Chemotherapy remains a primary treatment modality for various malignancies. However, resistance to chemotherapeutic drugs is a major obstacle to curative cancer therapy. Lysosomes are acidic organelles that participate in cellular digestion. However, there is rising interest in lysosomes because of their involvement with cancer. For example, extracellular secretion of lysosomal enzymes promote tumorigenesis; cytosolic leakage of lysosomal hydrolases promote apoptosis; and weak chemotherapeutic bases diffuse across the lysosomal membrane and become entrapped in lysosomes in their cationic state. Lysosomal drug sequestration lowers the cytotoxic potential of chemotherapeutics, reduces drug availability to sites of action, and contributes to cancer resistance. This review examines various mechanisms of lysosomal drug sequestration and their consequences on cancer multidrug resistance. Strategies for overcoming drug resistance by exploiting lysosomes as subcellular targets to reverse drug sequestration and drug resistance are also discussed. In addition to this housekeeping function, lysosomes perform diverse cellular processes. For example, they are involved in macroautophagy, chaperone-mediated autophagy, cholesterol homeostasis, and degrading receptor tyrosine kinase receptors and growth factors-6. Lysosomal hydrolases play opposing roles in neoplastic cells. Lysosomal proteases that are secreted extracellularly promote tumor invasion and metastasis-9. Specifically, cathepsins are used by tumor cells to degrade extracellular matrix components such as fibronectin, elastin, and laminin, thereby facilitating invasion, angiogenesis, and metastasis,11. In contrast, cytosolic translocation of lysosomal proteases induces apoptosis and cell death,13. Due to the dual role that lysosomes have in cancer, mounting evidence suggests that they are attractive targets for oncological therapies,15.Lysosomes are acidic organelles that contain over 50 digestive enzymes that can degrade all macromolecules. They are the major cell digestive organelles. It is well documented that the drug transporter permeability-glycoprotein (P-gp) contributes to MDR. Al-Akra and his team demonstrated that lysosomal P-gp plays an important role in conferring drug resistance. Additionally, P-gp has been observed to localize to lysosomes. In spite of novel approaches to address MDR, it continues to be a major cause of unsuccessful cancer treatments,21.Drug resistance is the primary reason that cancer treatments fail and patients die. Multidrug resistance (MDR) occurs in cancer cells that develop the ability to resist various drugs that are structurally and pharmacologically unrelated. Mounting evidence has demonstrated that certain hydrophobic weak base chemotherapeutics such as the anthracyclines doxorubicin, daunorubicin, and mitoxantrone, imidazoacridinones, the receptor tyrosine kinase inhibitor sunitinib, and pyrimethamine preferentially accumulate in lysosomes-30. Lysosomal sequestration markedly reduces drug concentrations and thereby directly decreases the effects of anticancer drugs on their intended targets, the nucleus and cytoplasm. This lysosomal scavenging of oncologic drugs potentiates MDR. Therefore, understanding the molecular mechanism underlying lysosomal sequestration of chemotherapeutic drugs should provide insights to circumvent this clinical problem.Mounting evidence shows that lysosomes play a role in MDR. A major limitation of chemotherapeutic drugs is that they become trapped or sequestered in acidic organelles, such as the lysosomes. Lipophilic chemotherapeutic agents with weak base properties readily diffuse across cell membranes. However, when these drugs enter the acidic lumen of lysosomes, they become protonated and are trapped in the lysosomeset al. (2000) were one of the first groups to demonstrate that lysosomotropic agents disrupted the lysosomal membrane, resulting in cytosolic leakage of the acid hydrolases, specifically the cathepsins, and apoptosis. Subsequently, various studies have confirmed that lysosomal membrane permeabilization (LMP) induces apoptosis,32,33.Lysosomes can also initiate the intrinsic apoptosis pathway in response to treatment by lysosomotropic agents. Li . Additionally, the hydrophobic weak base siramesine induced LMP in cancer cells in vitro and in vivo,36. Consistent with this approach, bovine α-lactalbumin and oleic acid was shown to kill various cancer cell lines  by a mechanism involving LMP.Since LMP is known to trigger apoptosis in various cancer cells, drug screens to identify agents that induce LMP may prove to be effective cancer therapies to overcome drug resistance induced by lysosomal drug sequestration. For example, a recent study detected 175 compounds that induced death in HCT116 colon cancer cells. Notably, over half of the 11 compounds that induced apoptosis in p-53 deficient cells did so by LMP and cathepsin-mediated cell death-40, promotes stability in tumor cell lysosomes. Mousavi et al. (2003) reported that PI3K regulates the size, maturation, and activity of lysosomes. Tumor cells can abolish LMP by overexpression of cytosolic protease inhibitors,43. Cancer cells also protect themselves from LMP by translocating Hsp70 from the cytosol to the lysosomal lumen where it stabilizes lysosomal membranes by promoting the activity of acid sphingomyelinase,45. Support for this role of Hsp70 comes from observations that depletion of this protein triggers a tumor-cell-specific lysosomal cell death program.The success of cancer cells to develop resistance to chemotherapeutics also involves mutating pro-apoptotic pathways and lysosomal-mediated death pathways while upregulating cell proliferation pathways. Tumor cells use various methods to modify their lysosomes in an effort to evade cell death. The increased activity of phosphatidylinositol-3’-kinase (PI3K), which is characteristic of many tumors. Notably, adriamycin was observed to concentrate in the lysosomes of drug-resistant cells but not in lysosomes drug-sensitive cells. Presumably these weak bases are freely transported to the lysosomes by passive transport due to their hydrophobic composition. The pKa values, predominantly above 7.0, for these drugs confirms that they are weak bases. Likewise, the intracellular efficacy of these compounds can be decreased in vitro at acidic pH,25. Many of the drugs used to treat malignancies are weak bases, and it has been demonstrated by several reports that they become sequestered in lysosomes. These include the following drugs: daunorubicin, doxorubicin, lapatinib, vincristine, and nintedanib,27,36,48. Once they cross the lysosomal membrane, these weak base chemotherapeutics become trapped through protonation in the lysosomal lumen,50. The use of the term “drug trapping” in lysosomes can be misleading. The ionized form, in most cases, is in equilibrium with the neutral drug in the cytosol. Furthermore, the ionized version of the drug can rapidly cross the lysosomal membrane by passive diffusion when the cytosolic concentration of the drug decreases due to transport into the bile, metabolism, or diffusion back into the plasma. Support for this comes from the observation of rapid reversibility of lysosomal trapping of a lipophilic drug seen when rats are asphyxiated with carbon dioxide, which modestly acidifies the blood and causes a decrease in tissue levels and an increase in plasma drug levels. These results suggest that lysosomes function as a reservoir pulling the drug from its target site and do not indefinitely trap cytotoxic drugs.Lysosomes have been shown to sequester lipophilic, weakly basic chemotherapeutic drugs via a non-enzymatic and non-transporter mediated mechanism,52. The multikinase inhibitor nintedanib has been investigated in clinical trials for the following tumors: non-small cell lung cancer, colorectal cancer, prostate cancer, and pancreatic cancer-56. One study reported that nintedanib was sequestered in lysosomes, thus lowering its cytosolic concentrations and its fibroblast growth factor receptor inhibition potential. Another report found that intracellular levels of imatinib are primarily determined by lysosomal sequestration. Lysosomes have also been reported to have indirect effects on drug sequestration. Kalayda et al. reported that abnormalities in the lysosomal compartment promote sequestration of cisplatin away from the nucleus due to faulty localization of transport proteins. Taken together, these data suggest that anticancer drug resistance is modulated by lysosomal sequestration, as summarized in In contrast, localization of chemotherapeutics to the acidic lumen of lysosomes does promote MDR. Support for this comes from several lines of evidence. Lysosomal accumulation of sunitinib was detected in hepatocellular carcinoma cells, renal cancer cells, and colon cancer cells. Likewise, another study found that lysosomal sequestration of doxorubicin in MCF-7/adriamycin breast cancer cells decreased the levels of the drug in the nucleus. Indeed, it was reported that cells with a higher number of lysosomes were more resistant to sunitinib compared to cells with lower numbers of lysosomes. Conflicting evidence comes from a study showing a cisplatin-resistant ovarian cancer cell line containing considerably fewer lysosomes than wild-type cells.Chemotherapeutic drugs are distributed between cytosolic and nuclear compartments intracellularly. Since lysosomes are not the intended target sites of these drugs, their entrapment in lysosomes effectively decreases their therapeutic effects at the wild type targets, such as nuclear DNA. Support for this notion comes from a study that showed that daunorubicin accumulation in lysosomes resulted in decreased nuclear concentrations of daunorubicin and drug resistance. TFEB activity appears to be regulated through its phosphorylation by mammalian target of rapamycin complex 1 (mTORC1). mTORC1 was shown to exert its kinase activity on lysosomal surfaces where it phosphorylates TFEB, thereby inactivating the transcription factor. TFEB can be dephosphorylated by calcineurin, which transforms it to its active form and facilitates its nuclear translocation. TFEB-mediated lysosomal biogenesis is induced by various stimuli, namely cell starvation, inhibition of mTORC1, and abnormal lysosomal storage. Interestingly, lysosomal stress also modulates lysosomal gene expression. A recent report showed that doxorubicin and mitoxantrone triggers TFEB-associated lysosomal biogenesis, thus further enhancing lysosomal sunitinib entrapment and MDR. A different study found that exposure of 786-O renal cancer cells and HT-29 colorectal cancer to various tyrosine kinase inhibitors increased the number of lysosomes. Taken together, these data lead credence to the notion that lysosomal drug sequestration induces lysosomal stress and TFEB-mediated lysosomal biogenesis. The number of lysosomes in cancerous cells may prove to be an important consideration when selecting treatment options for cancer patients. A possible solution may be to target lysosomal biogenesis by finding ways to circumvent the nuclear translocation of TFEB. Support for this comes from a study that found that interaction of TFEB with active rag heterodimers promoted recruitment of TFEB to lysosomes, leading to mTORC1-dependent phosphorylation and inhibition of TFEB.Lysosomal biogenesis is typically a response to cell stress and is regulated by the translocation of transcription factor EB (TFEB) from the cytosol to the nucleusAnother mechanism by which lysosomes participate in drug sequestration involves ATP-binding cassette transporters , such as doxorubicin, danorubicin, vinblastine, and imatinib become localized and trapped inside lysosomes,68. The pH difference between the cytosol and lysosomal lumen is the driving force for lysosomal drug sequestration and is regulated by proton-pumping vacuolar-ATPase proteins. This mechanism prevents such drugs from reaching their pharmacologic cytosolic concentrations and contribute to survival of the tumor cells. Lysosomal P-gp-mediated MDR can be overcome by using specific P-gp inhibitors or a combination of lysosomotropic agents with anticancer drugs. Support for this comes from work by Shiraishi et al. (1986) demonstrating that chloroquine partially reversed the resistance of multi-drug-resistant KB carcinoma cells to the P-gp substrates adriamycin, daunomycin, vincristine, vinblastine and actinomycin D. Furthermore, it has been shown that P-gp inhibitors valspodar and elacridar or silencing P-gp with siRNA reversed lysosomal sequestration of doxorubicin, leading to its redistribution to its intended target, the nucleus. A recent report demonstrated that cell-surface P-gp is degraded by the lysosomal pathway and suggests that this pathway could be exploited to induce cell death in P-gp expressing tumors. Lastly, lysosomal P-gp mediated resistance to sorafenib was reversed in hepatocellular carcinoma cells that were incubated with verapamil after drug pre-incubation. However, the majority of clinical trials using P-gp inhibitors to suppress drug resistance have failed to show improved survival or remission rates.P-gp is often a representative ABC-transporter, present in many malignant cells and a molecular target in cancer therapies. Indeed, the majority of the intracellular concentration of imatinib was found not in the cytosol, rather it was localized to lysosomes. An additional obstacle is the fact that ABCA3 mediated resistance is correlated with an increase in lysosomal-related organelles.Other transporter proteins have also been implicated in promoting lysosomal drug sequestration. The ABC transporter A3 (ABCA3) was shown to contribute to lysosomal sequestration of imatinib and to potentiate resistance to this drug2+-dependent process whereby lysosomes fuse with the plasma membrane and release their contents to the extracellular space. Interestingly, lysosomal exocytosis is also regulated by TFEB and overexpression of TFEB is correlated with increased exocytosis. It has been hypothesized that exocytosis of lysosomal sequestered drugs is another mechanism that contributes to reducing the concentration and efficacy of these drugs. Support for this comes from a study that found lysosomal exocytosis triggered in murine macrophages by treatment with agents that induced lysosomal alkalization. These data lead credence to the notion that drugs that are sequestered in lysosomes are not trapped there indefinitely and are extruded via lysosomal exocytosis. Furthermore, lysosomal exocytosis of cathepsins B, D, K, and L have been shown to promote cell motility, angiogenesis, and metastasis,77. Cathepsin D stimulates mitogen activated protein kinase signaling and angiogenic gene expression.Lysosomal exocytosis promotes MDR in malignant cells. Lysosomal exocytosis is a CaWe hypothesize that several mechanisms may reverse lysosomal drug sequestration. In the section below, we discuss putative strategies that might enable drugs that are trapped in lysosomes to translocate from lysosomal lumen to cytosol +-ATPase inhibitor. Bafilomycin A1, however, is too toxic for in vivo use and a more appropriate alkalinizing agent is chloroquine. One study administered chloroquine to mice and inhibited lysosomal function by raising the lysosomal pH. A different study found that chloroquine potentiates the cytotoxic effects of doxorubicin in liver carcinoma cells. Similarly, treatment of lysosomes in resistant cells with monensin, bafilomycin A1, or concanamycin A was sufficient to change the distribution of adriamycin to mimic that of drug-sensitive cells. Additionally, prevention of subcellular trapping of nintedanib by lysosomal alkalinization abolished drug resistance. These approaches appear plausible because pH gradient differences exist between MDR cancer cells and their wild-type drug sensitive counterpart cell lines. These findings suggest that the use of well-tolerated alkalinizing agents may circumvent lysosomal drug sequestration and thereby increase cytotoxic drug efficacy.Disrupting the acidification of lysosomes in multidrug-resistant cells has been shown to sensitize them to chemotherapeutics. A possible mechanism to reverse lysosomal drug accumulation of weak chemotherapeutic bases is by treatment with lysosome alkalinizing agents such as bafilomycin A1, a vesicular H. Chloroquine has been reported to promote cytotoxicity and to act synergistically with chemotherapeutic drugs. Chloroquine is a lysosomotropic agent that triggers destabilization of the lysosomal membrane in various tumor cells. In one study chloroquine was used to restore sensitivity to cisplatin in refractory non-small-cell lung cancer cells. In another report, chloroquine was shown to potentiate the cytotoxic effects of topotecan by inhibiting autophagy. The thiosemicarbazone, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), has been shown to accumulate in lysosomes of tumor cells where it induces LMP. Once in the lysosome, Dp44mT binds to copper forming a complex capable of producing cytotoxic reactive oxygen species (ROS) which triggers LMP.Another approach to abolish lysosomal drug sequestration is by using lipophilic drugs that become scavenged in lysosomes yet can induce LMP,86. Evidence for this comes from a report that conjugated doxorubicin to polyamidoamine dendrimers via hydrazone. This complex was shown to release doxorubicin to the nucleus and induce cell death. Additionally, degradable peptides, which are digested by lysosomal hydrolases, have been conjugated to cancer drugs and overcame lysosomal trapping.Conjugation of acid-labile chemicals to chemotherapeutic drugs has also been utilized to overcome lysosomal drug sequestration. Hydrazone is a commonly used linker molecule for this purpose because of its stability at cytosolic pH and its ability to hydrolyze at lysosomal pH. Another study combined sunitinib with phototherapy to combat lysosomal localization of the drug. However, this approach is of limited value because of the superficial and local treatment options of phototherapy.Lysosomal photodestruction of weakly basic chemotherapeutics that are also fluorochromes is another approach to reverse lysosomal sequestration. Photodestruction of imidazoacridinone-loaded lysosomes in MDR cancer cells resulted in cell lysis via formation of ROS. When tumor cells are exposed to cellular stress, they produce increased amounts of ROS resulting in upregulation of P-gp expression. As a result, P-gp actively pumps Dp44mT into lysosomes where it binds to copper, thereby forming ROS and triggering lysosomal membrane destabilization and apoptosis. Lysosomes in neoplastic cells presumably have higher concentrations of copper compared to normal cells due to their increased requirement for metals. The higher levels of copper are presumably needed by tumors for angiogenesis and metastasis,95. Moreover, P-gp inhibitors such as elacridar abolished LMP induced by Dp44mT. These findings suggest that Dp44mT only uses P-gp and not other ABC transporter proteins to overcome lysosomal drug sequestration. Taken together these results indicate that the design of novel metal-binding, P-gp substrate drugs like Dp44mT may be used to treat multidrug resistant tumors by targeting lysosomes.A recent study investigated the effects of glucose availability in cancer cells on reversing Pg-induced lysosomal drug entrapment. Seebacher and co-workers demonstrated that the anti-tumor agent and P-gp substrate Dp44mT induces LMP rather than lysosomal sequestration in response to glucose-induced stress. This finding suggests that DOX-NS was not recognized as a P-gp substrate. Another study added a monoclonal antibody 2C5, which recognizes various tumor cells via tumor cell surface-bound nucleosomes, to doxorubicin liposomes. The antibody-doxorubicin liposome complex significantly induced nuclear accumulation and cytotoxicity of doxorubicin in a doxorubicin-resistant colon cancer cell line.Nanomedicine is another mechanism that shows promising results to overcome P-gp mediated MDR. A study showed that doxorubicin-loaded nanospheres (DOX-NS) evaded MDR and delivered a high concentration of the drug to the nucleus and cytosol. Additionally, cancer cells display higher metabolic rates and turnover of iron-containing proteins that sensitize them to ROS-induced LMP. A novel imaging technique that can stably track lysosomes for at least 120 h, irrespective of pH changes in the organelle, is now available and can be used to monitor lysosomes in cancer cells. This new lysosomal tracing method is desirable over conventional acidotropic probes, which tend to dissipate in stressed lysosomes. The above characteristics of lysosomes in cancer cells should be fully exploited to trigger lysosomal-mediated cell death After long-term treatment with weakly basic anticancer drugs, lysosomal drug sequestration can occur. Lysosomes are attractive subcellular targets for creation of novel anticancer treatments for the following reasons. Tumor cells have larger, more active lysosomes, which make them more susceptible to lysosomal membrane degradation compared to lysosomes in non-neoplastic cells. The enlarged compartment allows for a significant amount of drug to be scavenged from sites of action. Evidence for this comes from studies that demonstrated that drugs that localize to lysosomes can reach intracellular concentrations that are markedly higher than drug concentrations in the surrounding medium,103. Similarly, lysosomal exocytosis of drugs reduces their intracellular concentrations and cytotoxic effects. There is a dire need to develop new strategies to overcome MDR in cancer treatment. Lysosomes, with acid hydrolases that can trigger the intrinsic apoptotic pathway and trigger caspase activation, serve as attractive targets for novel anticancer treatment modalities. Specifically, the lysosomes of tumor cells exhibit alterations that are not observed in normal cells: increased cathepsin activity, shifts in different endolysosomal populations, and modified lysosomal trafficking. Further studies are warranted to fully exploit the unique differences in cancer lysosomes compared to their normal cell counterparts to sensitize tumor cells to cell death. The results of such projects should provide more effective strategies to bypass lysosomal-mediated drug resistance.We have reviewed the important role played by lysosomes in MDR by three mechanisms: (1) lysosomal entrapment of weakly basic chemotherapeutics; (2) P-gp-mediated lysosomal sequestration; and (3) lysosomal exocytosis of anticancer drugs. Lysosomal potentiation of MDR is an issue that is compounded by the following factors. As described above, several of the weakly basic chemotherapeutic drugs are also P-gp substrates that are involved in P-gp-mediated lysosomal entrapment. Upregulated biogenesis of lysosomes in cancer cells leads to enlargement of the lysosomal compartment


Prostate cancer is the most common type of cancer and the second leading cause ofcancer death among men in the US. It preferentially metastasizes to bone, with up to 90%of men with metastatic disease having bone metastases. The presence and volume of bonemetastases are associated with worse prognosis and the potential for skeletalcomplications, such as pathologic fractures, spinal cord compression, or the need forsurgery or radiotherapy to bone. These skeletal-related events (SREs) collectivelyrepresent a clinically meaningful outcome that is often measured in clinical trials. Upto one-half of men with metastatic castration-resistant prostate cancer (mCRPC), theadvanced and often fatal stage of disease, experience SREs, which are associated withconsiderable morbidity, decreased survival, and increased health care utilization andcosts. Therefore, understanding strategies to optimize bone health and prevent skeletalcomplications remains a critical area of ongoing research and implementing them acomponent of high-quality prostate cancer care.1 In a subsequent phase 3 study of 1904 men,denosumab was superior to zoledronic acid, delaying the time to first SRE by 3.6 months.2 Zoledronic acid was more often associated with acute phasereactions and required monitoring of kidney function; denosumab conferred a higher riskof hypocalcemia. Rates of osteonecrosis of the jaw were comparably low. Internationalguidelines endorse the use of either agent for the treatment of men with mCRPC, althoughneither agent has independently been associated with an overall survival benefit in arandomized study, and some argue that the marginal benefit of denosumab must be weighedagainst its dramatically higher cost .4 Moreover, these pivotalstudies were conducted before contemporary drug approvals, including those ofabiraterone acetate, enzalutamide, radium-223, and cabazitaxel, each of which has beenassociated with extended survival and reduction in the risk of SREs. Thus, the benefitsof zoledronic acid and denosumab in combination with contemporary drugs for thetreatment of mCRPC remain incompletely understood.Over the past 20 years, 2 bone resorption inhibitors (BRIs) have emerged asalternatives for the prevention of skeletal complications among men with mCRPC:zoledronic acid (an intravenous bisphosphonate) and denosumab .These agents differ mechanistically, with zoledronic acid preferentially inhibitingosteoclast proliferation and denosumab inhibiting an important factor in osteoclastmaturation. In a placebo-controlled study of 643 men, zoledronic acid decreased the riskof SREs by 36% and delayed the time to first SRE by 167 days.5 report theresults of an international, multicenter, retrospective cohort study of men with mCRPCwho initiated abiraterone acetate with prednisone as first-line therapy with or withoutthe addition of a BRI (zoledronic acid or denosumab). Among 745 men with a medianfollow-up of almost 2 years, the authors found an overall survival benefit associatedwith the addition of a BRI (zoledronic acid or denosumab) to abiraterone with prednisonetherapy , a benefit that persisted withmultivariable adjustment and was most pronounced among men with high-volumedisease.5Francini et al7 Moreover, this studysupports the hypothesis that BRIs may be particularly important for men with high-volumemetastases,7 a hypothesis thatwarrants prospective testing in future studies.How should we interpret the significance of these findings? First, this studyhighlights the importance of bone-targeted therapy in current practice for men withmCRPC and bone metastases. Although data from a randomized study are lacking, multipleretrospective studies and post hoc analyses of phase 3 studies have suggested that theaddition of a BRI to contemporary therapies for men with mCRPC may prolong survival inaddition to preventing skeletal complications.5 exposes real-world practice patterns across 8 major hospital systemsin the US, Europe, and Canada. In a study population encompassing men with mCRPC andbone metastases for whom bone-targeted therapy is endorsed by international guidelines,more than 70% of participants were prescribed abiraterone acetate with prednisone aloneand thus did not receive an indicated BRI.5 This gap is particularly noteworthy given the study's mainfinding of improved overall survival with concomitant bone-targeted therapy andhighlights the need for implementation work  to bringevidence-based therapies to the patients who need them. Moreover, the authors found noassociation between the specific bone agent used (zoledronic acid vs denosumab) andoverall survival.5 Although thisfinding must be considered preliminary given the limitations of a retrospective study,it adds to data suggesting that these agents are comparably beneficial; thus, decisionsbetween them should focus on clinical factors, such as kidney function, patientpreference, and cost.Second, the study by Francini et alAs the authors admit, an important limitation of their study is itsunderrepresentation of patients from racial and ethnic minority groups, who oftenpresent with particularly aggressive prostate cancers and stand to benefit from thesetherapies. Although this limitation may, to some extent, reflect the geographiccatchments of the institutions involved in the study, underrepresentation of racial andethnic minority groups is endemic in cancer clinical trials, and the pivotal clinicaltrials leading to regulatory approvals of zoledronic acid and denosumab are noexception. A renewed focus on equitable participation in clinical research isneeded.5 should becommended for assembling a large international contemporary cohort of men with mCRPC toprovide insight into the important challenge of optimizing bone health in thispopulation. Perhaps what we learned most from this study is that most of the time, menare not receiving guideline-concordant bone-targeted care. Should they be? The answer isstill yes.Francini et al


Epigenetic alterations are one of the key hallmarks of aging. How epigenetic changes broadly affect euchromatin-heterochromatin balance, three-dimensional chromatin conformation, histone composition and global transcriptome in aging have not been addressed. Using murine liver as a model organ and an integrated multi-omics approach, we find that aged cells assume a repressed chromatin state characterized by condensed chromatin, broad heterochromatinization, global suppression of the transcriptome, and inability to self-interact over long distances. Our findings reveal that global chromatin alterations pose a barrier and promote a cellular state that is refractory to injury sensing and repair.


There has been a dramatic increase in clinical studies examining the relationship between disorders of gut-brain interactions and symptoms evoked by food ingestion in the upper and lower gastrointestinal tract, but study design is challenging to verify valid endpoints. Consequently, mechanistic studies demonstrating biological relevance, biomarkers and novel therapeutic targets are greatly needed. This review highlights emerging mechanisms related to nutrient sensing and tasting, maldigestion, physical effects with underlying visceral hypersensitivity, allergy and immune mechanisms, food–microbiota interactions and gut-brain signaling, with a focus on patients with functional dyspepsia and irritable bowel syndrome. Many patients suffering from disorders of gut-brain interactions exhibit these mechanism(s) but which ones and which specific properties may vary widely from patient to patient. Thus, in addition to identifying these mechanisms and the need for further studies, biomarkers and novel therapeutic targets are identified that could enable enriched patient groups to be studied in future clinical trials examining the role of food in the generation of gut and non-gut symptoms. A large proportion of patients suffering from disorders of gut-brain interactions (DGBIs) report that the ingestion of food triggers symptoms, but this relationship had received little attention for many years. However, this has changed dramatically in the past decade with a dramatic increase in published studies, particularly those examining functional dyspepsia (FD) and irritable bowel syndrome (IBS). Collectively, clinical studies overwhelmingly support that dietary factors can strongly influence DGBI symptoms, yet their interpretation is often complicated by the challenges of conducting dietary studies. These include patient and researcher bias, difficulties in blinding, placebo and nocebo responses, and low-quality study design. Moreover, an inability to identify patients where a diet is mechanistically implicated could lead to many patients enroled in studies that would ultimately not benefit and inclusion of these patients in the analysis could potentially dilute out a positive benefit of the dietary intervention. Thus, understanding mechanisms are critical to: (i) validating the results of clinical trials, (ii) identifying potential biomarkers to enrich treatment groups, and (iii) providing novel treatment therapies that might avoid the inconvenience and possible detrimental effects of restrictive diets. To address this need, we examine current mechanisms being explored, possible biomarkers and identifies knowledge gaps for future studies. The main focus is on FD and IBS as mechanistic studies have largely examined patients with these disorders to date.The gastrointestinal (GI) tract displays 3 types of sensory modalities: mechanosensitivity, chemosensitivity, and thermosensitivity—all of which can be activated by the ingestion of food and can contribute to gut-brain signals controlling food intake Figure  1). The. The1). In terms of nutrient chemosensing, the upper small bowel expresses a large number of specialized receptors which can detect all the main categories of nutrients. The presence of glucose, fatty acids, and amino acids is monitored through large numbers of specific receptors or transporters ,7,8. TheBesides nutrient sensors, the gut mucosa expresses transient receptor potential channels, which serve as receptors for a number of tastants. Examples are the TRPV1 receptor for capsaicin, the TRPM8 receptor for menthol and the TRPA1 receptor for cinnamaldehyde . The mucSeveral DGBIs are characterized by or associated with alterations in these sensory processes. Enhanced nutrient sensing of lipids has been reported in FD, where duodenal lipid exposure leads to enhanced release of CCK, altered gastric sensorimotor control and increased postprandial symptoms. These events are in part inhibited by a lipase inhibitor, orlistat, or by the CCK receptor antagonist dexloxiglumide –14. In aIn FD, increased sensitivity to capsaicin administration has been reported, suggesting hypersensitivity to TRPV1 receptor activation . In healMost of the available evidence suggests no alteration in digestion or absorption of nutrients in patients with DGBIs compared to the general population. However, well-known and widely available maldigestion/malabsorption syndromes may more easily lead to symptoms in subjects with DGBIs, presumably related to abnormal sensorimotor function in the GI tract and abnormal gut-brain interactions ,24.Lactose malabsorption is a well established and common entity that might lead to symptoms such as abdominal pain, bloating and diarrhea after lactose ingestion, i.e. lactose intolerance . The disThe involvement of fructose malabsorption in the generation of symptoms in patients with DGBIs has also received significant attention recently . FructosCongenital sucrase-isomaltase deficiency is an inherited deficiency in the ability to hydrolyze sucrose, maltose, short 1–4 linked glucose oligomers, branched (1–6 linked) α-limit dextrins, and starch. Exposure to these nutrients in affected patients provokes osmotic diarrhea with pain, bloating, and abdominal distention . In addiDGBI disorders express combinations of a number of pathophysiological alterations, including motility disturbances, visceral hypersensitivity, altered mucosal and immune function, altered gut microbiota, and altered central nervous system processing . These pIn vitro experiments demonstrated that this exaggerated response was suppressed by both a CCK antagonist and atropine, suggestive of disordered enteric nervous system function explaining this abnormality , or non-celiac gluten sensitivity, manifests with both GI and extra-intestinal symptoms after ingestion of wheat. Despite its high reported prevalence, which varies between 0.6% and 13%, the pathophysiology of NCWS is poorly understood . Apart fRecent emerging evidence indicates a role for food-induced mucosal alterations in the pathogenesis of upper GI alterations and symptoms. A number of studies used confocal laser endomicroscopy to demonstrate duodenal mucosal reactions to food allergens in patients reported to have IBS –72. In aThe landmark discovery that mast cells are activated in the colon and small bowel of IBS patients, that they are closely associated with intestinal nerves and their mediators, particularly histamine and proteases, and that their activation causes visceral hypersensitivity –86 has dSystemic food allergy had been implicated and while careful clinical testing ruled out this cause, clinical clues suggested that food could cause a local immune response in IBS patients. For example, injection of food allergens into the cecum of patients with food sensitivities, evoked a local weal and flare reaction despite low levels of serum IgE for those antigens  and studRecent novel studies provided direct evidence by demonstrating that common IBS triggers can break oral tolerance to food antigens ,89. SelfThe compelling evidence that lowering dietary FODMAPs in IBS patients significantly reduces abdominal pain and bloating has provided a strong rationale to examine underlying mechanisms . While cex vivo mouse colons and recording mechanosensitivity of afferent nerves. These studies show that IBS fecal supernatants signaling from the lumen to nerve terminals in the intestinal wall cause visceral hypersensitivity. This effect was blocked by histamine antagonists in some patients and proteases inhibitors in others. Importantly, following a low FODMAP diet in these patients this visceral hypersensitivity action was lost. Histamine has also been directly implicated in combined clinical and reverse translational studies where abdominal pain and urinary histamine levels decreased in a subset of patients after lowering intake of these carbohydrates , a gene that can be found in 2 different isoforms in both Gram- and Gram+ bacteria, but is far more efficient in Gram- Proteobacteria. Klebsiella aerogenes was identified as the main histamine producer in IBS-HH microbiota, producing >100× more histamine than any other bacterial isolate. K. aerogenes is higher in IBS-HH patients and its specific HDC gene is present in the stool in many but not all IBS patients. Together, these data strongly suggest that in a subgroup of IBS patients, specific microbiota are a driver of visceral hypersensitivity that is dependent on histamine signaling. Further studies are needed to identify specific bacteria or communities that produce other neuroactive mediators and how specific foods influence their production.The evidence that production of neuroactive mediators based on food-microbiota interactions may be individualized suggests that corresponding differences in microbiota may underlie these findings ,99–101. 2 signaling to nociceptive nerves , nutrient infusion deactivated the pain neuromatrix while activating the “default mode network” in the midbrain, which was associated with changes in the plasma levels of ghrelin and PYY Figure  104). T. T104). This review highlights the emerging evidence that specific mechanisms underlie food-induced symptoms in patients suffering from DGBI, and highlights that most of the work has focused on FD and IBS Figure . IncreasGuarantor of the article: Stephen Vanner, MD, MSc.Specific author contributions: All authors contributed equally to the writing of the manuscript and have approved the final submitted draft.Financial support: S.V. is supported by Canadian Institutes of Health Research (CIHR) and Crohn's and Colitis Canada (CCC) operating grants.Potential competing interests: None to report.


Congenital heart disease (CHD) is the most predominant birth defect and can require several invasive surgeries throughout childhood. The absence of materials with growth and remodelling potential is a limitation of currently used prosthetics in cardiovascular surgery, as well as their susceptibility to calcification. The field of tissue engineering has emerged as a regenerative medicine approach aiming to develop durable scaffolds possessing the ability to grow and remodel upon implantation into the defective hearts of babies and children with CHD. Though tissue engineering has produced several synthetic scaffolds, most of them failed to be successfully translated in this life-endangering clinical scenario, and currently, biological scaffolds are the most extensively used. This review aims to thoroughly summarise the existing biological scaffolds for the treatment of paediatric CHD, categorised as homografts and xenografts, and present the preclinical and clinical studies. Fixation as well as techniques of decellularisation will be reported, highlighting the importance of these approaches for the successful implantation of biological scaffolds that avoid prosthetic rejection. Additionally, cardiac scaffolds for paediatric CHD can be implanted as acellular prostheses, or recellularised before implantation, and cellularisation techniques will be extensively discussed. Congenital heart disease (CHD) is the most prevalent birth defect globally . AnnuallEarly diagnosis and advances in cardiac surgery and interventional cardiology have significantly increased the survival of paediatric patients with CHD over the past several decades ,7. This Despite these remarkable improvements, many interventions in CHD remain palliative, such as the Norwood procedure and overall Fontan palliation of single ventricle physiology, or coarctation of the aorta palliation ,10. A paSurgical replacement options for CHD patients include the use of valve substitutes, patches, and conduits. For instance, heart valve failure requires surgical replacement in 70% of cases . These oIn addition, CHD patients frequently need multiple open-heart replacements of failing valves and/or conduits, thus being exposed to the risks of additional surgical interventions ,17. Not The necessity to overcome such limitations of current clinically available prostheses for the treatment of paediatric CHD has prompted significant research into the development of novel bio-scaffolds. The search for readily available and biocompatible replacement parts endowed with growth and adaptive remodelling capacity, as well as durability over the patient’s lifetime, is ongoing in this field of paediatric research.A review of the technical and bioengineering aspects relating to the fabrication of biological scaffolds for application in paediatric CHD is not presently available, despite this being a rapidly evolving area of research. This review, therefore, aims to provide a comprehensive overview of the techniques used to produce biological scaffolds for paediatric patients with CHD, discussing future tissue engineering (TE) based approaches to treat one of the fastest-growing populations in cardiology .Since its beginnings in the early 1990s , TE has Scaffold choice is crucial in TE, being a platform onto which cells are deposited and guiding tissue formation. An optimal scaffold mimics both the structural and mechanical properties of the native tissue, as well as supporting cell viability and growth . ScaffolBiological scaffolds are ECM biomaterials that have been shown to facilitate the constructive remodelling of many different tissues in both preclinical animal studies and in human clinical applications ,27,28. TXenogeneic and allogeneic biological scaffolds have made major contributions to the field of cardiac TE. Tissue-engineered biological structures can be fabricated in vitro, where cells are cultured onto the biological scaffold by seeding, injection, or perfusion prior to surgery ,38. TheyAlthough many natural biomaterials have reached the clinical trial phase, including alginate and collagen, decellularised ECM (dECM) biomaterials have unique structural characteristics which distinguish them from other biomaterials. These properties are necessary for the regeneration, repair, and remodelling of the defective regions in the heart. There are solid and soluble dECM biomaterials. Solid scaffolds can be used in their native structural form or as patches consisting of dry or hydrated sheets. Soluble dECM biomaterials are used as injectable hydrogels  and theiPreclinical studies in this area are numerous and focus on identifying alternative biological scaffolds possessing traits eluding current surgical options for CHD, including availability, growth capacity, and longevity matching the patient’s lifespan. Clinical use of commercially available ECM biological scaffolds for paediatric CHD aims to exploit the optimal structural and mechanical properties to achieve growth and biocompatibility . In factProcessing of biological scaffold materials to mask or remove foreign antigens is an essential initial step of the fabrication process prior to any clinical application, including the treatment of paediatric CHD. Multiple immunogenic antigens exist on foreign tissue from allogeneic or xenogeneic sources. For example, N-glycolylneuraminic acid (Neu5Gc) is a sialic acid molecule present in most mammals, including cows and pigs, but is not found in humans . As suchArguably, the most important xenoantigen discussed in the literature is Galα1,3Galβ1,4GlcNAc-R . The α-Gal epitope is a major xenoantigen responsible for hyperacute organ rejection from α-Gal donors to humans and is a complex barrier to trans-species implantation ,47. ThisNext, we discuss the processing of biological scaffolds by chemical fixation or decellularisation to reduce antigenicity.In order to reduce their antigenicity, biological grafts can be treated chemically. The principle behind the fixation of tissue is not to remove the cellular component, but to kill the cells and stabilise the structures by chemical crosslinking of cellular proteins .Low concentrations of aldehyde fixatives are the most widely used, for example, glutaraldehyde (GA) . GA creaGA-fixed animal pericardium, usually of bovine or porcine origin, is currently used as an off-the-shelf commercially available cardiac valved xenograft source for the replacement of defective structures in CHD patients ,52. ThouAs an alternative approach to increase the biocompatibility of xenografts for use in CHD, detergent and/or enzyme treatments have been optimised with the aim to decellularise the structure and therefore reduce antigenicity ,68,69,70Decellularisation of biological tissues prior to transplant into the cardiac environment has the potential to surpass previously used fixation techniques, allowing for enhanced biocompatibility via complete removal of xenoantigens, the absence of GA fixative traces, ECM conservation, and subsequent in vitro or in vivo recellularisation ,71. ThisDecellularisation techniques for biological tissue can be broadly categorised into three groups: chemical, enzymatic, and physical, with the best results often achieved using a combination of approaches . At theiAchieving the perfect balance of full decellularisation while maintaining mechanical properties is challenging, demanding a multifaceted approach optimised to the specific material and clinical application. To date, three key criteria defined by Crapo et al.  are comm® decellularised porcine pulmonary valves is a cautionary tale against the application of incompletely decellularised xenografts in paediatric CHD patients [® processing is a patented decellularisation technology used on the tissue of both animal and human origin to produce acellular valve replacements with endogenous repopulation capacity [® decellularised porcine heart valve was introduced as the earliest tissue-engineered alternative to conventional xenograft valves in Europe. In 2002, four children received model 500 (n = 2) or model 700 (n = 2) SynerGraft® valves [® samples and only partial decellularisation of the porcine collagen matrix as shown in [An early report documenting the disastrous clinical results following the implant of SynerGraftpatients . SynerGrcapacity ,75. The ® valves . Sadly, shown in . This caAll methods described in the literature are imperfect. Alterations to ECM composition, integrity, and organisation is inescapable to some measure, but the extent of disruption to macromolecules, such as proteins, GAGs, and collagens, is dependent on numerous interlinking factors. Thus far, achieving 100% cell removal with absolute preservation of ECM ultrastructure composition and macromolecular properties has not been reported.Chemical decellularisation techniques include the use of surfactants, such as the detergents sodium dodecyl sulphate (SDS) ,64,65,66Once successfully fixed or decellularised, biological scaffolds for CHD can be used as acellular prostheses, or as a platform for cell seeding prior to implantation. We will review both approaches in specific CHD applications, discussing both acellular and recellularised biological scaffolds. Biological bioactive materials have structural properties that are necessary to support cell health, function, and tissue repair through the presence of a pool of growth factors, matricellular proteins, and complex ultrastructural compositions . Even afAcellular scaffolds could be used in their natural structure , patcheBiological substitutes are either homografts or of animal origin ,82. HomoDifferent sources of homografts include tissue obtained from the pericardium, decellularised cardiac tissues, AM, and ECM . CardiovA widely used autologous biological graft in CHD is represented by the pericardium, which is commonly used for surgical correction of the pulmonary valve. Despite the diffused application, the autologous pericardium has shown a tendency to degenerate due to endothelial stress caused by blood flow through the artery . Another® pulmonary allografts  and standard cryopreserved pulmonary allografts  used in right ventricular outflow tract (RVOT) reconstruction across three centres, the SynerGraft® group had significantly a higher absence of conduit dysfunction (83% vs. 58%), suggesting that the SynerGraft® decellularisation technology decreases allograft immunogenicity, increasing longevity of the conduit [To replace the pulmonary or aortic valve, decellularised allogeneic heart valve tissue has been used in CHD treatment. The durability of these acellular scaffolds is dependent on endogenous cell recruitment, including smooth muscle cells (SMCs) and fibroblasts, which can lay down ECM. In a retrospective 10-year comparison of SynerGraft conduit .dECM AM is a widely used biocompatible scaffold material for CHD as it has excellent biological properties that promote wound healing and tissue regeneration through mechanisms such as angiogenic, anti-inflammatory, antifibrotic, and anti-bacterial activity ,96. ThisA vascular graft produced from a sacrificial fibrin gel with encapsulated donor fibroblasts remodelled when placed in a bioreactor. Following this, the ECM was decellularised to create an off-the-shelf ECM product and implanted as a pulmonary artery replacement in three lambs . This prIn a transcatheter aortic valve replacement animal study, tissue-engineered heart valves (TEHV) were fabricated with the use of a stent and human-cell-derived ECM . ResultsA 10-year follow-up of fresh acellular decellularised pulmonary homografts for pulmonary valve replacements in 131 patients compared these with cryopreserved pulmonary homografts and bovine jugular vein conduits . Data shIn summary, results as to whether in vitro or in situ recellularisation is better are conflicting . When coHomograft represents the preferred option for CHD surgical replacement due to the more accurate matching of native tissue, and because they are considered more durable and less immunogenic than xenografts. Nevertheless, their availability is inadequate for clinical CHD scenarios, particularly in the context of small-valved conduits for neonates and infants. This represents a significant challenge in terms of use in paediatric CHD . Xenogra® valves (MP+V), urinary bladder matrix (UBM), Proxicor®, and dECM hydrogels.Xenografts of porcine origin, include Matrix P valves (MPV), Matrix P plusMPV and the MP+V, comprising decellularised porcine valves with a GA-fixed equine conduit component, are commercially available xenogeneic heart valves used in CHD treatment . An analA preclinical trial assessed non-GA fixed, SDS decellularised porcine TEHV for use in trans-species implantation, with the view to apply these in CHD treatment in the future . Here, SdECM UBM is a biological scaffold harvested from either human or, more frequently, porcine sources. Initially, it is created from the removal of the bladders from the body where it is then mechanically delaminated, and the detrusor muscle is segregated from the lamina propria and urothelium. The lamina propria is then decellularised and used as various end products such as patches, injectable particulate suspensions, and hydrogels . Remling®  is a commercially available decellularised porcine SIS scaffold [ProxiCorscaffold ,107,108.scaffold  since 20scaffold ,111, facscaffold , valve rscaffold , endocarscaffold  through scaffold . Despitescaffold .® scaffolds from infants with CHD [A SIS-dECM patch, used to close ventricular and atrial septal defects in 73 paediatric CHD patients (average age 22 weeks) , showed with CHD . Assessmwith CHD . A shortwith CHD .dECM in the form of an injectable hydrogel scaffold is an alternative approach for use of biological scaffolds in CHD. Wu et al. reported the fabrication of TEHV through the encapsulation of NIH 3T3 fibroblasts in injectable hydrogels derived from decellularised porcine mitral valve chordae, decellularised aortic valves, and decellularised mitral valve leaflets, remarking on their potential use in TEHV due to their ability to grow and remodel with the somatic growth of the patient . This pr® and CardioCel. The Contegra® xenograft is an integrated valved conduit composed of the bovine jugular vein and is currently the surgical standard for RVOT substitution in paediatric CHD patients [® has been widely adopted by surgeons due to its availability, size range, low reported calcification incidence, and comparatively low cost [® implantation in comparison to homografts or porcine xenografts [® recipients [Bovine sources of xenografts for use in CHD include Contegrapatients . Since ilow cost . Studiesnografts ,125,126.cipients . Supravacipients .Tissue-engineered bovine pericardium (CardioCel) was implanted over a period of 24 months in 135 patients  suffering from various congenital heart defects such as septal defects, repair of pulmonary arteries, intra-atrial/intraventricular baffles, repair of systemic arteries, valve repair, repair of systemic veins, and Fontan procedure . ResultsDespite the diffused application of xenografts for CHD that has led to many commercially available products, animal-derived products still have limitations. In fact, clinical data show that long-term durability of the GA-fixed graft is limited by severe dystrophic calcification due to the permanence of fixative remnants having potent cytotoxic effects . In addiAnother strategy to produce tissue-engineered biological scaffolds for paediatric CHD is an in vivo method that employs the innate inflammatory response as a natural bioreactor. A non-degradable mould is implanted subcutaneously, encapsulated with deposited ECM, including collagen, elastin, and proteoglycans, and then explanted . The proA Japanese group used this method to produce pulmonary valved conduits via in vivo encapsulation of silicone rods implanted in the dorsal subcutaneous space of beagles for 4 weeks . The depAs mentioned, despite fixed and decellularised xenografts having the advantage of accessibility, lack of growth potential, limited remodelling capacity, absence of repair, and poor longevity due to their acellular nature are well-known complications, and particularly prominent in the paediatric CHD population ,82,133. Homeostatic maintenance of the network of interwoven signalling pathways in a living structure requires not only a scaffold, but also cellular crosstalk. Therefore, to facilitate the recapitulation of native-like properties, the second component of a tissue-engineered product is cellularisation of the prepared scaffold framework. Without a cellular component, somatic growth capacity, repair, and adaption are impossible . The reqIn vitro seeding of the scaffold with autologous or allogeneic cells prior to implantation can facilitate the maturation of the cell-laden product. Biological scaffolds that have gone through the decellularisation process to avoid immunogenic rejection often require recellularisation to achieve the reconstruction of blood vessels, in which the presence of an endothelial lining in the lumen is crucial to avoid thrombogenic events. In other cases, recellularisation with autologous cells is needed to provide the capability to grow, remodel, and release new ECM proteins to improve mechanical properties. In vitro seeding can be achieved by manual cell deposition, injecting cells onto a scaffold, or 3D bioprinting .The use of autologous cells in a personalised medicine approach will support immunocompatibility . DesirabThe method of static  seeding is an inexpensive and simple technique for directly depositing cells onto a scaffold  Figure A. HistorAfter this precursor study, the approach was widely explored and resulted in many in vitro and in vivo studies. A porcine dECM was used in an in vitro proof-of-concept model of engineered heart tissue. Human-induced pluripotent stem cell-derived cardiomyocytes and primary cardiac fibroblasts were seeded on the decellularised matrix and exhibited significant electrical and functional characteristics . The engOther in vitro studies showed the use of biological grafts reinforced with synthetic material to improve the structural strength. Human umbilical artery-derived SMCs seeded on reinforced porcine blood-derived fibrinogen exhibited unique topographical and biochemical cues resulting in biologically inspired arrangement of ECM with an increased presence of elastic-fibre proteins . In an iZhao et al. reported the use of novel polyurethane/SIS patches seeded with hypoxia pre-treated urine-derived stem cells in comparison to commercial bovine pericardium patches in the reconstruction of RVOT in 40 rabbits . The steRecent studies described the use of progenitor cells for CHD applications, showing high performance in terms of adhesion, survival, and proliferation when seeded on decellularised porcine pulmonary artery  and cardOur group reported the use of decellularised swine SIS graft seeded with swine MSC-derived vascular SMCs for the replacement of pulmonary arteries in piglets . After 6In the attempt of exploring a novel alternative for CHD corrective surgery, freeze-dried AM-based scaffolds were tested as vascular grafts. When implanted in the left pulmonary artery of piglets, the cell-seeded multiple-layered AM-based graft showed promising results in terms of suitability and biocompatibility for vascular repair due to the development of newly formed endothelium in the intima, a SMC-rich medial layer, and an adventitia containing new vasa vasorum .® conduits to reconstruct the left pulmonary artery of 9-week-old piglets [Recently, a prosthetic graft composed of pericytes from neonatal swine was seeded onto Proxicor piglets . The in The manual seeding of MSCs derived from allogeneic Wharton’s jelly on SIS was compared to commercial unseeded SIS for the reconstruction of the main pulmonary artery in growing piglets . After 6An alternative model to the pig is the rodent model ,150. In The main drawbacks of this technology include inefficiency, impracticality compared to other technologies, and random deposition of cells resulting in a heterogenous distribution of cells across the tissue-engineered scaffold  Table 3Table 3.Cell injection is a simple technique which entails loading a cell suspension into a syringe and directly injecting it into a scaffold through a hollow needle, infiltrating deep into the scaffold  Figure B.Patel et al. reported the use of a two-stage cell seeding technique in addition to perfusion culture on a chitosan bioengineered open ventricle scaffold to fabricate a two-stage perfusion cultured ventricle to be potentially used in the treatment of hypoplastic left heart syndrome . This waThe drawbacks of this technique include inconsistency of cell suspension across the layers of the scaffold and exposure of cells to high shear rate and pressure during the injection process. Additionally, this slow and time-consuming process is only efficient for small-scale seeding  Table 3Table 3.The major challenge facing TE applied to paediatric CHD is the inability to fabricate functional constructs with full vascularisation and synchronous contractile activity. This limitation is due to the following factors; (i) partial tissue survival post-transplantation; (ii) generation of engineered tissue of clinically relevant sizes; (iii) optimising the mix of cardiac cell types; (iv) difficulty in sourcing cells from patients; (v) the immature phenotype of stem cell-derived cardiac cells; (vi) fate and safety concerns of potential undifferentiated stem cells; and (vii) immunogenicity which will require immunosuppression .In the last decade, 3D bioprinting emerged as a potential approach to overcome these limitations. In fact, bioprinted structures have the capability to play a key role in supporting cells by mimicking the mechanical characteristics of native ECM through overall patch geometry, size, survival, and function C. FurtheThe advantages of using the 3D bioprinting seeding technique include the automated approach for fabrication of the reproducible, complex 3D tissue microstructures that could biomimic the native physiological environment in various sizes and shapes. Furthermore, the deposition of multiple cell types, biomaterials, and biomolecules in precise locations in a layer-by-layer assembly with homogenous distribution of cells across the tissue constructs in a timely manner distinguishes this technology from the conventional fabrication methods . However3D bioprinting has been explored for the fabrication of heart valves cellularised with tissue-specific cells, such as aortic root sinus (ARS) SMCs, and aortic valvular interstitial cells (AV-VICs)  or MSCs MSCs encapsulated in GelMa/poly (eythelene glycol) diacrylate hydrogels supported by polycaprolactone were bioprinted to fabricate an anatomically accurate paediatric aortic heart valve with the ability to grow and remodel . AssessmAn in vitro validation was also performed on 3D bioprinted cardiac tissue grafts from human induced pluripotent stem cells (hiPSCs)-derived cardiomyocytes cell spheroids . BiomechBejleri et al. reported the use of a 3D bioprinted patch consisting of a hybrid bioink (cardiac dECM hydrogel and GelMA) loaded with paediatric human cardiac progenitor cells (hCPCs) in the potential repair of damaged myocardium associated with CHD . In vitrThe application of cardiac bioinks with optimal chemical-mechanical characteristics that best recapitulate the cardiac microenvironment has not yet been fulfilled . The devThe clinical translation of fabricated tissue-engineered scaffolds exhibiting growth potential and remodelling characteristics for reconstructive surgeries for paediatric CHD patients has been slow over the last few years ,166. TheWhen considering specific applications for paediatric CHD patients, the growth potential of genetically engineered scaffolds with somatic growth is a key consideration. Considering acellular or recellularised biological scaffolds, there are advantages and disadvantages associated with each . AcellulIn terms of scaffold biodegradability, degradation waste products should be body-friendly in addition to being used as an immunomodulatory tool that can trigger enhanced healing and integration with the host tissue . This isA better understanding of the process from implantation of the tissue-engineered scaffold to functional new tissue formation in the host could be achieved by advanced computational modelling and detailed animal studies. Through the inclusion of various input parameters, such as connections between different cell types, implanted scaffold factors, and cytokines, in computational modelling software, a better prediction of the combined effect of these inputs and the host environment on new tissue formation could be obtained. In an effort to improve clinical outcome, prediction of the effect of changes in scaffold parameters could be explored computationally in the short and long term . When coThere is little progress in terms of granting regulatory approval for clinically applicable tissue-engineered products, mainly due to a lack of consensus on classification and suitable surveillance programs. The classification used to define implanted materials and testing techniques falsely forecasts the efficacy of the tissue-engineered products. It is important for regulatory bodies to ease the process by standardising the pipeline and consequently aid the acquisition of approvals necessary to accelerate the safe, efficient use of tissue-engineered products in national and international markets ,174.It is imperative that the field of TE continues to grow and progress until palliative interventions evolve into curative treatments. In order for this to happen, technological advances and a deeper understanding of the mechanisms involved in the host environment are needed. This will allow the design and fabrication of scaffolds that recapitulate the native environment, with a particular focus on the safety and efficacy of those products in preclinical and clinical settings. Such scaffolds must possess growth potential and suitable haemodynamic characteristics, along with a resistance to thrombosis, calcification, inflammation, and stenosis. To overcome the several challenges associated with the clinical translation of tissue-engineered products from bench to bedside, experts from various fields, including clinicians, biomedical engineers, software engineers, and biologists, among others, must work collaboratively.


Older adults residing in long-term care (LTC) are especially vulnerable to the COVID-19 pandemic. Federal and local health officials have issued strict visitation guidelines, including family caregivers. Given that family caregivers are essential in the well-being for older adults in LTC, these measures have had an enormous impact. As little is known about the experiences of family caregivers, the purpose of this study was to explore how the COVID-19 pandemic impacted family caregivers’ roles, mental health, and adaptation. Semi-structured interviews (N=25) were conducted with family caregivers of older adults in LTC  via phone/Zoom. An interview guide led the question asking process and participants were asked open-ended questions about the impact of COVID-19 related protocols on their caregiving, mental health, and sources of social support. Interviews were transcribed verbatim and analyzed in Nvivo, guided by Grounded Theory methods. The majority of participants (76%) identified as a child of their care recipient. Findings highlight that most participants experienced numerous changes to their caregiving tasks, such as assisting with activities of daily living (ADLs), limited monitoring for their loved ones, and a reduction of social support provided to the care recipient. Family caregivers also reported other changes in their roles that resulted in increased stress and mental health concerns. These concerns were discussed in detail, including ways in which family caregivers adapted to their new roles and managed their stress. Findings from this study will inform interventions geared to better support family caregivers, particularly during times of crisis.


Modern PCR-based analytical techniques have reached sensitivity levels that allow for obtaining complete forensic DNA profiles from even tiny traces containing genomic DNA amounts as small as 125 pg. Yet these techniques have reached their limits when it comes to the analysis of traces such as fingerprints or single cells. One suggestion to overcome these limits has been the usage of whole genome amplification (WGA) methods. These methods aim at increasing the copy number of genomic DNA and by this means generate more template DNA for subsequent analyses. Their application in forensic contexts has so far remained mostly an academic exercise, and results have not shown significant improvements and even have raised additional analytical problems. Until very recently, based on these disappointments, the forensic application of WGA seems to have largely been abandoned. In the meantime, however, novel improved methods are pointing towards a perspective for WGA in specific forensic applications. This review article tries to summarize current knowledge about WGA in forensics and suggests the forensic analysis of single-donor bioparticles and of single cells as promising applications. This review focuses on the suitability of whole genome amplification (WGA) for forensic DNA profiling that uses current standard technologies. To be able to appreciate both the possible applications and the limitations of WGA in forensic DNA analysis, it will first be necessary to explain the basics of forensic DNA profiling from which the obvious fields of application of WGA will be motivated. Then, the technical principles of WGA will be described in conjunction with a critical discussion of published work applying WGA in forensics. By this means, the common deficiencies of WGA in a forensic context will be established, which finally will lead to the identification of fields of application where improved WGA methods may be promising.Forensic DNA profiles are based on the analysis of a standardized set of short tandem repeat (STR) loci that are highly polymorphic in the human population. In the European Union, 15 different loci are currently analyzed; in the North American CODIS system, this standard set is complemented by five additional loci . STR locTechnically, the set of forensic STR loci is analyzed using multiplex PCR, and the amplified fragments are sized using capillary electrophoresis (CE) ,6. One oIn modern forensic STR typing, commercial reagent kits are used that have been validated on the commonly used CE devices. Sizing standards and allele standards included in these kits allow for semi-automatic evaluation of electropherograms by software that assigns allele numbers to peaks for each locus. Of particular importance for this process of “allele calling” are threshold settings which preclude allele assignment for peaks that result from analytical noise or from typical technical artifacts .One important type of technical artifacts that typically occur in STR analysis are so-called stutter peaks, which result from the propensity of the repeat units to slip by one or more units during the elongation step of PCR amplification ,9. As a Modern commercial STR kits are highly sensitive and can establish full profiles from as little as 125 pg of genomic DNA ,12,13. , such asTo comply with stochastic effects, LCN DNA methods involving lower reaction volumes and more PCR cycles are applied in two or three replicates in order to identify those peaks as reliable that are reproduced by at least two assays . This coThe differences between WGA and simply increasing the cycle number of an ordinary PCR are not immediately obvious, and if DNA loci are missing right from the start, WGA will not be able to overcome resulting stochastic effects. However, WGA may reduce the risk of generating unspecific amplification products. Similarly to nested PCR protocols , the firMost DNA-containing traces have been exposed to the environment; thus, DNA integrity may be compromised by environmental influences, such as humidity, heat, acidic or oxidizing conditions, UV exposure, or enzymatic degradation (reviewed in ). TypicaAs DNA degradation will result in a lower number of copies of STR loci that can be amplified, one strategy might consist in the preamplification of genomic DNA by WGA in order to increase the number of the few copies that are still intact. To these ends, the suitability of WGA methods for environmentally exposed DNA traces needs to be evaluatedWGA methods can be classified as PCR-based or as based on multiple displacement amplification (MDA) . In addiPCR-based principles use either mixtures of primers with randomized sequences that can bind to many DNA loci, and by this means in theory will amplify all parts of the genome A 26,27],2726,27]MDA is based on the high-fidelity DNA polymerase phi29 that exhibits high processivity (synthesizing continuous DNA of up to 20 kb) and has strand displacement activity . MDA-basDegenerate oligonucleotide primed-PCR (DOP-PCR) was among the first WGA protocols developed . It is aDifferently from DOP-PCR, primer-extension-preamplification PCR (PEP PCR) uses primers of 15 nucleotides that are completely degenerate, and amplification starts at low-stringency annealing temperatures which are continuously raised in the subsequent PCR cycles A 27]. T. T27]. TAdaptor ligation-mediated PCR methods B differ Most MDA methods rely on phi29 polymerase in conjunction with random primers. Related methods use a primase instead of random primers that is coupled to a DNA polymerase with strand displacement activity ,41. GeneAll WGA methods tend to display some bias in terms of amplification of genomic DNA loci (reviewed in ); thus, Generally, PCR-based methods can better deal with low quality DNA (damaged or fragmented), because unlike MDA, PCR does not rely on long, undisrupted templates; however, the rolling circle MDA variants are suitable for fragmented DNA templates too. Like normal PCR amplification, the PCR-based protocols are prone to stutter artifacts when analyzing STR loci , whereasThe adaptor ligation-mediated PCR methods exhibit a fundamental problem if applied to only few copies of template DNA. Unlike the random primers used in DOP-PCR, PEP PCR, or MDA, which allow for initiating DNA synthesis multiple times from various locations without harming the template, the fragmentation of template DNA is irreversible, and thus any STR amplicon disintegrated during fragmentation (or located within a fragment too long for successful PCR amplification) will inevitably be underrepresented later on. This effect will be particularly apparent in the analysis of single cells where each STR allele is present only once.Already early in their development, the potential of WGA methods for forensic DNA analysis has been recognized ,33, and Several later studies have then tested the various available WGA methods for their potential to improve the STR typing success of problematic DNA samples using contemporary commercial kits for forensic STR analysis. These studies are generally difficult to compare, because they analyze DNA from different sources and of different amounts, and often evaluate STR typing success and sensitivities in different ways. Moreover, the contemporary STR typing kits used differ in their sensitivities, which sometimes do not reach the limits of the STR kits nowadays in use. In this section, the most significant findings will be summarized, paying attention in particular to sensitivity of the methods, technical artifacts, and success in typing degraded DNA samples.Two studies reported sensitivities down to to 10 pg input DNA for iPEP, GenomiPhi MDA, and the commercial adaptor ligation method GenomePlex ,47; howeOn the other hand, adaptor ligation PCR seems best suited for analyzing degraded DNA samples, as shown in two studies comparing STR typing success after the application of PEP PCR, DOP-PCR, adaptor ligation PCR, two MDA protocols, and the rolling circle MDA methods, to DNA extracted from heat-treated human muscle samples ,50. WithTo summarize these studies, WGA methods when applied to forensic STR typing, while generally increasing the analytical sensitivity, impose several novel problems: Profiles often display pronounced imbalances and ADOs that affect STR loci and alleles in a non-predictable fashion and are related to WGA-inherent bias. In addition, particularly with the PCR-based methods, high rates of stutters and ADIs are seen. These phenomena render STR profiles from unknown donors hard to interpret and lower the statistical power of the evidence. For example, in the case of an apparently homozygous locus (showing just one peak on the electropherogram), it cannot be decided whether a second allele is actually missing. ADIs or high stutters cannot be distinguished from normal alleles, and may thus mislead the interpretation as well. Furthermore, pronounced intra- or inter-locus peak height imbalances hamper the interpretation of mixed profiles, because peak heights no longer reflect the true amount of template DNA. Thus, although additional genotype information from LT DNA or damaged DNA can be obtained by WGA, the generated artifacts may mislead the interpretation of STR profiles, which strongly argues against the use of WGA in forensic casework.In light of the inability of WGA to significantly improve the STR typing of the typical forensic DNA samples, Barber and Foran (in a study comparing MDA and I-PEP PCR) in 2006 concluded that, “WGA appears to be of limited forensic utility unless the samples are of a very high quality” —which, hIn the meantime, however, novel WGA protocols have been developed, aiming in particular at sensitivities on the single-cell level, and at the same time reducing amplification bias; and several WGA kits have been commercialized that have been optimized for single-cell analysis, particularly for usage in clinical settings. These novel methods and kits have again sparked interest in the application of WGA in forensic DNA analysis, but so far only few of them have been tested in a forensic context.The ADOs, LDOs, and pronounced AIs reported after WGA-based preamplification were observed at DNA amounts well above the stochastic threshold, and thus cannot be fully explained by stochastic sampling effects. Rather, they point towards amplification bias, which is typical of WGA applied to low template DNA concentrations, as random events during the initial amplification become exacerbated due to the exponential nature of the amplification process .Bst polymerase during a first quasi-linear preamplification step, preceding the subsequent PCR amplification [Several WGA protocols have been established that aim at reducing bias by including non-exponential amplification steps. Among those low-bias methods are the multiple annealing and looping based amplification cycles (MALBAC) method, which uses the fication , and thefication . The LIAfication . Recentlfication .Bst polymerase that was reported to be less sensitive and more prone to stutters than phi29 polymerase when amplifying STR loci [Two studies have been published that tested MALBAC in conjunction with forensic STR typing kits, both analyzing DNA extracted from human peripheral blood ,60. EvenSTR loci .High proportions of ADOs and ADIs were also reported for the methodically related PicoPLEX kit when applied to single cells . Thus, aWith the high sensitivity of modern STR typing kits, the occurrence of mixed DNA profiles derived from more than one individual has increased, because now minute DNA amounts can be detected that have been left by other individuals who may not even be related to the actual crimes . STR proThere are various ways to deconvolve the peaks on electropherograms of mixed STR profiles , and modern software-assisted methods have increased the statistical power of the confounded information . To be aAs a way of avoiding mixtures right from the start, methods have been suggested that physically deconvolve mixed trace material by isolating bioparticles  that contain the genomic DNA of exactly one donor individual ,68. The The feasibility of micromanipulating and genotyping single cells from forensic trace material, such as chewing gums, cigarette butts, swabs, touched skin, and fabrics, has been demonstrated in several studies ,71,72,73In the studies by Li et al. ,72, buccThus, if in a forensic context, single-cell WGA methods were able to deliver on their promise, i.e., to enable the genotyping of single cells, their application might lead to a further improvement by increasing the template DNA of one- or two-cell subsamples to amounts that can more reliably be analyzed with modern forensic STR kits, even allowing for replicate analysis of the subsamples.The principal suitability of single-cell WGA methods for forensic STR analysis of single cells was tested in several recent studies. Analyzing single cells has the advantage of stochastic sampling effects being less likely, because from whole cells, complete diploid genomes can be extracted and then be subjected to WGA. In modern single-cell WGA kits, this is accomplished by carrying out cell lysis, DNA extraction, and WGA in the same tube.In a study from 2018, the low-bias single-cell method PicoPLEX was compared to a commercial single-cell DOP-PCR kit (DOPlify), a single-cell MDA kit (Repli-g), and an adaptor ligation method (Ampli 1) . In thatThe Repli-g single-cell MDA kit was tested in two further studies for its suitability for forensic STR typing. The study by Maruyama et al. (2020) reported that at least 20 micromanipulated buccal cells were required for successful STR-typing, whereas from single cells, most alleles remained undetected . AnotherA recently emerging, potential forensic application of WGA is in the STR analysis of micromanipulated single sperm cells. In rape cases, the DNA from vaginal swabs will in most cases be derived from both sperm cells from the perpetrator and vaginal cells from the victim, and differential extraction protocols aiming at separating the sperm fraction from the victim DNA fraction often remain unsatisfactory . The anaStudies addressing single sperm cell analysis using conventional forensic STR analysis, however, reported that at least 20 sperm cells are required to establish complete STR profiles ,81,82. SThe successful STR analysis of single micromanipulated sperm cells after application of the Repli-g single-cell MDA kit has been demonstrated in a recent study in which individual sperm cells were isolated using an adhesive-coated tungsten needle tip . ConsensDespite the shortcomings of classical WGA methods in forensic STR analysis, the latest single-cell WGA methods have opened up a new perspective for WGA in mixture deconvolution based on the analysis of single cells or small bioparticles. Methodologically, the successful application of WGA in the analysis of single sperm cells has already been demonstrated and now awaits further validation using forensic real-world samples. Applications of single-cell WGA to other bioparticles or to single cells (or 2-cell subsamples) micromanipulated from forensic traces still need to be tested. Furthermore, stimulated by biomedical interests , sev