti-fibrotic effect of EZH2 inhibition is related to the inhibition of expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) and deactivation of multiple intracellular signaling pathways, including TGFβ/Smad3, AKT and ERK1/2  is a purine metabolic disorder, in which the blood uric acid level is higher than the normal level due to the increase of uric acid production and/or the decrease of uric acid excretion. HUA is an independent risk factor for CKD progression  and has in vitro and attenuates ECM protein deposition and α-smooth muscle actin expression in obstructed kidneys  is a chronic autoimmune inflammatory disease characterized by loss of immune tolerance to autoantigens, production of autoantibodies, formation of immune complexes and deposition in different parts of the body, causing inflammation and multiorgan damage . LN is oSimilar to DNA methylation, histone modifications can lead to abnormal gene expression and contribute to the pathogenesis of SLE . It has Aging is an irreversible phenomenon characterized by gradual decline of cell function and gradual structural changes of many organ systems. Age-related changes in kidney function include anatomical and physiological changes . The hisEpigenetic histone modification plays a role in aging , especiaCurrently, renal transplantation is the most effective treatment for ESRD. However, acute rejection (AR) is a common adverse reaction after kidney transplantation, usually occurring weeks to months after transplantation. Rejection after transplantation is caused by the recipient’s immune system. Recognition of the graft as a foreign body stimulates secretion of various inflammatory factors to attack the graft. Eliminating the foreign body reaction and maintaining the stability of the internal environment would improve the long-term survival of the transplanted kidney. Transplantation rejection includes T-cell-mediated rejection and antibody mediated rejection . Acute TEnhancer of zeste homolog 2 plays an important role in maintaining T cell numbers and functions. It has been documented that EZH2 is essential for the expansion of T-effector cells  as well Enhancer of zeste homolog 2 is highly expressed in renal tumors and many kidney diseases. Abnormal expression or activation of EZH2 can lead to development and progression of renal tumors and several kidney diseases as indicated in this review. The molecular mechanisms of EZH2-mediated renal pathology are associated with renal tubular cell injury, podocyte dedifferentiation, renal interstitial fibroblast proliferation, production of multiple cytokines/chemokines and infiltration of inflammatory cells. Because EZH2 regulates expression of diverse genes and activation of multiple signaling pathways associated with pathogenesis of disease, its functional role may vary with cell types, tissues and disease models. In this context, EZH2 activation has been shown to contribute to renal tubular cell death, but protects against podocyte injury. Therefore, further studies are necessary to elucidate the detailed mechanistic actions of EZH2 in the pathogenesis and progression of kidney diseases.Given that preclinical studies have demonstrated that EZH2 inhibitors attenuate some renal diseases in animal models, EZH2 inhibitors alone, or in combination with other drugs may provide beneficial effects to ameliorate or prevent kidney diseases. This is encouraged by recent approval of tazemetostat (EPZ-6438), one of EZH2 inhibitors, for treatment of adult patients with relapsed or refractory (R/R) follicular lymphoma by the FDA . Among tTL and CY drafted the article. SZ edited the manuscript. All the authors reviewed the manuscript and approved is for publication.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.


Toxoplasma gondii is an exceptionally successful parasite that infects a very broad host range, including humans, across the globe. The outcome of infection differs remarkably between hosts, ranging from acute death to sterile infection. These differential disease patterns are strongly influenced by both host- and parasite-specific genetic factors. In this review, we discuss how the clinical outcome of toxoplasmosis varies between hosts and the role of different immune genes and parasite virulence factors, with a special emphasis on Toxoplasma-induced ileitis and encephalitis. Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis, a worldwide zoonotic disease that can affect virtually all mammals and birds, including approximately one third of humans  and asexual multiplication in intermediate hosts . Infection of intermediate hosts occurs by ingestion of food or water contaminated with oocysts, which are secreted with feline feces, or meat containing tissue cysts. Upon ingestion, sporozoites within oocysts or bradyzoites within tissue cysts are released in the small intestine and invade intestinal epithelial cells. Here they transform into tachyzoites, the fast-replicating stage responsible for symptoms during the acute phase of the disease, which can vary in severity depending on the host and parasite strain. After invasion of the host cell, Toxoplasma establishes its niche inside the host cytosol by forming a parasitophorous vacuole (PV), inside which it replicates . In the house mouse (Mus musculus -M.m.- domesticus) type I strains are very virulent (LD100 of 1 parasite), whereas type II and III are less virulent strains  species has been mapped to chromosome 11, which contains the immunity related GTPases (IRGs), a family of proteins that are induced by interferon gamma (IFNγ) and which coat and subsequently vesiculate the parasitophorous vacuole membrane (PVM), leading to the death of the parasite , which encodes for the NLRP1 inflammasome sensor that causes rapid cell death and disruption of the replicative niche of the parasite upon activation , ocular toxoplasmosis (OT) or congenital toxoplasmosis  by receptors known as pattern recognition receptors (PRRs). The toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs) are the most extensively studied  , cyclic GMP-AMP synthase (cGAS) or retinoic acid-inducible gene I (RIG-I). It is likely that only when the PVM is destroyed, parasite-derived nucleic acids are released and detected by cytoplasmic nucleic acid sensors. Indeed, human peripheral blood mononuclear cells (PBMCs) do not secrete cytokines upon treatment with Toxoplasma derived RNA or DNA unless pre-stimulated with IFNγ  and the caspases 1 and 4, all of which lead to the secretion of IL1β and IL18, a process known as inflammasome activation  to secrete IFNγ. Although DC-derived IL12 is important for secretion of IFNγ Toxoplasma , the CD8 T cell response is initiated when they encounter epitopes presented by major histocompatibility complex (MHC) I on APCs. The degree of CD8 T cell activation depends on the effective killing of intracellular parasites within the APCs, after which the immunodominant epitopes derived from Toxoplasma in murine cells is the induction of members of the p47 and p65 family of GTPases . By acting cooperatively, these cause the disruption of the PV, leaving the parasites exposed to the host cell cytoplasm  induces the production of inflammatory cytokines such as IL12 and IL1β, and elicits classically activated macrophages (M1)  phosphorylates the transcription factors STAT3/6, driving macrophages toward an alternatively activated (or M2) state that is associated with increased IL10 expression and a Th2-polarized response   against the overgrowing gram negative bacteria improves the immunopathology by reducing the cytokine storm . This progressive inflammatory disease is characterized by cyst rupture, tachyzoite conversion and parasite replication within the CNS, which often results in a fatal outcome in the absence of treatment  to reach the brain or other distant organs , CXCL10 or CCL2  and its growth is inhibited by the upregulation of IDO through the synergistic action of IFNγ and TNFα  and IRGA6 (IIGP1), which disrupt the PV , an enzyme produced in response to the structural chitin present in the cyst wall  or H-2d  haplotypes are resistant to the infection, harbor fewer cysts in the brain and rarely show signs or excessive inflammation  located at the C-terminus of GRA6 elicits a strong and protective CD8 immune response through its presentation exclusively by the H-2 Ld  or with the H-2b or k alleles in susceptible mice. Hence, the recognition of the GRA6 HF10 peptide by CD8 T-cells may account for the differences in virulence among mouse strains during chronic toxoplasmosis, and might also explain why subdominant responses, such as CD4 T-cells, could not be sufficient to control the parasite. In humans, some studies pointed out that the MHC II Human Leukocyte Antigen (HLA)-DQ3 and DQ1 could be genetic markers of susceptibility and resistance, respectively, to cerebral toxoplasmosis in AIDS and congenitally infected patients  does not seem to be important for the chronic stage of infection in these strains  when co-cultured with monocytes. Namely, GRA15 induces the expression of IL1β by monocytes that in turn promotes iNOS expression in the brain cell lines, which inhibits IDO1-mediated tryptophan degradation, thus facilitating parasite proliferation (Bando et al., In addition to ROPs, GRA proteins have also been described to play an important role in the chronic phase of infection, some of them in a strain-specific manner. For example, Bando et al., showed that type II GRA15 suppresses IFNγ-mediated anti-in vivo, most research on the mechanisms of intracerebral Toxoplasma control and immune response have been conducted in vitro or in mice. Thus, caution is needed when extrapolating results obtained with these models, as there are significantly different genetic backgrounds and immune mechanisms involved. Moreover, given the strong influence of both the host and the parasite strain on the outcome of chronic toxoplasmosis, it is possible that specific combinations can uniquely affect the course and pathology of the disease. Nevertheless, a further understanding of the association between strain, immune response and clinical manifestation is needed, especially in severe TE or ocular toxoplasmosis. This, together with an accurate diagnosis would in turn allow an individualized monitorization and improved treatments in risk groups.In summary, an effective control of TE depends on IFNγ and CD8 T-cells, while a correct immunoregulation is critical to control excessive inflammatory response in the brain. Of note, since human studies cannot be performed Toxoplasma infections in livestock that causes important economic losses, it also poses a risk to humans by direct exposure to contaminated animal products. The variable outcome of infection is strongly dependent on the Toxoplasma strain producing the infection, the host and its immune response. In the last decades advancements have been made to understand the mechanisms underlying these differences. Although in this review we focused on the current knowledge of host and parasite factors affecting the outcome of the disease and the control of the parasite, most studies have been performed in murine models and using laboratory adapted strains that do not reflect natural conditions of infection. In addition, most effectors that are important in mice for the control of Toxoplasma play no role in other animal species, including humans; thus, the results obtained in these models have to be carefully considered and extrapolations are not always possible.Toxoplasmosis is a disease that varies enormously in its clinical form, ranging from asymptomatic cases to lethal consequences. In addition to the importance of Toxoplasma strains extremely virulent in some hosts and totally controlled in others? What is the most critical component of the immune response that makes some hosts resistant to Toxoplasma infection? Arguably, many other factors besides the parasite strain and the host also play an important role and account for these differences, such as the infective dose, the immunological status or genetic individual differences. Moreover, although we have focused on toxoplasmic ileitis and encephalitis, Toxoplasma also causes other important diseases in humans such as ocular toxoplasmosis, congenital infection or abortions in pregnant women. Each one of the clinical forms deserve individual investigation to uncover the most relevant host and parasite factors affecting the outcome of infection. Finally, experimental models beyond the murine ones would greatly improve our understanding of the host-parasite relation in toxoplasmosis and, more importantly, would help to elucidate the mechanisms of resistance in humans, with potential applications in terms of new vaccine or drug targets.There are still many questions to be answered: for instance, why is there an intrinsic variability between individuals from the same species? Why are some DM, DA-S, and JPJS have contributed to the conceptualization, writing, and editing of this manuscript. All authors contributed to the article and approved the submitted version.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.


As the maternal–foetal interface, the placenta is essential for the establishment and progression of healthy pregnancy, regulating both foetal growth and maternal adaptation to pregnancy. The evolution and functional importance of genomic imprinting are inextricably linked to mammalian placentation. Recent technological advances in mapping and manipulating the epigenome in embryogenesis in mouse models have revealed novel mechanisms regulating genomic imprinting in placental trophoblast, the physiological implications of which are only just beginning to be explored. This review will highlight important recent discoveries and exciting new directions in the study of placental imprinting. The placenta is essential for healthy pregnancy because it supports the growth of the baby, helps the mother’s body adapt, and provides a connection between mother and the developing baby. Studying gene regulation and the early steps in placental development is challenging in human pregnancy, so mouse models have been key in building our understanding of these processes. In particular, these studies have identified a subset of genes that are essential for placentation, termed imprinted genes. Imprinted genes are those that are expressed from only one copy, depending on whether they were inherited from mom or dad. In this review, I describe recent novel approaches used to study the mechanisms regulating these imprinted genes in mouse models, and I highlight several new discoveries. It has become apparent that the regulation of imprinted genes in placenta is often unique from other tissues and that there are species-specific mechanisms allowing the evolution of new imprinted genes specifically in the placenta. The cells that make up the placenta perform a diversity of functions in pregnancy, including invasion into the maternal uterus, remodelling maternal vasculature, mediating nutrient and waste exchange between mother and foetus, producing pregnancy-supporting hormones, and modulating the maternal immune system to tolerate and support pregnancy. Although the placenta primarily comprises cell types that arise from the conceptus, maternal immune and endometrial cells also contribute to its development and differentiation –3. SignaImprinted genes are those that are expressed monoallelically based on parent of origin, and over 100 imprinted genes have been identified to date in mice and humans , a numbeDifferentially methylated regions (DMRs) inherited from the germline typically regulate the monoallelic expression of imprinted genes. In total, 24 imprinted germline DMRs (gDMRs) have been identified in the mouse genome: 21 maternal gDMRs inherited from the oocyte and three paternal gDMRs inherited from sperm . Many ofThe investigation of imprinted genes using next-generation sequencing techniques necessitates distinguishing the maternal and paternal alleles. Two main approaches have been employed in mouse models to facilitate this, including the comparison of reconstituted embryos with only either maternal DNA (parthenogenetic or gynogenetic) or paternal DNA (androgenetic) ,13 and tAfter genome-wide erasure in primordial germ cells, DNA methylation is reestablished during gametogenesis through the differential targeting of de novo DNA methyltransferase 3A (DNMT3A) and the essential catalytically inactive cofactor DNMT3L ,16. In oAfter the embryo implants into the maternal uterus and initiates gastrulation, there is genome-wide reacquisition of DNA methylation through the targeting of de novo DNMTs: DNMT3A and DNMT3B . NotablyCdkn1c) , SPARC-related modular calcium binding 1 [Smoc1]) or drove expression of noncoding RNAs. It remains to be tested whether a subset of these imprinted ERVs can also function as enhancers for nearby genes or whether the noncoding RNAs themselves are functionally significant.The mechanisms underlying this lineage-specific imprinting are only beginning to be uncovered. Notably, a number of noncanonically imprinted domains, identified as monoallelic H3K4me3 peaks, overlapped endogenous retroviral (ERVs) insertions, many of which were sites of imprinted transcription initiation . These nFig 2C)  and pleckstrin homology like domain family A member 2 [Phlda2]) have been recently studied in detail. Using a mouse model carrying a large deletion near Ascl2, which results in its partial loss of imprinting, it was shown that impaired expression of Ascl2 resulted in a failure to properly establish the junctional zone [Ascl2 also resulted in impaired junctional zone development and an increase in stored glycogen due to a striking misallocation of glycogen cells to the labyrinth [Ascl2 is associated with foetal growth restriction [Although knockout models are valuable in revealing gene function, to understand the importance of imprinting in regulating gene dosage, more subtle perturbations are required. The consequences of manipulating gene dosage of two placental-specific imprinted genes in the nal zone ,74. Convabyrinth . Glycogeabyrinth . Consisttriction ,75.Phlda2, the levels of Phlda2 expression were shown to be essential for correct formation of the spongiotrophoblast, appropriate levels of glycogen accumulation [Fig 3B) [Phlda2 expression were shown to result in changes in the maternal brain and, consequently, altered postnatal care [Using mouse models expressing two, one, or no copies of mulation , and foemulation . The pla[Fig 3B) . As a sttal care .Together, these findings suggest that placental-specific imprinting is key in the regulation of nutrient acquisition and foetal growth in mouse development. Nevertheless, the functions of the majority of placental-specific imprinted genes remain unexplored, and future work may reveal novel mechanisms for imprinting in regulating placentation and development.The role of placental-specific imprinting in human development and placentation is unclear and remains challenging to study because of the inaccessibility of early embryonic development and the species-specific nature of placental imprinting. A recent study identified an association between aberrant imprinting at a number of placental-specific imprinted loci and intrauterine growth restriction, suggesting that these genes may regulate foetal growth, but determining whether these changes are the cause or consequence of poor growth will require further study . New tecIntriguingly, widespread placental-specific imprinted DNA methylation has been observed in humans, with more than 100 maternal gDMRs identified in placental trophoblast –91, a fe


However, the volatility of these respiration products complicates quantifying their rates of production in the field. Attempts to circumvent the challenges through closed system incubations, from which gases cannot escape, have been used to investigate bulk in situ geochemistry. Efforts towards mapping mechanistic linkages between geochemistry and microbiology have raised concern regarding sampling and incubation-induced perturbations. Microorganisms are impacted by oxygen exposure, increased temperatures and accumulation of metabolic products during handling, storage, and incubation. We probed the extent of these perturbations, and their influence on incubation results, using high-resolution geochemical and microbial gene-based community profiling of anaerobically incubated material from three wetland habitats across a permafrost peatland. We compared the original field samples to the material anaerobically incubated over 50 days. Bulk geochemistry and phylum-level microbiota in incubations largely reflected field observations, but divergence between field and incubations occurred in both geochemistry and lineage-level microbial composition when examined at closer resolution. Despite the changes in representative lineages over time, inferred metabolic function with regards to carbon cycling largely reproduced field results suggesting functional consistency. Habitat differences among the source materials remained the largest driver of variation in geochemical and microbial differences among the samples in both incubations and field results. While incubations may have limited usefulness for identifying specific mechanisms, they remain a viable tool for probing bulk-scale questions related to anaerobic C cycling, including CO2 and CH4 dynamics.Mechanisms controlling CO To produce a melt curve, a cycle of 2 min at 95°C was run, and then a final cycle of 15 s at 60°C. The cycle threshold (Ct) values were recorded and analyzed using ViiA7 v1.2 software, and 16S rRNA gene copy numbers were calculated for each sample. These calculations accounted for the genome size  and 16S rRNA gene copy number (7) of the standard. To account for variance in genome copy number of the 16S gene between different microbial lineages for more accurate cell numbers, the resulting qPCR counts were processed using CopyRighter /[δ13CH4+1000]), a larger alpha indicates hydrogenotrophic methanogenesis, while lower alphas are associated with acetoclasty. As previously reported by Hodgkins et al. [2 reduction more favorable. Nevertheless, although there were differences in magnitude between the field alphas and incubations for some comparisons, the general trend in alphas across the thaw gradient was preserved wherein collapsed palsa > bog > fen. Furthermore, the differences in alpha between the field and incubations, though significant, were small in magnitude and generally did not alter interpretations. The incubations correctly reflect the dominant hydrogenotrophic processes in the collapsed palsa, the mixture of hydrogenotrophic and acetoclastic methanogenesis in the bog, and the high proportion of acetoclastic methanogenesis in the fen. Collectively, the similarity in trends of alpha across habitats between the field and incubation samples, the similarity of trends in production rates relative to field emission rates and the similarity of CO2:CH4 ratios in the incubations compared to the field samples, supports the use of closed-system incubations for reproducibly studying anaerobic carbon cycling processes–and most specifically and climate-relevantly, CH4 production dynamics–in the field.Another often-explored process in peatland ecology is the pathway by which methane is produced. Acetoclastic methane production (i.e. disproportionation of acetate into COeatlands . To furtorewater . Because extent) . This iss et al.  for Stor2 and CH4 production in peatlands is driven primarily by substrate availability in the dissolved organic matter (DOM) pool [254), while the Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) measurements provide detailed identification of individual formula comprising the DOM. Spectral slope, which is the ratio of the first derivatives of the log transformed UV-vis spectra from 275–295 nm over 350–400 nm, is inversely correlated with average molecular weight and is a commonly used metric because of the speed and wide accessibility of spectroscopy measurements. In all habitats  the spectral slope (inferred molecular weight) was significantly different in the field relative to the incubations (see We have shown previously that COOM) pool ,67,69,70254, which is the Specific UV Absorbance at 254 nm divided by the dissolved organic C concentration (sensu34), is correlated with bulk aromaticity. SUVA254 was higher at the start of the shallow bog and fen incubations relative to the field  measures the fluorescent optical properties of the bulk DOM; local excitation-emission maxima within the 3D spectra can be used to identify different fluorescent groups . Integra254 results during the collapsed palsa incubations  to simultaneously identify thousands of individual organic compounds present in the samples . AlthougWe plotted the H/C vs. O/C ratios of assigned compounds, i.e. van Krevelen diagram, for each/site depth combination . In this2 and CH4 production, From the FTICRMS-identified formulae, we are able to calculate a number of measures of organic matter quality. The average nominal oxidation state carbon  reflect2 and is interpreted as decarboxylation. This comparison was used to identify the dominant chemical transformations  by which compounds were being transformed in the field as well as in the incubations  within each habitat type. Considered together with the high percent of compounds identified in the field that are also in the incubations samples, this suggests that despite differences in some of the bulk characteristics of the organic matter, the transformations, and therefore, decomposition processes for secondary metabolites (>100 Da) are surprisingly similar in the field and incubation samples. Thus, we can infer that chemical mechanisms working on the secondary metabolites occurring in the incubations largely reflect the dominant transformation mechanisms in the field.The top five transforms accounted for ~30% of the total number of transforms and were similar in all three habitats, though their rank of importance differed slightly among all three sites. In comparing the field and incubation transforms within each habitat type, the percentages for each transform type are overall very similar, typically within 1 standard deviation overlap, and notably follow similar rank order . Thus, though there were visible trends in the inferred cell numbers, they were not generally significantly different in the incubations compared to the field.To infer the abundance of microbial cells, the 16S rRNA gene abundances were measured, and corrected using the community composition profiles (below) for known variation in 16S rRNA gene copy number among microbial lineages see ; this trCommunity profiles from 16S rRNA gene amplicon sequencing recovered 1538 distinct microbial lineages across all samples. The communities appeared well-sampled at the sequencing depth and clustering applied, based on leveling rarefaction curves for most samples. Post-processing reads per sample averaged roughly 40,000 . While community functional profiles predicted from 16S rRNA composition via PICRUSt are imprecise (particularly for habitats with less genomic representation in utilized reference databases), several studies have demonstrated ~80% agreement with those inferred from metagenomes across diverse soil environments ,79. In aDuring the incubation period itself, in all habitats and depths, richness and diversity decreased over time  as it hahigher diversity metrics for the incubation samples  generally showed  samples . This in samples . A seconhe field  were theactivity  and loweactivity , allowinnum spp. , the remHigher diversity has been hypothesized to confer more stability, and offers more opportunity for functional redundancy , and indProteobacteria) (as duplicate averages for each time point) were examined  were calculated between all communities in this study and used to represent community composition by principal coordinates analysis PCoA; . SamplesWe also examined each habitat separately to remove the overriding impact of the habitat differences . After hSimilar to the results from OTU ordination, principal coordinate analysis of the rarefied functional profiles predicted from the 16S rRNA sequences see  revealedTo look at the functional profiles of these microbiota at higher resolution, we examined the individual KEGG metabolic functional modules for each rarefied sample with at least 400 predicted KEGG functions see . A heatmThe statistical significance of community compositional differences was assessed within each habitat and depth for all comparisons  via PERMANOVA of the weighted Unifrac distances . The 4-mPrevious work assessing storage impacts on microbial communities from soil, human skin, and human guts, after much shorter sample storage than ours (3 or 14 days) and a range of temperatures (-80°C to 20°C), found no significant impact . However2:CH4 production rates or pathways), the disturbance must cause a lasting change in the microbial community composition such that the resulting community is not functionally similar to the original community. In their meta-analysis, Allison and Martiny [Changes in community composition will only affect ecosystem process rates if the microbial community is (1) sensitive to the disturbance, (2) not resilient, and (3) the resulting community is functionally dissimilar to the pre-disturbance community ,94. In o Martiny  found ev Martiny . AdditiomcrA in 6 targeted metagenomes , and then mostly recovered by Day 50, though with more Methanobacteria. This suggests that Methanobacteria may be more sensitive to O2 exposure relative to the other lineages present as they decreased somewhat due to partial oxygen exposure during storage, before recovering by Day 50. In the shallow fen, the overall methanogen abundance increased over time even as the cell numbers and overall community diversity stayed flat  system relative to the field. For most comparisons, alphas, which indicate methanogenic pathways, were not significantly different in the field relative to the incubations. When differences were significant, the differences were slight such that the incubations correctly reflected the overall trend of hydrogenotrophic methanogenesis dominating in the collapsed palsa, a mixture of hydrogenotrophic and acetoclastic processes in the bog, and strongly acetoclastic in the fen. The preservation of this trend in the incubation samples suggest that such incubations are useful for studying environmental controls on the differences in methane production.By a number of complementary techniques, the variation in geochemical parameters appears to be driven more strongly by habitat and depth differences than by the effects of incubation. Changes over time in the incubations can be explained by expected changes to the DOM during decomposition, such as declining NOSC as labile material is used up. In many cases, the incubation COAt various levels, examination of the microbial taxa detected in the incubations and the community structures reproduce field observations that spatial differences, by habitat and depth, were the strongest drivers of microbial community differences across the Mire. In turn, geochemical parameters were most strongly separated according to habitat types. Of the optical measurements diagenetic alteration of the material , humic:f2 and CH4 dynamics among different peatland habitat types. Therefore, incubations remain a powerful tool for understanding habitat differences at this site.Microbial cell abundances were not significantly different from the field except in the deep bog incubations. Richness and diversity could only be significantly differentiated from the field in the shallow bog incubations. The KEGG functional analysis revealed that the majority of functional modules were similar between the field and incubation samples for a given site./depth. An examination of the microbial communities at high taxonomic resolution (OTU level) did reveal important differences between the incubations and the field that likely reflect the perturbations imposed by collection and handling of the sample, and depletion of substrates in the closed system. However, these perturbations did not seem to affect the large picture geochemistry of how decomposition occurred. Nor was their strong evidence of the build-up inhibitory compounds as evidenced by continued high production rates throughout 100 days of incubation. These results–that changes among habitats across the thaw gradient greatly exceeded variation introduced by incubation perturbations–indicate that closed-system incubations reasonably approximate conditions in the field for exploring ecosystem-scale questions about differences in COex situ incubations. This is a critical finding as it suggests that despite differences in the specific microbial community, critical C cycling processes can still be conserved. This is good news for researchers wishing to scale from field observations to general global principles controlling C cycling. That differences among the different habitats was maintained in the incubations also suggests that abiotic factors have a strong control on C cycling processes. Thus, on the timescale of this study, it appears that environmental factors confer greater amounts of variation to peat decomposition than storage or ‘bottle effects’ and that such incubations remain a useful tool for studying differences in CO2 and CH4 production dynamics across the thaw gradient despite the perturbations induced by manipulation of the samples.Though the microbial communities in this study were altered post-storage, with compositional differences at the OTU level, increases in richness and diversity, and temporal trends during incubation time, habitat is still the strongest driver of separation seen in these incubations. This result, suggests that the functional capacity of the communities to carry out large-scale geochemical processes is maintained during S1 Table(CSV)Click here for additional data file.S2 TableNumbers are counts of transform occurrences within each sample.(CSV)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(CSV)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


Bioprinting process involves the precise deposition of multiple layers of different cell types in combination with biomaterials capable of generating 3D bioengineered tissues based on a computer-aided design. Bioprinted cancer models containing patient-derived cancer and stromal cells together with genetic material, extracellular matrix proteins and growth factors, represent a promising approach for personalized cancer therapy screening. Both natural and synthetic biopolymers have been utilized to support the proliferation of cells and biological material within the personalized tumor models/implants. These models can provide a physiologically pertinent cell–cell and cell–matrix interactions by mimicking the 3D heterogeneity of real tumors. Here, we reviewed the potential applications of 3D bioprinted tumor constructs as personalized in vitro models in anticancer drug screening and in the establishment of precision treatment regimens.After cardiovascular disease, cancer is the leading cause of death worldwide with devastating health and economic consequences, particularly in developing countries. Inter-patient variations in anti-cancer drug responses further limit the success of therapeutic interventions. Therefore, personalized medicines approach is key for this patient group involving molecular and genetic screening and appropriate stratification of patients to treatment regimen that they will respond to. However, the knowledge related to adequate risk stratification methods identifying patients who will respond to specific anti-cancer agents is still lacking in many cancer types. Recent advancements in three-dimensional (3D) bioprinting technology, have been extensively used to generate representative bioengineered tumor   It is now well established that the soluble factors released from cancer and stromal cells can influence the cell viability/proliferation, cell-cell adhesion, cell migration, mechanotransduction, and signaling of cells within the tumor tissue, which is difficult to replicate in traditional 2D cell culture models. Recent developments have demonstrated that tumors can successfully grow across the 3D microenvironment/extracellular matrix (ECM), resulting in gradient exposure of cancer cells to oxygen and nutrients in vivo conditions increasing the percentage of non-proliferating viable hypoxic tumor and/or cancer stem cells in vitro personalized drug screening. The most frequently utilized 3D cancer models for drug testing include multicellular tumor spheroid model (MCTS), multilayered cell cultures, organotypic slices of cancer tissue, and cell seeded scaffolds Unlike iIn this review, we provide a comprehensive summary of the collective findings in relation to various bioprinted cancer models utilized for chemotherapeutic drug screening. 2D and 3D cancer models are critically evaluated and comprehensively compared, in terms of their ability to recapitulate physiological tumors and their microenvironment. Various strategies used for bioprinting of 3D cancer models including inkjet, micro extrusion, and laser ablation technologies as well as cancer and stromal bioinks, and biomaterials, are discussed. This review clearly outlines current challenges and prospects for 3D bioprinting technologies in cancer research by focusing on the clinical application of these technologies for chemotherapeutic drug screening and the development of personalized treatment regimens for cancer patients.2in vivo and in vitro models cannot be necessarily used for. Patient-derived cell-based tumor organoids and xenografts have shown some promise in advancing precision medicine, enabling the development of personalized chemotherapeutic regimens Cancer patients display a high degree of inter-patient variation in terms of clinical outcomes, prognosis, and response or tolerance to medication Therefore, testing of multiple chemotherapeutic drugs on patient-derived bioprinted cancer models could identify the most effective combination of chemotherapeutic drug candidates for a individual patient. These bioprinted cancer models can also complement human clinical trials where the effectiveness and mechanisms of drug candidates can be tested on individual basis taking into the account not just the molecular subtype of the tumor but also the differences in age, sex and ethnicity as well as identifying the most optimal doses of chemotherapeutic agents 3in vitro systems 2D cancer models have contributed significantly to our basic understanding of cancer biology over the past several decades. These simple cancer models have also played a significant role in pharmaceutical testing. However, considerable differences in the molecular signaling, protein and gene expression, cell phenotype, cell migration, cell viability, cell proliferation and drug response, have been observed between 2D and 3D cancer models ,30,31. 3in vivo models. For example, 3D cell refinements within organoids have shown potential as an alternative method to study drug pharmacokinetics within the tumor tissue therefore addressing the gap in transition between 2D cell culture and animal models et al used 3D multicellular DU-145 prostate cancer cell line based spheroids that could effectively simulate the prostate tumor microenvironment to evaluate the cytotoxic effects of ruthenium complexes On the other hand, 3D cancer constructs represent physiological cancer tissues better, in terms of tumor microenvironment and cellular behavior with characteristic spatial distribution of cells in vivo tumor tissues, such as high growth rates of cancer cells, aggressive invasiveness, angiogenesis, metastasis, high resistance to anticancer drugs In pursue of personalized drug screening, multicellular organoids can be generated using different types of patient-derived cells isolated from biopsy tissues in a co-culture system capable of creating spheroids. The major advantage of these spheroid systems is that the screening can be performed with very small quantities of chemotherapeutic candidates 43D bioprinting is an innovative biomanufacturing platform, which allows the deposition of living cells, signaling molecules and biomaterials using computer-aided design (CAD) to generate tissue engineered constructs with highly controlled tissue architecture ,52. BiopUtilizing bioprinting technology, many complex internal tissue structures including channels and stroma with varying dimensions can be generated Based on instrumentation approaches, bioprinting approaches can be classified into droplet, extrusion and laser-based bioprinting depending on their deposition mechanism 4.1For generating a personalized bioprinted construct, patient-specific cells and their microenvironment need to be recapitulated in the printed construct. The success of the fabricated construct is highly dependent on the selection of appropriate cells for bioprinting. Multiple types of cancer cells including primary cancer cells, CTCs, and stromal cells including fibroblasts, endothelial cells and stem cells can be used for printing personalized tumor construct. Of note, stem cell-like phenotype was observed in personalized bioprinted tumor construct using a gelatin-alginate-Matrigel bioink containing patient derived intrahepatic cholangiocarcinoma cells Apart from cancer-associated cells, a bioprinted tumor construct should contain stromal cells to mimic a natural tumor microenvironment of human tumors where the cancer cells are encapsulated within different types of healthy cells. Selection of these normal cells in bioinks should be based on the tumor characteristics and bioengineered accordingly. For instance, a brain tumor construct should be based on patient-derived tumor cells of interest mixed with surrounding healthy brain cells including neurons, glial cells, astrocytes, and microvascular endothelial cells ,81. Howe4.22+ or other divalent cations are commonly used due to fast gelation and minimum adverse effects on cells Bioinks are the building blocks of bioprinted constructs, which are generally composed of a suitable hydrogel, multiple types of cells, nutrients, and growth factors. The hydrogels used in bioinks should be biocompatible, and their gelation should be easily controllable in vivo tumors [Cancer bioink: Cancer bioinks are composed of patient-derived cancer cells , suitable biopolymer , growth factors  and other nutrients (e.g. cell culture medium). For this purpose, a non-globular protein, fibrin, important for blood clotting o tumors ,100. Scho tumors ,104,105,In vivo, endothelial cells or endothelial progenitor cells are recruited by tumor immune cells and others to initiate tumor angiogenesis through an array of secreted signaling molecules. Generally, tumor blood vessels have special features including extensive branching and loose intercellular junctions making them leakier than normal blood vessels. Thus, incorporating endothelial or endothelial progenitor cells along with key angiogenic factors such as VEGF or EGF in stromal bioink is crucial for recapitulating tumor growth Stromal bioinks: In addition to primary cancer cells, tumor tissue is also composed of heterogeneous group of healthy stromal cells including endothelial cells, mesenchymal/hematopoietic stem cells, fibroblasts and other tissue specific cells ,107. Sim4.3in vivo tumor architecture and microenvironment Briefly, the process of bioprinting starts with the CAD design of the structural architecture of the specific tumor tissue. The bioink can have multiple types of patient-derived malignant and healthy cells . The cells are mixed with other components of bioink  at the ratio required to mimic the In vitro tumorigenesis facilitated by cell division, proliferation and differentiation during maturation of bioprinted constructs is a critical step in the post‐printing stage on cancer constructs. Achieving rapid maturation of bioprinted construct is key especially when these are utilized for screening of personalized anti-cancer therapies. This is time sensitive and requires identification of the most suitable chemotherapy regimen that could be administered to the cancer patients. Nevertheless, accelerated tissue maturation is challenging and further research and optimization is required to progress the development of personalized bioprinted tumor constructs for precision chemotherapeutic screening. Past studies indicated that tissue maturation can be achieved in static culture systems to some extent in vitro maturation and maintenance. Viability of different types of cells within the constructs can be tested by calcein-AM based staining which can label live and dead cells by staining live cells green. Depending upon the 3D model, different types of immunohistochemistry or immunofluorescence assays can be performed to understand the expression of various proteins important in the development and maintenance of cancer progression, synthesis of ECM components and membrane proteins Several physicomechanical measurements and biological assays are required to properly characterize bioprinted cancer constructs before using these for personalized drug testing. Stiffness of the construct can be measured by nanoindentation using an Atomic Force Microscope (AFM) Personalized bioprinted tumor constructs after maturation can be used to test an array of potential chemotherapeutic agents. In static culture systems, the chemotherapeutic agents can be administered to a tumor construct within the cell culture medium to determine the effects on cell viability and tumor growth reduction. Bioprinting can be performed in multi-well plates, generating large numbers of homogeneous organoids thus increasing the throughput of screening ,118. In 55.1in vivo tumor microenvironment. Initial studies in this field were focused on the development of bioprinted breast cancer spheroids using widely available breast cancer cell lines and synthetic hydrogels Breast cancer is the most frequently diagnosed cancer in women worldwide, with approximately 2.09 million new cases and 627,000 deaths in 2018, estimated by the WHO 5.2in vitro models of brain tumors, this should be taken into the account in vivo tumor microenvironment and represent promising personalized GBM platforms for chemotherapeutic screening. The results from a study investigating temozolomide treatment for GDM, showed that 3D tumor models containing glioma stem cells were more resistant to temozolomide treatment compared to 2D monolayer, thus likely recapitulating physiological tumor response more closely About 10–40% of brain tumors are metastatic at the time of diagnosis and hence when developing 5.3Skin cancer or melanoma is one of the most common forms of cancers especially in Caucasian people consisting of different cancer types including non-melanoma skin cancer (NMSC), malignant melanomas (MM), Merkel cell carcinomas (MCC), basal cell carcinomas (BCC) and cutaneous squamous cell carcinomas (cSCC), that affects over 300,000 people annually across the globe 5.4in vitro models and personalized drug screening Colorectal cancer  is associated with one of the poorest survival rates and it is often diagnosed at the metastatic stage 5.5in vitro cervical cancer models for investigating mechanisms and treatment options for preventing cervical cancer progression. It was observed that HeLa cells migrated to different extents depending on the channel diameter and no effects were reported on fibroblast migration β) induced (EMT) in an advanced bioprinted cervical tumor model shows promising results for developing future therapeutic strategies towards preventing or treating cervical tumor metastasis. A HeLa/hydrogel grid construct comprising alginate, gelatin, Matrigel and HeLa cells showed rapid proliferation, formation of spheroids and displayed tumorigenic feature in the 3D-printed construct. The down-regulation of epithelial marker, E-cadherin, and up-regulation of mesenchymal markers including snail, vimentin and N-cadherin, were reported in the 3D-printed model with the supplementation of TGF-β. Furthermore, the TGF-β induced EMT was inhibited following treatment with disulfiram and EMT pathway inhibitor C19 in a dose-dependent manner Cervical cancer continues to be the leading cause of death in women despite recently introduced vaccination and screening, hence with an urgent need for improvement in personalized screening, and treatment efforts 5.6in vivo tumors and microenvironment and include strategies for recapitulating surrounding fibrous tissue containing barrier proteins in vitro. Most pancreatic cancer cells do not form cohesive and compact spheres when the original hanging drop method is used. However, a modified hanging drop method following addition of methylcellulose polymer was capable of growing uniform and reproducible spheroids for all five of the tested human pancreatic cancer cell lines; Panc-1, BxPC-3, Capan-1, MiaPaCa-2, and AsPC-1 Pancreatic adenocarcinoma is the second most lethal cancer in the world. Advance stages of pancreatic cancer have very poor prognosis with a five-year survival rate ∼2–5% cases 5.7in vitro tumor model composed of epithelial ovarian cancer (EOC) cells, reporting a substantial transition in histological features of 3D constructs compared to 2D tumor models, which are hallmark features of primary tumors.Ovarian cancer is the seventh most lethal cancer in women 5.8Based on histological classification, there are two main types of lung cancers- I) small cell lung cancer and II) Non-small cell lung cancer 5.9in vitro human liver cancer model is a highly promising approach to better understand the disease mechanism, and serve as a drug screening platform, eventually improving the treatment of disease TM showed colony forming ability, high proliferation and survival in vitro drug screening. A novel 3D biomimetic HCC model composes fibrotic stromal compartment and vasculature was developed, which can mimic bio-physical properties of a fibrotic, cirrhotic and HCC liver. This model showed chemotherapeutic drug resistance commonly seen in HCC patients. This 3D tumor construct could provide a reliable new platform to investigate multifocal HCC that contribute to early stages of cancer metastasis in vivo tumor heterogeneity and high throughput chemotherapeutic testing Hepato-cellular carcinoma (HCC), the fifth most common malignant cancer represents about 90% of primary liver cancers and constitutes a major global health problem 6in vitro and in vivo cancer models to recapitulate the genetic makeup, molecular biology, and physiology of cancer cells in human tumour tissues impedes their applicability in the screening of chemotherapeutic drugs and precision medicine. Despite significant progress that has been made in the development of a number of new anti-cancer therapies using traditional cancer models, these systems have limited applicability in screening of personalized treatments as these are unable to reliably reproduce the heterogeneity of tumour tissue and the variation in cancer molecular and cellular characteristics from individual patients. In this context, 3D constructs containing patient-derived cells that are propagated in vitro within similar microenvironment as human in vivo tumours, are more reliable for generating personalized disease models. Although there has been a substantial progress and emerging potential of bioprinted cancer models for these applications, a number of challenges still remain, limiting translation to clinical and industrial applications. Although large scale manufacturing of bioprinted constructs is still in its infancy, manufacturers including Cellink and their partners, Prellis Biologics, are in the process of developing bioprinting equipment that can potentially address the requirements of industry and the clinic. Holograph X bioprinter is intended to facilitate the in-lab manufacturing of vascularized tissue constructs for organ transplant, via holographic projection printing. Bioprinting of patient-derived tumour constructs for screening of anticancer agents for the purpose of precision medicine is still in the development and it will take a number of years before it can be used in the clinical setting. From the very beginning, these technologies should comply with the good manufacturing practice (GMP) standards and the regulatory requirements, in order to standardize and optimize the bioprinting process of patient-specific tumours. Future studies should focus on the development, fabrication, and application of bioprinted tumour constructs in compliance with GMP standards. These bioprinted platforms should also be adapted accordingly for high throughput screening of chemotherapy and other anti-cancer agents by taking into the account the ease of use and the cost. Hence, future research should also focus on the rapid and automated determination of cellular parameters including cell viability and cell cycle within the 3D construct in real-time. Integration of organotypic constructs within simulated physiology of microfluidic platforms are challenging with the potential to incorporate all the required characteristics of human in vivo tumours.Limitations of current in vitro and cost-effectiveness for high-throughput screening and application in the low resourced clinical setting. The design and development of a fully integrated bioprinting mechanism is key for the commercial translation of bioprinting technology for precision medicine purposes in small to medium size laboratories. Future studies should focus on the approaches that help to miniaturize the bioprinted constructs suitable for automated workflow systems that are less labour -intensive and cost-effective.Published evidence in the field of bioprinted cancer models indicates that bioprinted cancer models are highly promising platforms for evaluation of personalized anti-cancer treatments. Nevertheless, there are still substantial gaps in the knowledge in terms of various aspects of tumour bioprinting including rapid fabrication of 3D cancer models, their maintenance The accurate control over physicomechanical and biological characteristics of a bioprinted construct is possible using advanced 3D printing technologies. Suitable material for bioprinting is required to precisely be placed with the preferred spatial and temporal control. Bioprinting technologies like inkjet have drawbacks in terms of material viscosity whereas others such as microextrusion may need materials having specific crosslinking mechanisms or shear-thinning properties. Nozzle gauge is one of the processing parameters determining the shear stress to which cells are exposed to along with the time necessary for the material to be placed to produce a 3D structure. For instance, inkjet printing involves the use of materials with a rapid crosslinking time to enable the generation of a complex 3D structure whereas microextrusion can make use of highly viscous materials to keep a 3D shape after deposition with final crosslinking that occurs following fabrication. A recent report indicated that thermal inkjet bioprinting has impact on gene expression including those involved in key drug resistance, cell motility, proliferation, cell survival, and differentiation. These are important factors to consider and monitor as part of the process of bioengineering tumour constructs by bioprinting.The cell viability, a critical factor for cancer modelling, can be affected by the shear forces generated during extrusion bioprinting. Recent approaches focusing on LAB methods may be a solution for maintaining cell viability in the future, bearing in mind that photoinitiator and UV light used in LAB need further optimising to minimise their effect on cell viability. Less toxic photoinitiator or visible light photocrosslinking approaches may solve these issues. Several other crosslinking approaches are also used for bioprinting to provide the sufficient mechanical strength and relatively long degradation rate to the bioprinted construct. A wide range of natural polymers including gelatin, alginate, chitosan, and collagen, have been used as a bioink for printing tumour constructs because of simplicity of their crosslinking mechanisms. However, ionic, or chemical crosslinking agents used for hydrogel systems can also affect cell viability and phenotype of the tumour construct. Recent advancements with introduction of photocrosslinkable methacryloyl groups into these biopolymers is showing some progress towards better preservation of cell viability and phenotype. However, fine tuning the methodologies for bioprinting cancer and stromal bioinks capable of recapitulating the properties of physiological tumour tissues and their microenvironment in adequate spatial and temporal manner needs to be further explored. These models can also recapitulate the heterogeneity of the tumors using different materials and cells simultaneously within the same bioprinted construct. As a drug screening platform, bioprinting should be applicable to several cancer types including breast cancer, GBM, cervical cancer, lung cancer and ovarian cancer.in vivo. Generating bioprinted constructs with well-developed vasculature is particularly important in GBM, tumor characterized by pronounced angiogenesis. Unsurprisingly, a study investigating angiogenesis within MCTS of undifferentiated melanoma cell line, NA8-MCTS, revealed that the invasion and network formation of HMEC-1 cell line within NA8-MCTS increased following co-culturing with HMEC-1 in vivo environment, cellular secretion of enzymes, hormones, cytokines, or other agents can influence the microenvironment of the growing tumour. Hence, all these cellular and molecular factors need to be taken into the account when designing and bioprinting tumor constructs.One of the key challenges in cancer bioprinting is the difficulty in developing well-established vascular network within tumors, which is necessary given that tumor tissues contains large number of highly branched capillary networks that facilitate tumour growth. Thus, connecting the bioengineered tumour tissue with surrounding stromal tissue is necessary for mimicking these cellular communications that are present in vitro and in vivo findings have demonstrated the significance of the interaction between the tumour cells and host stromal cells including endothelial cells, fibroblasts, adipocytes, mature immune cells, and circulating immune cell progenitors. Incorporation of various types of healthy cells and specific cancer cells is a major challenge since it is far from simply combining similar types of cells. However, recapitulating cellular communications is vital for paracrine and autocrine molecular signalling that affects key hallmarks of cancer. These include interactions with the surrounding matrix, host cells, and receptors or ligands of both cancer and stromal cells. For instance, CAFs that affect tumor microenvironment are the most common component of cancer stroma mainly in breast and pancreatic cancer, MRC-5 fibroblasts and OVCAR-5 in human ovarian cancer cell models, a set of cancer and stromal cells including U87MG glioblastoma cells, MM6 monocyte/macrophages, glioblastoma stem cells, glioma associated stromal cells (GASCs) in GBM and iPSCs in colorectal cancer models have been used for personalized drug screening. Understanding biology of specific types of tumors well will aid the development of suitable bioprinted platforms with high predictive power, capable of drugs screening and precision medicine.Although the bioprinted cancer models share a common 3D conformation, each display its own intrinsic properties. For example, a variety of The effectiveness and purpose of tissue constructs can be designed, and hence improved, before it is printed using computer models. Therefore, design stage is key for generating optimal and specific 3D cancer tissues representative of physiological tumours. Rapid developments in bioprinting instrumentation along with progress in bioink preparation and bioprinting process can significantly improve the ability of the construct to closely mimic cancer tissue and its reliability to test chemotherapeutic drug efficacy. Moreover, regulatory agencies should have standardized guidelines for the requirement of processes and procedures to accelerate the clinical translation of these bioprinted tissue models and enable personalized treatments that will improve outcomes for the patients.7in vitro models, including cell supportive biological material and 3D bioprinting, have rapidly progressed. Conventional cancer models have a number of limitations due to the lack of capacity to mimic the complexity of tumours, thus restricting their utilisation for chemotherapeutic drug screening. Attempts to diligently mimic the tumour microenvironment appear to be feasible with the introduction of latest technologies such as bioprinting and microfluidic chips. Past research shows that the 3D bioprinting has a huge potential to construct tissue models that can recapitulate tumour microenvironment at molecular, cellular, and physiological level. Bioprinted breast, brain, skin, colorectal, pancreatic, and other cancer models were highly successful in mimicking tumour anatomy, physiology, and molecular biology. Patient-derived 3D bioprinted cancer models could be successfully used for in vitro drug screening of anti-cancer drugs in order to develop personalized treatments. Ongoing and future studies are focusing on the development of integrated microfluidic/bioprinted constructs in a lab-on-a-chip format to minimize the cost and facilitating high throughput testing of a large number of available chemotherapeutic agents enabling selection of the most suitable drug combination for a particular patient and hence personalized medicine approach.Over the last two decades, the tools used to create 3D cell cultures, organoids, and other 3D Conceptualization: R.A (Robin Augustine). Investigation: R.A (Robin Augustine)., N.K.S., Resources: R.A. (Robin Augustine). Writing—original draft preparation: R.A (Robin Augustine)., N.K.S. Writing—review and editing: R.A (Robin Augustine)., N.K.S., R.A (Rashid Ahmed)., A.N., L.M., A.A.Z. Visualization: N.K.S., R.A (Robin Augustine). Supervision: R.A., A.H. Project coordination: R.A. Funding acquisition: A.H. All authors have read and agreed to the published version of the manuscript.Authors declare that there is no financial/personal interest or belief that could affect the results, discussions or conclusions which are reported in this work.


SHANK  proteins are postsynaptic core scaffolds involved in excitatory synapse development, function, and plasticity. The three members of the SHANK family  differ in their temporal and regional expression patterns in the central nervous system and non-neuronal tissue during development. Mutations in the cognate genes are associated with various neuropsychiatric conditions, such as autism spectrum disorders, Phelan-McDermid syndrome, intellectual disability, schizophrenia, and bipolar disorder. Exactly how defects in SHANK proteins contribute to these conditions is currently under active investigation.Eltokhi et al. observed that the large phenotypic diversity of patients carrying SHANK2 mutations is reflected in the phenotypic diversity displayed by the various Shank2 mouse models. Hassani Nia and Kreienkamp reviewed the effects of Shank3 mutations on the gene, protein, and synaptic signaling. Both reviews provide a comprehensive state-of-the-art overview on SHANK2 and SHANK3 different scales. An example of how mouse models can closely mimic human Shankopathies was provided by the comprehensive characterization of the Shank3Q321R knock-in model conducted by Yoo et al..The present Research Topic provides an overview of current knowledge on the involvement of SHANK genes in neuropsychiatric disorders. Shankopathies are explored at different scales: genes, proteins, cells, synapses, neural circuits, behaviors, and environment. For example, The other contributions address two major questions for Shankopathies: (i) Are there specific modulating factors for Shankopathies and (ii) Which brain regions are specifically affected by Shankopathies?Sungur et al. explored whether Shank1 mutant mice and control mice were equally sensitive to amphetamine and methylenedioxymethamphetamine. In Shank2 mutant mice, Ey et al. demonstrated that the abnormal social behavior of Shank2 knockout mice was associated with a deficit in social motivation, but with an intact social recognition phenotype. They also highlighted that methylphenidate is ineffective in restoring typical activity levels in hyperactive Shank2 knockout mice. By contrast, Berkel et al. examined the role of an inherent modulating factor; namely, sexual hormones, on Shank1, Shank2, and Shank3 gene transcription and protein levels. The role of sexual hormones as transcriptional fine-tuners, especially shortly before or after birth, might explain sex-related differences in patients and animal models. Finally, zinc was identified as a potentially major modulating factor in Shankopathies, given its role in recruiting Shank2 and Shank3 at synapses, as well as having a key role in the gastro-intestinal tract, as reviewed by Hagmeyer et al.. At the molecular level, the mode of regulation of AMPA receptor (AMPAR) subunit composition by Shank2, Shank3 and zinc was explored by Ha et al.. Zinc regulated the switch during development of AMPARs lacking GluA2 to AMPARs containing GluA2, while Shank2 and Shank3 also regulated this process. At the scale of the organism, Fourie et al. tested the effect of long-term zinc supplementation in alimentation of the Shank3ex13Δ−16 mouse model. Importantly, some aspects of synaptic transmission and some behavioral traits, such as self-grooming, were rescued. Taken together, the studies on zinc presented in this Research Topic present evidence that zinc is a promising potential new therapeutic agent for Shankopathies.Concerning the first question, three putative modulating factors were examined; namely, pharmacological intervention , sexual hormones, and zinc. Shank mutations at the different scales of observation. Yoo et al. highlighted that a conditional Shank3 knockout  in GABAergic neurons (enriched in the striatum) induces a strong reduction of excitatory synaptic inputs onto dorsal striatal neurons as observed in global Shank3ex14Δ−16 mutant mice. However, the conditional knockout leads to milder social deficits and stereotyped behaviors than global Shank3ex14Δ−16 mutant mice. Using magnetic resonance imaging (MRI), Schoen et al. examined mouse brain anatomy during development showing that total brain volume, cerebellar volume, and cortical thickness were unaffected in homozygous and heterozygous Shank3 mutant mice and the prenatal zinc deficiency model compared to wild-type controls. By contrast, the striatal volume and the globus pallidus volume were increased in both models, while the changes in the thalamus were reduced in the Shank3 mouse model and increased in the prenatal zinc deficiency mouse model. Transcriptomic analyses allowed  to identify brain region-specific modulation of gene expression in different Shank3 models, across age classes and between brain regions . This analysis highlighted an important modulation of the expression of myelin-related and ribosome-related genes. At the protein level, Heise et al. found that Shank2 and Shank3 mutant mice displayed a reduced expression of receptors specific to excitatory synaptic transmission in the striatum and in the thalamus . These models were compared to another mouse model of neurodevelopmental disorders, the Cntn4 mutant that displays increased surface expression of glutamatergic receptors (GluA1 and GluA2) in the striatum or downregulation of GABAergic receptors (GABAA) in several brain regions. Finally, the involvement of the reward system was examined directly and indirectly. In the direct approach, Bariselli et al. knocked down Shank3 in the ventral tegmental area in mice and observed a dysfunction in social-seeking behavior. The indirect approach concerned the characterization of a Shank2 model in another species, namely the Shank2ex31Δ mutant rat, characterized by Modi et al.. Shank2ex31Δ mutant rats displayed an atypically high motivation to collect food reward, in parallel with social deficits, impaired learning, increased activity, and repetitive circling, combined with increased striatal activity and decreased hippocampal function.Regarding the second question on brain regions specifically affected by Shankopathies, different approaches were used to investigate the effect of SHANK mutations, as well as providing prospects for new potential therapeutic strategies, targeting specific brain regions or modulating factors.In summary, this Research Topic provided a multi-scale overview of the current state of knowledge on Shankopathies. The research studies contribute to the understanding of the spectrum of disorders related to All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Most myeloid lineage cells express the receptor and coreceptors that make them susceptible to infection by primate lentiviruses (SIVs and HIVs). However, macrophages are the only myeloid lineage cell commonly infected by SIVs and/or HIVs. The frequency of infected macrophages varies greatly across specific host and virus combinations as well as disease states, with infection rates being greatest in pathogenic SIV infections of non-natural hosts ) and late in untreated HIV-1 infection. In contrast, macrophages from natural SIV hosts  are largely resistant to infection due to entry and/or post-entry restriction mechanisms. These highly variable rates of macrophage infection may stem from differences in the host immune environment, entry and post-entry restriction mechanisms, the ability of a virus to adapt to efficiently infect macrophages, and the pleiotropic effects of macrophage-tropism including the ability to infect cells lacking CD4 and increased neutralization sensitivity. Questions remain about the relationship between rates of macrophage infection and viral pathogenesis, with some evidence suggesting that elevated levels of macrophage infection may contribute to greater pathogenesis in non-natural SIV hosts. Alternatively, extensive infection of macrophages may only emerge in the context of high viral loads and immunodeficiency, making it a symptom of highly pathogenic infections, not a primary driver of pathogenesis. Human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency viruses (various SIVs) are primate lentiviruses capable of generating chronic disease in humans and in nonhuman primates (NHPs). SIVs have been replicating in African nonhuman primates for at least 32,000 years , which hThe development of antiretroviral therapy (ART) and increased knowledge of HIV biology have saved millions of lives. Much of this progress was made possible by the use of NHP models of HIV infection. SIV infections of African NHPs  typically generate no disease or disease that is much less pathogenic than HIV-1 infections in humans , making All primate lentiviruses replicate extensively in CD4+ T-cells, and certain variants are also able to efficiently infect macrophages ,16,17,18This review will explore SIV and HIV-1 infection of myeloid lineage cells, particularly macrophages, which are the only myeloid lineage cell type infected at appreciable rates ,22,23. REntry is the first step in infection and is one of the primary determinants of cellular tropism. Primate lentivirus entry (reviewed by ) is faciGiven that entry involves the sequential binding of CD4 and a coreceptor, primate lentiviral infection is typically restricted to cells expressing CD4 and a coreceptor  at densities that support viral entry. Studies have repeatedly shown that antibodies or drugs altering viral interactions with CD4 inhibit viral entry and replication in vivo ,27,28,29Of their potential target cells, CD4+ T-cells are the primary cells in which HIV-1 and SIVs replicate. The preferential infection of CD4+ T-cells is clearly supported by studies of infected humans and NHPs in which the frequency of CD4+ T-cells containing viral DNA, RNA, or protein is high relative to the low frequency found in monocytes in the blood ,22 or myMyeloid lineage cells are granulocytes, monocytes, macrophages, and dendritic cells (DCs) . These cMonocytes and DCs have been proposed to be potential targets for SIV and HIV replication, but there is no evidence that they are frequently infected ,22,23. Wvpx gene but express the accessory protein Vpr, which may also inhibit SAMHD1 [Primate lentiviral infection of myeloid lineage cells is thought to be limited by the host protein SAMHD1 , which impairs reverse transcription of the viral RNA genome by reducing nucleotide pools within resting CD4+ T-cells and myeloid cells ,68. HIV-t SAMHD1 .vpx deletion mutant found that the Vpx expressing SIV infected macrophages at higher rates, suggesting that Vpx does enhance infection of myeloid lineage cells in vivo [Studies examining the role that SAMHD1 plays in infection of myeloid cells suggest that SAMHD1 restriction alone does not explain the extremely low levels of myeloid cell infection in vivo. One experiment comparing macaques infected with either an SIV expressing Vpx or with a  in vivo . However in vivo . The low in vivo , other pThe frequency of macrophage infection is highly variable in HIV-infected people but is consistently higher in pathogenic infections late in the disease. This is most clearly illustrated by studies examining HIV-1 populations in the immune-privileged CNS at end-stage disease. Immunostaining of brain tissue from such individuals has identified large numbers of productively infected microglia ,74,75,76Multiple studies have shown that the vast majority of HIV-1 variants throughout the body, and virtually all HIV-1 variants in the blood, are poorly adapted to infecting macrophages  and require high CD4 densities similar to the levels on CD4+ T-cells for efficient entry  resulted in reduced plasma viral loads . In contThere is now both in vitro and in vivo data indicating that SIV infection of macrophages is blocked in natural hosts, but not in non-natural hosts. The ability of SIV to enter macrophages from non-natural hosts depends on the surface density of CD4 , but stuSIVs are capable of generating a wide range of disease states, with highly variable levels of macrophage infection. Our understanding of how natural hosts are protected from the pathogenic effects of SIV infection remains unclear, but there is growing data suggesting that blocks to macrophage infection in natural hosts may contribute to this protection. However, the fact that some highly pathogenic SIV and HIV-1 infections do not generate high levels of macrophage infection suggests that extensive macrophage infection is not a requirement for the development of pathogenic infections. An alternative hypothesis is that extensive infection of macrophages is a symptom of highly pathogenic infections. Additional work is needed to untangle the mechanistic link between pathogenesis and infection of macrophages in primate lentiviruses.