Molecular imaging is an emerging technology that enables the noninvasive visualization, characterization, and quantification of molecular events within living subjects. Positron emission tomography (PET) is a clinically available molecular imaging tool with significant potential to study pathogenesis of infections in humans. in situ measurements of intralesional antimicrobial pharmacokinetics in multiple compartments, including privileged sites. Finally, the potential clinical applications of this promising technology are also discussed.Molecular imaging is an emerging technology that enables the noninvasive visualization, characterization, and quantification of molecular events within living subjects. Positron emission tomography (PET) is a clinically available molecular imaging tool with significant potential to study pathogenesis of infections in humans. PET enables dynamic assessment of infectious processes within the same subject with high temporal and spatial resolution and obviates the need for invasive tissue sampling, which is difficult in patients and generally limited to a single time point, even in animal models. This review presents current state-of-the-art concepts on the application of molecular imaging for infectious diseases and details how PET imaging can facilitate novel insights into infectious processes, ongoing development of pathogen-specific imaging, and simultaneous  Conversely, nuclear-medicine-based molecular imaging utilizes “tracers” or “probes” labeled with high-energy emission radionuclides, which can be used to target specific molecular pathways deep inside the body , a glucose analog that is selectively taken up by cells with a high rate of glucose metabolism, is a valuable clinical tool for predicting tumor response to treatment and patient survival . Therefore, target-specific PET probes for cancer are being developed to allow for a more specific diagnosis , a PET tracer used to detect hypoxic lung lesions in a mouse model of TB and confirmed using postmortem analyses. While no tracer accumulation was noted in nonhypoxic TB lesions in acutely infected or in control mice without lesions, a progressive, time-dependent tracer accumulation was noted in chronically infected mice, which are considered hypoxic. These findings were subsequently confirmed in TB patients using 18F-fluoromisonidazole (18F-FMISO) PET imaging  was introduced under the control of a strong mycobacterial promoter. TK phosphorylates 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)-5-125I-iodouracil (125I-FIAU), a nucleoside analog, leading to trapping and accumulation of 125I-FIAU in the M. tuberculosis Phsp60 TK strain. Thus, bacteria were specifically and noninvasively detected in experimentally infected animals demonstrating heterogeneous bacterial burdens in visible TB lesions  predicted reactivation TB with high accuracy  identifying reservoirs of the virus even in monkeys controlling the infection  and thus are a risk factor for transmission , or antin silico screen of 961 radiolabeled small molecules followed by bacterial uptake assays of promising candidates and identified several potential bacterium-specific imaging tracers such as para-aminobenzoic acid (PABA), which accumulated in all bacterial species tested, d-mannitol, which accumulated selectively in Gram-negative and Gram-positive bacteria but not mycobacteria, and d-sorbitol, which accumulated selectively in Gram-negative Enterobacteriaceae  which is the largest family of bacterial pathogens in humans. Together, these afford a set of tools for the differential imaging of bacteria in vivo.Mainstay clinical microbiology uses differential bacterial metabolism, such as selective growth media, to differentiate bacteria in the microbiology laboratory . Therefo18F-fluoromaltotriose , which can be easily synthesized from 18F-FDG, demonstrated that the tracer accumulated ∼1,000-fold more in Enterobacteriaceae than in mammalian cells. Moreover, 18F-FDS PET was able to specifically detect and differentiate live Enterobacteriaceae (infected lesion) from sterile inflammation (heat-killed bacteria) in a murine thigh myositis model as well as other animal models of infection , which target the bacterial folate pathway  . Therefo18F-fluoro-3-hydroxy-2-propoxy)methyl]guanine (18F-FHPG), a derivative of ganciclovir, in which one hydroxyl group is replaced with a radioactive fluorine atom (18F-FHPG is selectively phosphorylated by the herpes simplex virus (HSV) TK and becomes trapped within infected cells. This method was utilized to visualize HSV-affected brain regions in a rat model of encephalitis (64Cu-labeled simian immunodeficiency virus (SIV) Gp120-specific antibody has also been utilized to study the viral dynamics and localization of SIV in viremic and antiretroviral therapy-treated macaques using PET  also hold potential to provide a more specific tool for diagnosis , was associated with subsequent development of HIV-related immune reconstitution inflammatory syndrome (IRIS) after initiation of ART (PET is becoming a routine clinical tool and is proliferating in the United States and abroad, although its application to infectious disease is mostly limited to and cure . This inormation . Similarn of ART .124I-DPA-713, a novel tracer for macrophage-associated inflammation, has recently been translated to the clinic. 124I-DPA-713 PET studies in healthy volunteers demonstrate that 124I-DPA-713 clears rapidly from the lungs, with predominantly hepatic elimination, and is safe and well tolerated in healthy adults (11C-rifampin PET to study antimicrobial distribution and optimize treatments for TB meningitis (ex vivo tools.However, there is need for the development and clinical translation of more specific imaging tracers for infections. For example, y adults . Human sningitis  and pulmningitis  have alsningitis , 85 and


Wounds within the oral mucosa are known to heal more rapidly than skin wounds. Recent studies suggest that differences in the microRNAome profiles may underlie the exceptional healing that occurs in oral mucosa. Here, we test whether skin wound-healing can be accelerating by increasing the levels of oral mucosa-specific microRNAs. A panel of 57 differentially expressed high expresser microRNAs were identified based on our previously published miR-seq dataset of paired skin and oral mucosal wound-healing [Sci. Rep. (2019) 9:7160]. These microRNAs were further grouped into 5 clusters based on their expression patterns, and their differential expression was confirmed by TaqMan-based quantification of LCM-captured epithelial cells from the wound edges. Of these 5 clusters, Cluster IV  is most intriguing due to its tissue-specific expression pattern and temporal changes during wound-healing. The in vitro functional assays show that ectopic transfection of miR-31 consistently enhanced keratinocyte proliferation and migration. In vivo, miR-31 mimic treatment led to a statistically significant acceleration of wound closure. Our results demonstrate that wound-healing can be enhanced in skin through the overexpression of microRNAs that are highly expressed in the privileged healing response of the oral mucosa. Wound-healing in adult tissues is a complex event that generally ends with a scar. The elimination of scar formation remains an elusive goal in the wound repair field. One tissue that exhibits rapid wound-healing with reduced scar formation is the oral mucosa. Studies in at least 3 different models  support that enhanced wound closure is a shared feature of oral mucosal tissue across species ,2,3,4. ITo date, the available evidence clearly reveals the complexity of the highly orchestrated wound-healing process at the genomic and epigenomic levels. Our previous studies provided the dynamic time course-based profiles of both the transcriptome and microRNAome on a paired oral mucosa and skin wound-healing model, and showed that site-specific injury responses exist at each site ,10. Studp < 0.01) highly expressed microRNAs [number of mapped sequence reads greater than the average (5458) of the dataset] (p = 0.001), with these fractions remaining relatively constant during the time course of the wound-healing . In contrast, Cluster II and V do not contain any tissue-specific microRNAs, while Cluster III contains 16.7% tissue-specific microRNAs. To confirm the tissue specificity, an independent microRNA profiling analysis was performed on baseline unwounded skin and oral mucosal epithelium using a mouse microRNA microarray; this analysis identified 85 tissue-specific differentially expressed microRNAs (p = 0.003). Thus, of the 5 clusters, Cluster IV contains a group of tissue-specific and wound-healing associated microRNAs and was therefore subjected to additional validation studies. To assess the tissue specificity, the microRNAs from these 5 hierarchical clusters were matched with our previously reported panel of 53 tissue-specific differentially expressed microRNAs . As showicroRNAs . When coicroRNAs . The enrp-value < 0.05) and between time course series .Cluster IV consists of 8 microRNAs . Examination of the general expression pattern of this 8-microRNA cluster (represented as the average of the mapped sequence reads of the microRNAs in the cluster) showed that these microRNAs in this cluster generally exhibit relatively high expression levels in oral mucosa as compared to skin A. While TaqMan assay-based validation with LCM-captured cells was also performed on selected microRNAs from Cluster I , Cluster II , Cluster III  and Cluster V  . The resWe tested the functional effects of several differentially expressed microRNAs from each cluster  on wound-healing using in vitro proliferation assays and in vitro migration assay on skin (HaCaT) and oral epithelial (TIGK) cell lines. As showed in We then focused on evaluating the effects of miR-31 on wound closure using both in vitro and in vivo wound-healing assays. As showed in We further evaluated the effects of miR-31 in a mouse skin wound-healing model, where an animal origin-free lipid nanoparticle in vivo delivery system was used for introducing the miR-31 mimic to the wounds. As shown in Wound-healing is a complex process that is mediated by a highly diverse group of factors, including genomic and epigenomic regulators. Skin and mucosal wound-healing proceed through the same stages , yet wounds of the oral mucosa heal more quickly and with less scar formation. We recently reported the first dynamic microRNAome profiles of paired skin and oral mucosal wound-healing . TogetheBy focusing on highly expressed microRNAs, our current study defined a panel of differentially expressed microRNAs that are associated with skin and oral mucosal wound-healing. Based on their expression pattern, a cluster of 8 microRNAs was further identified which exhibit tissue specificity. This specific expression pattern was validated by both independent microRNA profiling on additional wound samples and TaqMan-based quantification in LCM-procured epithelial cells from the wound edges. Among these 8 microRNAs , miR-31 and miR-34c have previously been implicated in skin wound-healing ,17,18. mAmong the microRNAs we tested, the effect of miR-31 on wound closure was demonstrated consistently in both in vitro assays and in vivo wound-healing models. MiR-31 is a well-known tissue-specific regulator in cancer  e.g., t. By usinThe effectiveness of microRNA-based application in wound-healing treatment is currently under intense investigation. The microRNA-based therapeutic approaches are thought to have specific advantages over traditional methods. Notably, microRNA treatment can modify several pathways at one time, a feature that is attractive due to the complexity of wound pathology. With proper optimization, this approach may eventually develop into an advanced and relatively inexpensive therapeutic strategy. The novelty of the current study is in our attempt to harness regenerative healing capability from mucosal wounds and to “geno-copy” it into skin wounds. Future studies with impaired wound-healing models  will be essential to fully explore the translational value of our findings.Female Balb/c mice  were housed in a temperature-controlled environment (22–24 °C) on a 12 h/12 h light-dark cycle, and were provided with food and water ad libitum. For wound-healing studies in skin and oral mucosa, the previously established mouse excisional skin and oral mucosal wound-healing models were adapted with minor modifications ,10. In bMouse skin wounds were treated with microRNA mimics as described previously . In brieTotal RNA  was isolation from matched mouse skin and oral mucosal samples using a miRNeasy Mini Kit . Pooled skin and oral mucosal samples from three animals were subjected to microRNA profiling using the MRA-1002 miRmouse v21 microarrays  as described previously , and thet-test was used for between group comparisons, one-way ANOVA was used for time course data analysis, and two-way ANOVA was used for between time course series comparison. The Benjamini-Hochberg adjusted p-values were computed for multiple hypothesis testing. High expresser microRNA was defined as having the number of seq reads greater than the average of the dataset (5458) at any time point of the wound-healing time course. The first panel of tissue-specific differentially expressed microRNAs was based on this existing miR-seq dataset, where between group comparison made between unwounded skin and oral mucosal epithelium. This tissue-specific microRNA panel was reported in our recent publication [The previously published miR-seq dataset on paired skin and oral mucosal wound-healing time course (GEO accession number: GSE121996) was processed, mapped, and analyzed using a proprietary pipeline script, ACGT101-miR v4.2g  as described previously [https://software.broadinstitute.org/morpheus). MicroRNA targeted biological pathways were predicted by DIANA-mirPath (v3.0) [For validation, a second panel of tissue-specific differentially expressed microRNAs was generated based on an independent microRNA profiling analysis on unwounded skin and oral mucosal epithelium using a microRNA microarray. For microarray data analysis, normalization and differential analysis were performed with CyberT , using Vh (v3.0) , and theh (v3.0) ,48 was uEpithelial cells from the wound edges were captured with a LMD7000 Laser Microdissection system  as we previously described ,50.-delta delta Ct method [TaqMan MicroRNA Assays  were used for the real time PCR-based quantification of microRNA. Relative expression levels of the microRNA were computed with the 2t method , where sHuman keratinocytes HaCaT  and TIGK  were grown as previously described ,52. For


Extensive desmoplasia is a prominent feature of the pancreatic ductal adenocarcinoma (PDAC) microenvironment. Initially, studies demonstrated that desmoplasia promotes proliferation, invasion and chemoresistance in PDAC cells. While these findings suggested the therapeutic potential of targeting desmoplasia in PDAC, more recent studies utilizing genetically-engineered mouse models of PDAC, which lack key components of desmoplasia, demonstrated accelerated progression of PDAC. This contrast calls into question the paradigm that desmoplasia unilaterally promotes PDAC progression and the premise of desmoplasia-targeted therapy. This review briefly examines the major reports of the tumor-promoting and -restraining roles of desmoplasia in PDAC with commentary on the gaps in our current understanding of desmoplasia in PDAC. Additionally, we discuss the studies demonstrating the heterogeneous and multifaceted nature of desmoplasia in PDAC and advocate for future areas of research to thoroughly address the various facets of desmoplasia in PDAC, reconcile seemingly contradictory reports of the role of desmoplasia in PDAC progression, and discover aspects of desmoplasia that are therapeutically actionable. Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States . The disDesmoplasia consisting largely of dense extracellular matrix (ECM) and fibroblasts is a prominent feature of the TME in PDAC. Initially, the role of desmoplasia in PDAC was overlooked. However, later studies showed that during the development of pancreatic cancer, cancer cells expend a great deal of energy to promote the recruitment, proliferation and activation of fibroblasts. Subsequently, activated fibroblast deposit extracellular matrix and secrete numerous factors that profoundly affect the behavior of cancer cells. Indeed, the preponderance of data from early studies demonstrated the tumor-promoting role of desmoplasia by driving tumor cell proliferation, promoting invasive properties and suppressing anti-tumor immune response. Furthermore, pharmacologic inhibition of desmoplasia in combination with chemotherapy slowed PDAC progression to a greater extent than PDAC alone, thereby highlighting desmoplasia as a potential therapeutic target in PDAC -6. HowevThis review analyzes the major studies investigating the significance of desmoplasia in PDAC. We accentuate both the contribution of each study to the understanding of desmoplasia as well as their limitations, which warrant caution in interpretation and generalization of the impact of desmoplasia in PDAC. From these analyses, we hope to gain insight into the mechanistic role of desmoplasia in PDAC, investigate the emerging concept of fibroblast heterogeneity in PDAC and comment on the future of desmoplasia-targeted therapies in PDAC.in vitro, myofibroblast-conditioned media increased PDAC cell proliferation, migration, invasion, and decreased apoptosis. Additionally, CAFs increased expression of cancer stem cell and epithelial-mesenchymal transition markers in PDAC cells, further supporting the role of CAFs in metastasis and therapy resistance [Cancer-associated fibroblasts (CAFs) are a major constituent of the PDAC desmoplastic reaction and are responsible for most of the ECM deposition. Among various factors, PDAC cells secrete Transforming growth factor beta-1 (TGF-β1), Fibroblast growth factor-2 (FGF2), sonic hedgehog (SHH) and platelet-derived growth factor (PDGF) that drive fibroblast proliferation and ECM deposition, which in turn, profoundly affect the cancer cell behavior -10. Subcsistance , 13.in vitro. Further analysis demonstrated that TSLP is derived from CAFs; correspondingly, TSLP receptor-positive DCs are found only in the tumor and tumor-draining lymph nodes [ex vivo through STAT3 activation [CAFs have also been shown to promote immunosuppression in PDAC. CAF-derived thymic stromal lymphopoietin (TSLP) modulated dendritic cell (DC) cytokine profiles to favor Th2 polarization ph nodes . Depletiph nodes . Additiotivation .LSL-G12D; TGFBR2fl/fl; Ptf1a-Cre (KTC) mice enhanced STAT3-mediated signaling in PDAC cells and accelerated disease progression compared to KrasLSL-G12D; TP53R172H; Pdx-1cre (KPC) mice [ECM deposition is another prominent feature of desmoplasia that is thought to promote PDAC progression. For instance, collagen I potentiates proliferation and chemoresistance of PDAC cells and limits T-cell accumulation near cancer cells , 18. FurPC) mice . IncreasPC) mice , 21 and PC) mice , 23, andPC) mice . Cumulatfl/fl;KrasLSL-G12D/+;P53fl/+;Rosa26LSL-YFP/+) mice to deplete SHH expression in pancreatic epithelium. While SKPC pancreata lacked SHH, Indian Hedgehog (IHH) expression was increased. Despite this compensation, expression of Gli-1 in F4/80+ cells and pancreatic tumor decreased more than ten-fold indicating decreased HH signaling [SHH overexpression in PDAC cells promotes desmoplasia through paracrine signaling , 25, 26.ignaling . SKPC tuignaling . Unexpecignaling .fl/fl; Ptf1a-Cre; KrasLSL−G12D) and SKPC  murine PDAC models [LSLG12D; Pdx-1cre (KC) animals. Further, SKC mice demonstrated persistence of PanIN lesions following cerulein-induced injury to pancreatic parenchyma. Consistent with Rhim et al., SKPC mice showed reduced survival and increased vascular density compared to KPC mice. Pharmacological modulation of SHH signaling with cerulein-accelerated carcinogenesis demonstrated an inverse relationship between hedgehog signaling and presence of PDX-1-positive EPCAM-positive progenitor cells suggesting that SHH signaling may constrain the expansion of pancreatic progenitor cells following inflammation [Similarly, Lee et al. generated SKC  [−/−, BOC−/−) increased angiogenesis through upregulation of fibroblast-derived angiopoietin-1 and 2 and augmented tumor growth. However, complete loss of SHH signaling in fibroblasts  abrogated angiogenesis and tumor growth. Interestingly, suppression of SHH signaling in fibroblasts did not decrease desmoplasia or alter PDAC cell phenotype, which is consistent with findings from biopsies of a subset of patients treated with smoothened inhibitor, GDC-0449 [per se may mediate aggressive disease course in SKPC mice [−/−, BOC−/− tumors were larger compared to respective controls. Regardless, these findings are, to some extent, mirrored in an Ela-myc model of PDAC, in which deletion of Galectin-1, a promoter of SHH signaling in PDAC, resulted in decreased angiogenesis, desmoplasia, and prolonged survival of mice [Similarly, SHH deletion increased intratumoral vascular density, which was not the result of loss of SHH signaling in endothelium . These fCDON−/−) . Here, sGDC-0449 , 36. TheKPC mice . It is,  of mice . Finally of mice .−/− PDAC models replicate the findings from clinical trials of smoothened inhibitors in PDAC. Phase I trials of IPI-926 showed potential, but a phase II trial was halted due to decreased survival in patients receiving IPI-926 [−/− models helped elucidate the changes in the TME due to loss of SHH signaling and unraveled mechanisms that contributed to the failure of SHH inhibitors in clinical trials. However due to the pleiotropic effects of SHH deletion, it is challenging to tease out the intricacies of the interplay between SHH and desmoplasia in PDAC initiation and progression.Importantly, both SHH IPI-926 . Similar IPI-926 , 39. The IPI-926 , 39. RhiLSL-G12D;Tgfbr2fl/fl mice thereby sensitizing α-SMA-positive cells to ganciclovir and allowing temporal control of myofibroblast depletion [The α-SMA-positive CAFs (pancreatic stellate cells-PSCs PSCs / myofibroblasts) are an abundant population of CAFs in PDAC. To determine the contribution of α-SMA-positive CAFs to PDAC progression, Ozdemir et al. cloned human herpes virus thymidine kinase under the α-SMA promoter into Ptf1a-Cre;Krasepletion . Mice trepletion . In contepletion . Similarepletion . Additioepletion .n = 53) [LSLG12D/WT;Ptf1a-CRE;Tp53fl/WT;Ela-Tgfa) and other mouse models [As in experimental models, correlation of desmoplasia with clinical outcomes show conflicting results. Using gene expression analysis from 145 primary and 61 metastatic tumors, Moffit et al. identified normal and activated stroma gene signatures. Patients with activated stroma signatures had worse survival compared to patients with similar tumor types and normal stroma signatures . Anothere models , 40, 46.An important consequence of these three major studies which failed to support the pro-tumorigenic contribution of the desmoplastic reaction is the appreciation of fibroblast heterogeneity in the PDAC TME. While there is much speculation on the subject, only one study has demonstrated clear differences in the activity of fibroblasts based on the expression of FAP and α-SMA . Here, iin vivo. With such studies, a clearer picture of the complexity of the PDAC desmoplastic reaction will emerge along with an understanding of the duality of desmoplasia and its role in PDAC progression.Ultimately, it is too early to accurately discern the role of desmoplasia as a whole in PDAC progression. Moving forward, an improved understanding of fibroblast biology including the cellular origins of fibroblasts in PDAC and the transcription factor networks driving fibroblast phenotypes both in physiological and pathological conditions will be critical. This understanding can be leveraged for the generation of murine models to specifically identify and characterize heterogeneous CAF populations present in the PDAC TME. Additionally, such insight will allow the generation of mouse models with conditional deletion of ECM components to begin to address the role of ECM in PDAC progression Finally, while various mouse models have suggested that desmoplasia may play a tumor-restraining role in PDAC, the potential of desmoplasia as a therapeutic target has not diminished. Recent work demonstrating the presence of multiple CAF populations indicates that in the PDAC TME pro-tumorigenic and tumor-restraining components may coexist. Therefore, the discovery and characterization of distinct populations of CAFs may yield promising new targets for therapeutic intervention. Similarly, the activities of CAFs in PDAC have yet to be fully characterized. As with targeted therapy against cancer cells, it may not be advisable to deplete an entire fibroblast population from a tumor, but rather target a specific subset or activity of these cells to undermine the tumor supporting role or augment the tumor-restraining activities. The direct modulation of the extracellular matrix properties using a recombinant PEGylated hyaluronidase showed promising results in phase II clinical trials and is now in phase III testing . While t


We previously found that UC-MSCs attenuate brain injury by secreting neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and hepatocyte growth factor (HGF). However, how these factors contribute to neuroprotection remains unknown. In this study, we aimed to investigate to what extent UC-MSC-derived HGF and BDNF contribute to neuroprotection using a Transwell co-culture system of neonatal cortical neurons damaged by oxygen-glucose deprivation. The influence of HGF and BDNF were determined by investigating neurons in both the presence and absence of UC-MSCs as these cells consistently secrete both factors and can be blocked by neutralizing antibodies. In the co-culture, UC-MSCs significantly improved neuronal injury, as indicated by an increase in immature neuron number, neurite outgrowth, and cell proliferation. Co-culture of damaged neurons with UC-MSCs also exhibited a reduction in the number of neurons displaying signs of apoptosis/necrosis. The neuroprotective actions of UC-MSCs were partially reverted by neutralizing antibodies. Together, our findings reveal that UC-MSC-secreted HGF and BDNF have neuroprotective effects on damaged neurons. Further studies should address the existence of other potential neurotrophic paracrine factors.Several studies have reported that human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) restore neurological damage  Mesenchymal stromal cells (MSCs) can be isolated from several sources, including bone marrow, cord blood, adipose tissue, the placenta, and the umbilical cord (UC) –5. Amongin vitro in this study.We previously reported that intravenously administered UC-derived MSCs (UC-MSCs) attenuate intraventricular hemorrhage-induced injuries, and brain-derived neurotrophic factor (BDNF) and hepatocyte growth factor (HGF) concentration were elevated in serum and cerebrospinal fluid in some part of UC-MSCs administered mice . TherefoGuo et al.  reportedin vitro, and verified our previous results.In this study, we focused on whether BDNF and HGF secreted by UC-MSCs exert neuroprotective effect in addition to the neurorestorative effect 2. Fibroblast-like adherent cells that migrated from the UC tissue fragments were harvested using TrypLE Select (Life Technologies), and were defined as passage 1 UC-MSCs. The harvested cells underwent four passages, after which they were used for experimental analyses as well as previous report  were taken from euthanized, pregnant mice in sterile conditions. Fetal brains were removed and cortical tissues were dissected under a microscope. The meninges were then removed and cortical tissues were chopped into small pieces. Cells were dispersed followed by mechanical trituration using Neuron Dissociation Solutions  and filtered through a 70 μm pore-size cell strainer. Cells were then resuspended in neurobasal medium (GIBCO) supplemented with 2% B27 (Invitrogen) and plated onto Poly-L-Lysine Culture Dishes . Cells were cultured in a humidified incubator at 37°C with 5% CO2, and half of the medium was replaced with fresh solution every 3 days. To reduce contamination by glial cells, 10 μM cytosine arabinofuranoside (Sigma-Aldrich) was added for 24 h on the 4th day of culture. We cultured cortical neurons for 7days, and OGD procedure was performed.All experiments were carried out in accordance with the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo. Cortical neurons from B6 Albino mice  were prepared according to previous reports , 13. Bri2, 5% CO2)  at 37°C. The OGD condition was maintained for 4 h, after which cells were re-oxygenated in the original medium and placed in a normoxic chamber . After injury by OGD was completed, co-culture with UC-MSCs was started immediately. Neurons were co-cultured with UC-MSCs according to previously reported methods  were added to UC-MSC culture media in order to deplete HGF and BDNF, as previously reported , 15, 16:The expression of neural protein markers and a mitotic marker was analyzed by immunocytochemistry. Briefly, neurons were fixed with 4% paraformaldehyde for 30 min at room temperature, blocked in 5% skim milk and 0.3% Triton-X 100 , and incubated overnight with primary antibodies at 4°C. The primary antibodies included mouse anti-human microtubule-associated protein 2 , rabbit anti-growth associated protein-43 , rabbit anti-BrdU , and mouse anti-phospho histone H3 (S10) . Secondary antibodies used were donkey anti-mouse IgG heavy and light chain-specific (H&L) (Alexa Fluor® 488) , and donkey anti-rabbit IgG H&L (Alexa Fluor® 594) . Nuclei were counterstained with 4',6-diamidino-2-phenylindole , and images were acquired using a fluorescence microscope  and NIS-Elements microscope imaging software version 4.10 according to previously described imaging methods –20. NeurProteins were extracted from the cells according to the manufacturer instructions as previously described , 10. ProTo evaluate whether co-culture with UC-MSC attenuated the death of cortical neurons after OGD, cell proliferation was determined using the Cell Proliferation ELISA kit, BrdU  according to the manufacturer's instructions. Briefly, neuronal BrdU incorporation was quantitatively evaluated using OD450 reader measurements (Bio-Rad iMark Microplate Absorbance Reader Version 1.02.01).To evaluate caspase activity in cortical neurons after OGD, cells were incubated in fluorochrome-labeled inhibitor of caspases (FLICA) solution  at 37°C for 1 h. After three washes, cells were labeled with propidium iodide (PI) and immediately imaged using a fluorescence microscope. Cell nuclei were counter-stained with Hoechst 33342. PI labeling was used with FLICA to identify four populations of cells: living ; early apoptotic ; late apoptotic ; and necrotic . Unstained live cells were labeled with only Hoechst 33342, and necrotic cell membranes appeared compromised and stained with PI in red. Cells in the late apoptotic phase were dually stained with FAM-FLICA (green) and PI, and cells in the early apoptotic stage were stained only with FAM-FLICA. Apoptotic/necrotic cells were counted using a microscope , and the proportion of apoptotic/necrotic cells /total number of cells ×100% was calculated .P-values of 0.05 or less were regarded as statistically significant.Values are expressed as mean ± standard deviation (SD) from three independent experiments. Differences between groups were analyzed with JMP 10.0.2 software . Groups were compared using one-way analyses of variance (ANOVAs), followed by Turkey's tests. n = 3, Figure Primary cultures of neonatal cortical neurons exhibited typical morphology, showing a neurite outgrowth-forming network Figure  that couUnder control conditions, both immature  and mature  neurons could be observed Figure . After OTo investigate the effect of HGF and BDNF on neurorestoration, cortical neurons were stained with the mitotic marker, anti-phospho histone H3 and counterstained with DAPI Figure . In the Next, we examined the neuroprotective effect of HGF and BDNF on neuronal apoptosis/necrosis after OGD using FLICA labeling. We identified four populations of cells: living ; early apoptotic ; late apoptotic ; and necrotic  Figures . QuantitRecently, UC-MSCs have attracted attention for their potential in treating neurological disorders, as several studies using neurological disease models have reported improvements after UC-MSC transplantation, and clinical studies using UC-MSCs to treat traumatic brain injury and cerebral palsy have already been implemented –26. The Consistent with a previous report , we confin vitro OGD model of primary cortical -neurons, as indicated by shortened neurites, a reduction in the number of network-forming neurites, fewer developing neurons, decreased cell proliferation, and increased apoptosis/necrosis. Co-culture with UC-MSCs was sufficient to maintain MAP2-positive mature neurons showing extended neurites and cluster formations, whereas HGF and BDNF NAbs significantly attenuated the restorative effect of UC-MSCs on neurite elongation.In this study, we successfully generated an Firstly we investigated neurorestorative effect of UC-MSCs using phospho histone H3 and BrdU experiment. Higher number of phospho histone H3 and BrdU-positive mitotic cells were observed in UC-MSC co-cultured cells compared to cells in the OGD group, whereas the reverse effects of HGF and BDNF NAbs on cell mitosis were not significant in histone H3 and BrdU experiments. Next we confirmed neuroprotective effect of UC-MSCs on cortical neurons after OGD injury by apoptosis/necrosis assay. The results demonstrated that co-culture with UC-MSCs reduced the number of cortical neurons displaying signs of apoptosis and necrosis post-OGD and this improvement was attenuated by the addition of anti-BDNF and anti-HGF NAbs.These results suggest that HGF and BDNF secreted from UC-MSCs may support neuroprotection through anti-apoptotic effect rather than neurorestoration, and that there is the possibility of other UC-MSC-secreted trophic factors.HGF—a multi-functional growth factor originally reported as a potent mitogen for mature parenchymal hepatocytes in primary culture—plays an important role in tissue regeneration in the nervous system. It has been reported that HGF binds a tyrosine kinase receptor encoded by the human proto-oncogene, c-Met, and that HGF and c-Met are expressed in both the adult and fetal central nervous system , 30. FolSimilar to HGF, BDNF  is also expressed in the adult and developing brain, and plays a key role in the proliferation, survival, and differentiation of neurons –35. BDNFTo exclude the possibility of mouse-BDNF and mouse-HGF secreted from damaged cortical neurons in this study, we measured the concentration of mouse-BDNF and mouse-HGF in the supernatant of the cortical neurons with or without OGD. The result showed that mouse-BDNF and mouse-HGF were very low below detection limit (data not shown), but they should be taken into consideration in the present study. In addition, even if it seems more likely that UC-MSCs secrete those neurotrophic factors, we cannot exclude the possibility that UC-MSCs induce self-secretion of BDNF and HGF by injured neurons. These endogenous neurotrophic factors must be considered for future study.In conclusion, although the presence of other UC-MSC-secreted factors likely exist, the current study showed that UC-MSCs exert their neuroprotective effects partially through secretion of BDNF and HGF by inhibiting the apoptosis/necrosis of injured neurons. Considering that UC-MSCs have been administered to treat several neurological disorders, including cerebral palsy, traumatic brain injury, and hereditary spinocerebellar ataxia , 26, 37,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.


The increasing popularity of porphyrins and hydroporphyrins for use in a variety of biomedical  and technical  applications is also associated with the growing number of methodologies that enable their solubilization in aqueous media. Natively, the vast majority of synthetic porphyrinic compounds are not water-soluble. Moreover, any water-solubility imposes several restrictions on the synthetic chemist on when to install solubilizing groups in the synthetic sequence, and how to isolate and purify these compounds. This review summarizes the chemical modifications to render synthetic porphyrins water-soluble, with a focus on the work disclosed since 2000. Where available, practical data such as solubility, indicators for the degree of aggregation, and special notes for the practitioner are listed. We hope that this review will guide synthetic chemists through the many strategies known to make porphyrins and hydroporphyrins water soluble. The utility of porphyrins and hydroporphyrins (chlorins and bacteriochlorins) in a variety of biomedical applications has been demonstrated . These imeso-pyridyl and meso-p-sulfonatophenyl-substituents in (mostly) symmetric porphyrins. These porphyrins were based on Adler syntheses of tetrapyridyl- and tetraphenylporphyrin, followed by quarternization with a suitable alkyl halide or direct sulfonation, respectively. Albeit simple, both methods have drawbacks. They are difficult to translate to β-alkyl-substituted porphyrins, the sulfonation reaction conditions are harsh and therefore incompatible with many hydroporphyrin chromophores or substituents that convey desirable properties onto the porphyrin. This led to the development of a range of alternative methods to impart water-solubility that are mild, selective, and that generate the water-soluble porphyrin derivative late in the synthetic sequence, thus facilitating its isolation and purification.Initial efforts to impart water-solubility relied on the utilization of Rather than attempting to be comprehensive, we aim to present here a tutorial-style review that highlights recent (since ~2000) synthetic strategies. Where data are available, we compare the resulting solubilities, and report on other notable practical considerations. This summary extends a review on water-soluble porphyrins published in the year 2000  that proStrategy A encompasses the synthesis of a porphyrin macrocycle using building blocks bearing solubilizing groups (protected or not). Strategy B involves the synthesis of a porphyrin macrocycle with precursor functionalities, that can later be manipulated to hydrophilic groups. Strategy C employs synthetic modifications of an already-formed porphyrin to impart water solubility.The review is organized along the charge the solubilization groups introduce: Cationic , anionic  and neutral (polyethylene glycol (PEG) chains). Each section discusses the examples loosely in increasing order of complexity and the position of the solubilizing group. Three principle strategies for the introduction of solubilizing groups can be distinguished . StrategN-alkylation of meso-tetrapyridylporphyrins and their metal complexes  to another anion . Fo. Fo14. Tsitizers .19 to directly introduce arylamines to the β-positions via Suzuki couplings -porphyrins are long known, and their intercalation into DNA was studied in detail . U. U66 beain water .trans-AB porphyrins using a 2 + 2 dipyrromethane condensation strategy, using dipyrromethane 70 carrying a protected 2,4,6-triester-subsituted meso-phenyl group  groups were also introduced to porphyrins through amide linkers on one or more ications ,113.meso-Tetrakis(4-hydroxyphenyl)porphyrin (76) is a versatile starting material for the synthesis of hydrophilic derivatives since the p-phenolic oxygens can react with a variety of solubilizing groups. A4-Porphyrin 76 is synthesized through the Adler procedure, generally as its 4-methoxy derivative, that can be purified and handled easily. It is subsequently deprotected using BBr3  th th76, ChO-alkylation of the phenolic oxygens of a meso-4-hydroxyphenyl-substituted core-modified dithiaporphyrin. Like their aza-analogues, the requisite core-modified porphyrins were synthesized with a methyl-ether protecting group in the p-position of one or more phenyl groups and deprotected using the standard BBr3 method. The phenolic oxygens were then alkylated with ethyl bromoacetate. Saponification of the installed esters furnished the dithiaporphyrin 81 bearing carboxylic acid functional groups  groups were also introduced to meso positions of a porphyrin by alkylation of meso-tetrakis(bromomethylphenyl)porphyrin . T. T108) w0 mg/mL) . The promeso-positions of a meso-brominated PEGylated conjugated porphyrin dimer using a Suzuki coupling with a sulfonated arylboronic acid, a strategy similar to the introduction of pyridine functionalities described above 3, and subsequent hydrolysis of the intermediate with MeOH/H2O to afford phosphonate 116M. The free phosphonate functional groups were used to attach these derivatives to electrode surfaces . T. T3B por26 bearing two methyl-capped tetraethylene glycol (TEG) groups on opposite meso-phenylgroups was prepared. Leaving the PEG chains intact, it was converted to the bis-acetylene linked porphyrin dimer 134. Porphyrin dimer 134 was found not to be sufficiently soluble in aqueous media for biological application. Therefore, these materials were further derivatized with additional solubilizing groups, all while the PEG functional groups remained intact  or by semi-preparative reverse phase HPLC. Due to insufficient solubility of the compounds in regular reverse-phase HPLC solvents , DMF, DMSO, or THF were used. Also due to solubility issues, the authors could not measure logP values for these compounds in octanol:water. However, they did compare the relative polarity of some derivatives by means of their retention times under reverse-phase HPLC conditions chlorin (141) was rendered water-soluble by introducing PEG groups through two different etherification strategies. Tetraphenolporphyrin 141 was treated with p-nitrophenylcarbonate- or cyanuric chloride-activated methoxypolyethyleneglycol to afford the water-soluble PEGylated porphyrins 142 or 143, respectively porphyrin (76) can also be alkylated using various activated PEGs. For instance, this procedure has been employed to alkylate 76 by treatment with PEG-mesylates to form 144.   ca ca142] crphyrins ,144. Vitated PEG . The PEGer PEGs) ,143; porixtures) ,142.meso-arylporphyrins was also achieved by inversion of the Williamson ether synthesis reaction partners, i.e., involving a halogenated porphyrin precursor and PEGs bearing a free alcohol. An example is the PEGylation of tetra(p-bromomethylphenyl)porphyrin (147) with a methyl-capped PEG alcohol (Mn = 550) that afforded the tetra-PEGylated porphyrin 148 as the main product, along with partially PEGylated porphyrins (2Cl2) and were purified by HPLC.PEGylation of rphyrins . The pormeso-Aryl porphyrins bearing peripheral carboxylic acid functional groups can be PEGylated by amide formation using an amine-terminated PEG. For instance, Scolaro and co-workers prepared PEGylated derivatives of meso-tetrakis(4-carboxyphenyl)porphyrin (149) by treatment with Jeffamine M-600 (n = 7–9) (n = 7–9)  147]..meso-Ary149, Lovell and co-workers reacted it with a variety of homobi-functional PEG-diamines (average Mw ranging from 150 Da to 10 kDa) to synthesize a PEG-linked porphyrin mesh 151 chlorin with the acid chloride of the diblock copolymer. The polymer-functionalized porphyrin was purified by precipitation with Et2O, followed by a dialysis step in acetone. Vicente and co-workers prepared water-soluble porphyrin-peptide conjugates with a PEG linker on one meso-aryl group starting from an AB3 porphyrin 152 bearing one p-aminophenyl group.PEGylated porphyrins and hydroporphyrins with amide linkages can also be prepared starting with aminophenyl porphyrins. For instance, Peng et al. functionalized a chlorin with a poly-(ε-caprolactone)-polyethyleneglycol) diblock copolymer . Introdu152 was reacted with diglycolic anhydride, followed by amide-coupling using a PEG-amine and Boc-deprotection  was reported porphyrin. A BBr3-mediated deprotection was employed to deprotect the phenolic oxygens as the penultimate step of the synthesis. The phenol could then be PEGylated using either a short (n = 4) or long chain  PEG-mesylate . L. L165a),166 could also be functionalized with PEG-chains through amide linkages, whereby a short diamine established the requisite amine linkage sites. Amine intermediate 167 was then reacted with succinimidyl-PEG5000 to afford the water-soluble PEGylated protoporphyrin derivative 168  and (178) were also prepared using a regioselective meso-bromination strategy, followed by similar coupling sequences [Another representative example for the introduction of PEG groups via Suzuki coupling of dibromobacteriochlorin orin 176  135]. D. D19 wit19, various solubilization motifs could be introduced to this scaffold. Thus, ammonium  using a range of synthetic methodologies ,136. Onetrans-AB porphyrin 179  reduces the number of difficult chromatographic steps needed in the synthetic sequence; and these polar moieties and/or their water-solubility generally complicate their isolation and purification. Irrespective of the timing of the introduction of the solubilizing groups, multiple linking strategies are available. As far as flexibility and ease of implementation, PEGylation strategies stand out because they generate neutral chromophores of good solubility, the PEG chains are chemically stable, the versatility of the linking moieties that can be used to link PEG chains to the chromophore, and the facility of the work-up conditions once the PEG chains are established. A combined approach using both ionic and PEG groups may also have its merits in cases where the introduction of short PEGs was not enough to solubilize the macrocycle.Even though the toolbox for the synthetic chemist to affect water-solubility is already extensive, the continued development of solubilizing strategies that are mild, selective, and introduce chemically robust solubilization motifs to porphyrins is vital to the field for these versatile chromophores to realize their full potential in the many technical and biomedical applications that take place in aqueous media.


Immunotherapy is rapidly maturing towards extensive clinical use. However, it does not work well in large patient populations because of an immunosuppressed microenvironment and limited reinvigoration of antitumor immunity. The tumor microenvironment is a complex milieu in which the principles of physiology and anatomy are defied and which is considered an immune-privileged site promoting T cell exhaustion. Tremendous research interest exists in developing nanoparticle-based approaches to modulate antitumor immune responses. The increasing use of immunotherapies in the clinic requires robust programming of immune cells to boost antitumor immunity. This review summarizes recent advances in the engineering of nanoparticles for improved anticancer immunotherapy. It discusses emerging nanoparticle-based approaches for the modulation of tumor cells and immune cells, such as dendritic cells, T cells and tumor-associated macrophages, with the intention to overcome challenges currently faced in the clinic. Furthermore, this review describes potentially curative combination therapeutic approaches to provoke effective tumor antigen-specific immune responses. We foresee a future in which improvement in patient's surveillance will become a mainstream practice. Cancer is being treated by conventional therapies, including surgery, chemotherapy, and local radiotherapy. However, cancer metastasis and relapse lead to therapeutic failure. Cancer immunotherapy has shifted the paradigm by delineating an alternative treatment regimen in order to thwart the development of metastasis. The antitumor immune response can also be augmented by immunotherapies. It can specifically prevent the tumor metastasis as well as relapse by boosting the immune system, modulating the immune response, and inducing immunological memory while minimizing off-target adverse effects in situImmunogenic cell death (ICD) is associated with a series of well-defined sequential changes, including the constellation of alterations in cell surface of dying cells, such as heat shock proteins and calreticulin (CRT) as well release of soluble mediators, including high-mobility group box 1 protein (HMGB1) and adenosine triphosphate (ATP), which stimulates dendritic cells (DCs) and T cells Table 1. The overarching goal of this review is to provide insights into the potential of nanoparticles to improve the outcomes of cancer immunotherapy Figure .Nanoparticles (NPs) can effectively amplify immunomodulatory effects and modulate the immune response by integrating various molecules. NPs are capable of manipulating immune cells by facilitating the targeted delivery, and can be taken up and escape the endosome to release cargo, including drugs, antigens, and adjuvants at intended target sites (tumor and lymph nodes), while escaping the pathophysiological barriers, such as compact extracellular matrix, endonucleases degradation, and renal clearance, etc. The understanding of the mechanisms underlying the tumor cells to evade immunity as well as to deleterious antitumor immune responses is required to defeat cancer. It is imperative to modulate the tumor microenvironment in order to trigger an effective antitumor immune response by designing NP-based platforms, which have great potential to overcome the pathophysiological barriers.The immune system plays critical 'gatekeeper' functions that are capable of mounting adaptive antitumor response by activating immune cells, especially T cells. However, the tumor microenvironment (TME) is considered the immune-privileged site, which promotes T cells exhaustion by inhibiting their activation and functions reg) cells and myeloid-derived suppressor cells (MDSCs) participate in immunosuppression and tumor metastasis. Treg cells can suppress T cells by releasing transforming growth factor-β (TGFβ) and IL-10 while MDSCs promote immunosuppression by secreting immunosuppressive agents, such as reactive oxygen, arginase, and nitric oxide reg cells are not the only culprits in tumor metastasis, tumor cells can also exploit other immune cells, especially neutrophils, to “hitchhike” in the process of metastasis reg cells and thus promote the immunosuppression The studies bolstered the concept that immune cells may play a dual role either heroes or villains, where instead of rescue, immune cells support tumor metastasis reg cells, which ultimately activates MDSCs reg and MDSCs infiltration via IDO inhibitors are found to be an effective strategy to relieve immunosuppression The lymphocytic immune cells, including NK cells, are an integral part of the innate immune system that defend against tumor by adapting different mechanism, such as the receptors-ligands interactions reg cells) and cytokines (IL-10) are involved in T cell exhaustion The intrinsic and extrinsic negative regulatory pathways, for example, inhibitory receptors (PD-1), immunoregulatory cell types  containing the hydrophobic and hydrophilic domains, where PLGLAG, a peptide substrate of highly expressed proteinases, e.g., matrix metalloproteinase-2 (MMP-2) and a functional 3-diethylaminopropyl isothiocyanate (DEAP) were linked together to form hydrophobic domain, while hydrophilic domain constituted by a peptide antagonist of PD-L1 (DPPA-1). The delivered NLG919, a well-known inhibitor of IDO, co-assembled into micelle-like NPs under physiological conditions. In the acidic tumor niche, the unconsolidated hydrophobic core of NPs allowed MMP-2 to hydrolyze and cleave the peptide substrate, and precisely release modified DPPA-1 and NLG919. The targeted release of modified DPPA-1 and NLG919 ensured the blockade of immunosuppressive pathways, e.g., PD-L1 and IDO, respectively and ultimately rescued CTLs. The level of IL-2 and IFN-γ, as well as the frequency of natural NK cells, were significantly increased after treatment  NP-based delivery approaches enabled reversing immune suppression by inhibiting IDO pathway. Guangjun Nie's group designed a peptide-assembled nanostructure  An IDO pathway is considered a key regulator of immunosuppression. Engineered NPs enabled the reversal of immune suppression by blocking IDO pathway. The studies are suggesting a pronounced antitumor effect of the NP-based immunostimulatory formulations 3 NPs can serve to modulate the acidic tumor microenvironment as well as to precisely release immunomodulatory therapeutics.Macrophages and DCs are key players of the immune system that are capable of recognizing and presenting antigens to T cells. However, the overexpressing CD47 cancer cells can escape this recognition process by interacting with its corresponding ligand e.g., signal regulatory protein-α (SIRPα) on DCs, macrophages, and neutrophils 3) can modulate the immune response via blockade of CD47 as well as inducing phagocytosis of cancer cells. CaCO3 NPs play an active role in releasing the encapsulated anti-CD47 in the acidic environment by reacting with H+ and also act as proton scavenger to increase the pH value. The augmented T cell-mediated immune response and activation of TAMs (M1-type) were observed after treatment. The increased level of IL-12 and decreased level of IL-10 indicated the polarization of macrophages from M2 to M1-like TAMs, which was attributed to NP-based scavenging of H+ within the tumor. The immune response was further modulated by overcoming the other potential barriers, such as Treg, MDSCs, and hypoxia-inducible factor 1-ɑ (HIF1-ɑ)  A recent study reported that the solution of anti-CD47 antibody-conjugated calcium carbonate NPs  are responsible for initiating T cell-dependent immune responses. Among APCs, DCs are of paramount importance owing to their capability to elicit a T cell-mediated immune response. One of the major challenges in immunotherapy is the lack of activated immune cells in the tumors, especially DCs and T lymphocytes, which are mainly attributed to immunosuppressive pathways. Therefore, the activation of DCs by delivering both adjuvant and antigens in order to induce antitumor immunity, as well as the blockade of immunosuppressive pathways is emerging clinical strategies.Table . PAMPs are used to trigger immune responses by recognizing pattern recognition receptors (PRR) on the immune cells. Codelivery of antigen and adjuvant by NPs confers enhanced DCs-uptake, antigen cross-presentation, and efficient T cell-mediated response for cancer immunotherapy The NPs are being designed to deliver the cargo (such as antigens and adjuvants) through desired intracellular pathways, which allow targeted delivery to the immune cells. NPs have been engineered for lymph node-targeted delivery of various adjuvants, especially pathogen-associated molecular patterns (PAMPs). PAMPs, including cytosine-phosphate-guanosine oligonucleotides (CpG) and R848 can be delivered by NP-based formulations in vivo trafficking, a potent TLR9 agonist, i.e., immunostimulatory oligonucleotide (CpG) motif was functionalized with cholesterol (Cho-CpG), and thus designed highly stable, homogeneous, and ultrasmall nanodiscs (sHDL-Ag/CpG). The nanodiscs greatly improved the co-delivery of Ag and adjuvants in lymphoid organs and DCs maturation, which stimulated augmented antitumor T cell-mediated responses to inhibit tumor growth. The cross-priming of T cells could induce multivalent CD4+ T and CD8 α+-mediated T cell immunity. The CTLs frequencies were observed to be 47 and 31-fold higher than soluble vaccines and adjuvant of clinical trials (CpG in Montanide), respectively. However, the antitumor responses could not be sufficient to eliminate the tumor owing to TME induced immunosuppressive PD-L1/PD-1 pathways. Therefore, the immune checkpoint blockade strategy (anti-PD-1 and anti-CTLA) was applied to eradicate the MC-38 and B16F10 tumors Figure A strategy was suggested for augmented CD8α+ cytotoxic T lymphocyte responses in personalized nanomedicine, where Ag/adjuvant co-delivery was markedly improved by conjugating with high-density lipoprotein-mimicking nanodiscs. A nanodisc-based platform was developed by exploiting the intrinsic properties of high-density lipoprotein as a cholesterol nanocarrier and then decorated with tumor neoantigens, i.e., a cysteine-serine serine (CSS) linker coupled Ag peptides. In order to improve cellular uptake and in vivo owing to their binding affinity with endogenous albumin. Albumin/AlbiVax dissociated and liberated AlbiVax in the acidic conditions and thus elicited durable innate and adaptive immunity, and T cell-mediated memory. The therapy efficacy was further improved by combing nanocomplexes with anti-PD-1/Abraxane, which resulted in the eradication of established tumors in multiple tumor models The “albumin hitchhiking” approach, where cargo (antigen or adjuvant) containing amphiphiles-vaccines are linked to albumin-binding tail ensures precise delivery of cargo in the lymph node, T-cell priming, and thereby acquire remarkable antitumor efficacy Figure  a-cChen's group and co-workers have designed a self-assembled intertwining DNA-RNA nanocapsules capable of efficiently deliver neoantigens and adjuvants to APCs in lymph nodes and modulating immune response by synergistically targeting signaling pathways . DNA and RNA therapeutics were integrated into the single nanocomplex to develop biostable nanovaccines. CpG, STAT3-silencing short hairpin RNA (shRNA), and tumor neoantigens were incorporated in the same reaction system and self-assembled via rolling circle replication and transcription to form microflowers (MFs), termed as iDR-NC/neoantigen complexes (iDR-NC-MFs). The iDR-NC-MFs having shrinking capability due to the presence of the biocompatible PEG-grafted polypeptide (PPT-g-PEG) and therefore shrunk into iDR-NCs in acidic endolysosomes, and exhibited the enhanced proton sponge effect to facilitate cytosolic delivery of the cargo at the intended target. iDR-NCs also acquired tumor-specific neoantigen loading capabilities. The targeted delivery of iDR-NCs/neoantigen elicited APCs for sustained antigen presentation, about 8-fold more frequent neoantigen-specific CD8+ T cells than CpG and ultimately eliminated colorectal tumors by acquiring strong and durable antitumor immunity Figure dThe stimulation of potent tumor-specific T cells is considered as one of the major challenges in cancer immunotherapy. Therefore, the researchers explored the different synthetic compositions in an attempt to achieve a robust T-cell response. It is proposed that immunotherapeutic armamentarium can be expanded by designing stimulator of interferon genes (STING) pathway-activating NPs to improve the clinical performance of cancer immunotherapy Mesoporous silica rods (MSRs)-based formulations have been demonstrated for the recruitment of DCs in lymph nodes and augmented systemic helper T cells response in order to modulate immune function and elicit adaptive immune responses The effective stimulation of T cells to defeat tumor is the ultimate goal of cancer immunotherapy Figure a. CD19-specific CARs genes containing NPs were incubated with T cells in vitro whereas intravenously delivered to re-program T-cell in vivo. CAR did not fully express and induces such programming in other cells and therefore leukemia regression was effectively achieved in vivo . Reprogrammed T cells acted as a “living drug' to eradicate the tumor, and induced immunological memory since the expressions of these receptors were maintained even for weeks.The lack of cytotoxic T lymphocytes as well as regulation of the primed T cells is one of the main challenges of immunotherapy. NP-based DNA carriers can precisely program tumor-recognizing capabilities into circulating T cells and thus capable of inducing tumor regression. A simplified, cost-effective, and practical programming approach was introduced to generate “on demand” antitumor immunity, where polymeric nanocarriers functionalized with lymphocyte-targeting ligands precisely delivered leukaemia-specific CAR genes into T cells Figure b-c. The redox-responsive anti-CD45/IL-15Sa-NGs stimulated augmented T cell expansion in vitroFigure d. The anti-CD45/IL-15Sa backpacked T cells were intravenously injected in mice. About 8-fold higher cytokine dose and 16-fold specifically expanded T cells was achieved by programming the location and timing of T cell activation, which ultimately resulted in improved tumor inhibition It was demonstrated that the performance of T cell therapy can be reprogrammed by T cell receptor (TCR)-activatable NP drug delivery system. T cells are capable of modulating cell surface redox state when activated due to the transformed expression of their reducing enzymes. This discovery was exploited to achieve antigen-triggered release of adjuvant protein by designing reduction-responsive NPs. The activated protein nanogels (NGs) in T cell surface reduction potential were employed to 'backpack' supporting cytokines on T cells. The supporting cytokines, such as human interleukin 15 super-agonist (IL-15Sa) was selected as a drug cargo in this context. The disulphide cross-linker was cleaved by reducing conditions of T cell surface and released the cargo, i.e., intact cytokine in antigen-bearing microenvironments. The reducing conditions of TME, including glutathione, can also promote the release of IL-15Sa from NGs. However, T cell surface redox-mediated cytokine release was observed. A small quantity of anti-CD45 and poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) was incorporated into the NGs, where anti-CD45 kept the NG backpack intact on the surface by preventing internalization and PEG-PLL maximized the NG loading efficiency by providing a uniform positive charge The conjugated supporting drugs, including antibodies and interleukins, can secure T cells from immunosuppression Macrophages are one of the major components of the leukocyte infiltrate that is engaged with cancer in a dual yin-yang relationship by exhibiting M2-M1 phenotypes. M1-like phenotypes are correlated with antitumor responses and mainly driven by bacterial products and IFN-γ whereas M2-like phenotypes driven by IL-4 or IL-13 are responsible for tumor progression and adaptive immune suppression Figure . CDNP-R848 re-educated the TAM phenotype and thus ensured the reduced tumor and improved survival rate in CDNP-R848 treated mice after a single dosage. The synergistic treatment with anti-PD-1 further improved the therapeutic potential, which resulted in complete tumor regression in 2/7 mice while inducing antitumor memory TME comprised of a variety of cells, including TAMs, which play a critical role in the immune system. A continuum of macrophage phenotypes, such as M1 and M2 exists. The recent studies suggest that macrophages contribute to the immunosuppression in the TME while the reprogramming of TAMs from M2 to an antitumor M1 mode and inhibition of macrophage recruitment can provoke macrophage-mediated extracellular cancer death. Therefore, the polarization of TAMs or re-programming is considered a clinically approved therapeutic strategy •), which can induce apoptosis in the cancer cell. Therefore, the increase in caspase-3 expression and pro-inflammatory Th1-type responses was observed. Compared to control groups, about 11 and 16-fold higher level of hydrogen peroxide and hydroxyl radicals was observed in macrophages plus ferumoxytol-treated cells, respectively. Moreover, the increased expression of M1-related markers, i.e., TNF-α and CD86 and the decreased level of M2-related markers, such as IL-10 and CD206 after exposure to ferumoxytol, suggested induction of M1 macrophage polarization. This study demonstrated that tissue-resident macrophages could lose their M2 polarization while infiltrating macrophages was shifted towards M1 polarization. Ferumoxytol is therefore effective in inhibiting pulmonary metastasis and established liver tumors in a TAM-dependent manner. The NP-based gene-editing platform can also be designed that are able to reprogram immune responses. NLRP3 inflammasome, one of the endogenous damage-associated molecular patterns for the treatment of multiple inflammatory diseases can be targeted by using NPs. Cationic lipid-assisted nanoparticle (CLAN), a type of PEG-b-PLGA-based NPs has been demonstrated as a carrier for delivering Cas9 mRNA that guides RNA into macrophages and ultimately disrupts NLRP3. CLAN is capable of selectively targeting and reprogramming the macrophages since NLRP3 specifically function in macrophages Macrophage reprogramming has also been demonstrated by Saeid Zanganeh using iron oxide NPs (ferumoxytol) and suggested that ferumoxytol could be applied 'off label' to suppress metastasis as well as to improve the performance of macrophage-modulating cancer immunotherapy approaches Angiogenic factors, including vascular endothelial growth factor (VEGF), are highly expressed in TAMs and responsible for tumor progression and metastasis. A strategy presented by Conde et al. to reprogram TAMs where targeting immunotherapy was combined with silencing in the tumor microenvironment. Gold NPs having a peptide sequence (M2pep) selectively targeted TAMs and delivered a small interfering RNA (siRNA) to silence the expression of VEGF mRNA in the inflammatory M2 macrophages, which could transform immune response from an immunosuppressive to an immunostimulatory as well as tumor inhibition in lungs The cancer relapse even after combination conventional therapies, such as surgery, radiotherapy, and chemotherapy leads to therapeutic failure. It is imperative to introduce alternative strategies to defeat cancer. The focus of alternative strategies should be on the stimulation of immune response, which will induce immunological memory to overcome the cancer relapse ICD can be induced by the generation of ROS and endoplasmic reticulum (ER) stress by instigating the danger signaling pathways. The stress-specific mechanisms are involved in releasing key DAMPs, such as CRT and ATP NP-based strategies can be applied to exploit the ICD-inducing properties of conventional therapies for improving the therapeutic potential of cancer immunotherapy. NP-based combination therapies, including photothermal therapy, photodynamic therapy, radiation therapy, and chemotherapy with immunotherapy improve the therapeutic performance by enabling the simultaneous delivery of various therapeutic molecules Figure 9) Wang et al. designed a versatile micelleplex, which could enhance the therapeutic potential by combing multiple functionalities, such as ultra- pH-sensitive diblock (PDPA), pheophorbide A photosensitizer (PPa), and siRNA. The conjugated amphiphilic polycation, e.g., 1,2-epoxytetradecane alkylated oligoethylenimine (OEI-C14) having an intrinsic binding affinity with siRNA and thereby ensured the endosomal escape of the siRNA by promoting proton sponge. PDPA-OEI-C14-PPa (POP) micelleplex was precisely activated in acidic pH (6.2) while keeping intact in extracellular microenvironment. The siRNA-PD-L1-conjugated micelleplexes (POP-PD-L1) effectively induced PD-L1 blockade and thus rescued from immunosuppression by silencing PD-L1 expression on the tumor cells. The antitumor immune response was amplified by photodynamic therapy (PDT), where it effectively eradicated the tumor and distant metastasis in B16-F10 melanoma model by promoting cytokine secretion (TNF-α and IFN-γ) and the frequency of the tumor infiltrating lymphocytes (CD8+ and CD4+). The more apoptotic cancer death was observed after combination therapy than individual modalities and thus ultimately induced immunological memory owing to the presence of activated immune cells (2-PEG/C&D) can relieve tumor suppression whereas checkpoint-blockade (PD-L1 blockade) promotes the higher TNF-α secretion and augmented CD4+ and CD8+ T cells-mediated immune response than chemo-PDT therapy Figure 10) reg cells was observed when immunotherapy combined with PDT. Therefore, more than 90% of tumor regression was attributed to iron NP-based alleviated immunosuppression and T cells infiltration The doxorubicin-loaded lipoprotein-mimicking nanodiscs (sHDL-DOX) can exert antitumor efficacy by triggering ICD of cancer cells. Combination chemoimmunotherapy can sensitize tumor cells to immune checkpoint blockade and potentiate antitumor T cell-mediated responses. Elimination of established tumors (MC38 and CT26 colon carcinoma) in 80-88% of animals was achieved by delivering chemotherapeutic agents via nanodiscs. Survivors were protected against tumor relapse due to the induced antitumor memory TLR agonists and checkpoint blockade strategies are combined with photothermal and radiotherapy to achieve effective therapeutic efficacy and immunological memory Cancer immunotherapy has shifted the paradigm by delineating an alternative treatment regimen in order to thwart the development of metastasis. The studies have bolstered the concept that immune cells may play a dual role either heroes or villains in TME. On the other hand, there is circumstantial evidence that engineered NPs can promote the recruitment of immune cells in the tumor and overcome the anergy of tumor-specific T cells via blockade of immunosuppressive pathways. Besides modulating the immune response in TME, NP-based programming of immune cells improves the antitumor efficacy by enabling precise and persistent CTLs response. Programming of the immune system with engineered NPs has emerged as an effective strategy to improve antitumor immune response. TAMs contribute to promote tumor progression by taming protective adaptive immunity. The repolarization of TAMs in the tumor environment is, therefore, urgent to peruse. It is envisaged that the nanoparticle-mediated interplay between TME and immune system will significantly impact the clinical performance of cancer immunotherapy. However, a deeper understanding of mechanisms underlying the immune system and safety profiles of nanoparticles is required to avoid immune-related toxicity.The non-persistent and autoimmune responses, lack of tumor-specific antigens and CTL, variation in checkpoint expression, and limited response to checkpoint blockade among patients are key challenges for cancer immunotherapy. The TAA-specific and persistence responses of CTL are crucial for inducing prolonged antitumor immunity. These limitations pose an urgent demand for combination strategies to simultaneously target immunostimulatory and immunosuppressive pathways to ensure effective therapeutic outcomes. The better understanding of the mechanisms underlying the tumor cells to evade immunity as well as to deleterious antitumor immune responses is required to defeat cancer. The acclaimed success of nanoparticle-based formulations in thwarting cancer is sometimes over exaggerated; however, we foresee a future in which defeat of tumor metastasis will be a mainstream practice.Table 2). A challenge in these platforms, however, is that most of the NP-based formulations are currently in phase 1 or 2. A range of approaches to modulate the immune system have been discussed in this article. These approaches present several opportunities for clinical translation. Notwithstanding, the animal models are mostly used to evaluate these strategies, making it challenging to translate the results from these models to humans. The choice of the humanized animal model may promote the translatability from preclinical studies to clinics. Choosing the correct nanocarriers is also crucial for nanomedicine-mediated cancer immunotherapy. For example, FDA-approved pharmaceutical excipients  are likely to get into the clinic faster than the unapproved materials. The biosafety and reproducibility of the nanovector formulation are also critical for the clinical translation of NPs-based cancer immunotherapy. When considering all of these issues, the survival rate in patients can be dramatically improved by broadening the spectra of cancer patients respond to immunotherapy.A quiet large number of NP-based platforms are currently being evaluated in clinical trials for cancer immunotherapy (


To promote environmental sustainability and mitigate climate change that causes numerous families to suffer from natural disasters, a group of older residents’ volunteer to recycle usable materials by setting up recycling stations in their communities and transform recycled bottles into eco-friendly blankets for disaster survivors globally. This study examined long-term effects of the peer-led Tzu Chi Recycling Program (TCRP) on older adults’ compassion, psychological and physiological well-being. Using a quasi-experimental design, 1-year longitudinal data were collected from older adults at recycling stations (intervention group n = 36) and community centers (control group n = 36) in rural areas in Southern Taiwan. Findings from two-way repeated analysis of variance show that TCRP significantly improved older adults’ self-compassion, compassion for others, depression, hostility, happiness, and hypertension. Older adults built resilience through environmental volunteering in the TCRP as an environmental sustainability model for health promotion and social good


Based on three proof-of-principle applications we demonstrate that TAC-seq is an accurate and highly potential biomarker profiling method for advanced medical research and diagnostics.Targeted next-generation sequencing (NGS) methods have become essential in medical research and diagnostics. In addition to NGS sensitivity and high-throughput capacity, precise biomolecule counting based on unique molecular identifier (UMI) has potential to increase biomolecule detection accuracy. Although UMIs are widely used in basic research its introduction to clinical assays is still in progress. Here, we present a robust and cost-effective TAC-seq (Targeted Allele Counting by sequencing) method that uses UMIs to estimate the original molecule counts of mRNAs, microRNAs, and cell-free DNA. We applied TAC-seq in three different clinical applications and compared the results with standard NGS. RNA samples extracted from human endometrial biopsies were analyzed using previously described 57 mRNA-based receptivity biomarkers and 49 selected microRNAs at different expression levels. Cell-free DNA aneuploidy testing was based on cell line  genomic DNA. TAC-seq mRNA profiling showed identical clustering results to transcriptome RNA sequencing, and microRNA detection demonstrated significant reduction in amplification bias, allowing to determine minor expression changes between different samples that remained undetermined by standard NGS. The mimicking experiment for cell-free DNA fetal aneuploidy analysis showed that TAC-seq can be applied to count highly fragmented DNA, detecting significant  that tags RNA transcripts or DNA molecules with strings of random nucleotides, called unique molecular identifiers, to accurately quantify their numbers despite biases introduced by amplification ahead of sequencing. As a proof of principle, the researchers tested the ability of TAC-seq to detect biomarkers important for reproductive medicine. In endometrial biopsies, TAC-seq offered an accurate profile on levels of messenger RNAs and microRNAs linked to a woman’s chance of reproductive success. Proof of principle Down syndrome trisomy detection was carry out using simulated cell-free DNA testing, mimicking non-invasive prenatal genetic testing (NIPT). There are several methods available enabling detection of RNA- or DNA-based biomarkers but recently, next-generation sequencing (NGS) has become one of the favorite approaches because of its high sensitivity, high-throughput, and flexibility. However, despite of its advantages, the relatively high cost of NGS limits its wider application in healthcare. In addition, common NGS assays consist of multiple laboratory steps that often introduce technical biases limiting accurate quantification and, therefore, hinder the robust and clinically valid detection of biomarkers.4 (Templated Oligo assay with Sequencing readout) and MLPA-seq5 are advancement of the well-known MLPA6 (Multiplex Ligation-dependent Probe Amplification). Both methods overcome original MLPA multiplexing and detection limitations, and enable to apply sensitivity of NGS and analyze up to 20,000 RNA and 200 genomic DNA (gDNA) targets, respectively. Similar approaches are also RASL-seq7 for targeted multiplex messenger RNA (mRNA) analysis and another NGS and targeted ligation-PCR-based method DANSR  for cell-free DNA (cfDNA) detection in non-invasive prenatal genetic testing (NIPT). The authors of the latter method report that 384 loci per chromosome 18 and chromosome 21  are sufficient for aneuploidy discrimination, which makes it significantly less complex assay than so far widely used low-coverage whole-genome sequencing for NIPT.8 However, the strategy where ligation-PCR is combined with NGS ensures high level of multiplexing but suffers from the random ligation at low target nucleic acid levels, and on the polymerase-induced errors in both PCR and sequencing steps.11 As an outcome, above highlighted methods overestimate the original number of studied molecules and are biased by PCR uneven amplification.To overcome the challenges of precise target quantification on extended scale, NGS sensitivity together with high specificity of ligation-PCR have been compiled into common methods and assays. For example, NGS- and ligation-based TempO-Sequnique molecular identifiers  are applied. UMI is a string of random nucleotides used in library preparation that was recently introduced to genomic DNA analysis12 and also in single-cell transcriptome analysis.13 One specific motif out of a large pool of random sequences is incorporated into the original target molecule through oligonucleotides used in library preparation prior to amplification. Later, grouping by identical UMI clones eliminates PCR duplicates and detects the original number of biomolecules. So far, UMIs are widely used in research applications where relatively high PCR amplification is required, such as single-cell analysis and tumor mutation identification.17 Similarly to above-mentioned applications, UMIs can also be used in targeted ligation-PCR NGS assays to enable absolute quantification of studied biomarker molecules. Taking the previous into account, we developed a ligation- and NGS-based method, TAC-seq, targeted allele counting by sequencing method for original molecule counting of plasma cfDNA and RNA-based biomarkers.To overcome amplification bias in NGS and to maximize nucleic acid detection sensitivity, http://nipt.ut.ee/design/) and a computational workflow software to enable data processing  UMI thresholds.18 These results suggested that conservative UMI thresholds   RNA spike-in controls to validate the technical sensitivity and accuracy of the TAC-seq method. Altogether, 22 spike-in sequences were assayed at various concentrations, ranging from 1 to 3 × 1019 Ten endometrial biopsy samples were analyzed by TAC-seq and compared with the levels of selected 57 transcripts from full transcriptome RNA sequencing (RNA-seq) data. Principal component analysis of these data showed identical clustering of samples when applied to RNA-seq results and two different TAC-seq assays, carried out with high-  and low-coverage  sequencing  and distinguished the pre-receptive and receptive samples   and selected ERCC spike-ins (n = 5). We observed that although the assayed housekeeping genes represented 47.4% of all unique reads in this assay, the biomarker-based clustering probability was still high (AUTAC-low = 81%) and all of the targeted 57 biomarkers were detected  with low or moderate expression to low-coverage TAC-seq endometrium assay to ensure enough sequencing reads for all studied receptivity biomarkers.In low-coverage assay we expanded the assay to a 70-plex by adding housekeeping genes , we selected 49 miRNAs from endometrial tissue, which were previously analyzed by small RNA-seq and covered highly variable expression levels from 10 to 4,712 counts per million. TAC-seq detected all 49 assayed miRNAs over 16 analyzed endometrial samples with high reproducibility (Spearman 20 at positions one (G < 2%) and five (C < 3%), causing potential sequencing failure  and studied chr21.22  excess of chromosome 21 molecules at 10% fetal fraction level, applying UMI = 2 threshold physiological conditions. TAC-seq is an advanced ligation-based NGS method that differs from existing ligation-PCR assays such as MLPAr = 0.9958 on Fig. 24 The same applies to TAC-seq expression applications if lower RNA concentrations are used. Alternatively, it is possible to extend UMI sequences in both detector probes from current 8 to 12 nucleotides, ensuring 16.7 million possible combinations. At the same time we are aware that introducing significantly longer and random UMI strings into detector probes may increase the probability of probes self-pairing and unspecific ligation. However, UMI-related issues such as ‘‘saturation’’ and replication-caused novel, ‘‘phantom’’ UMIs25 that should be taken into account in assay design and data analysis.We detected very high sensitivity correlation over 22 analyzed ERCC spike-in input and detected molecules  as the locus is not detected if it locates closer than 25 bp to cfDNA fragmentation site.4 where recently described SplintR ligase26 for RNA/DNA hybrid is used to detect any, even fragmented RNA targets by specific detector oligonucleotides. In addition, SplintR ligase optimum working temperature is up to 37 °C that may limit the specificity of formed RNA/DNA probe complexes prior ligation. In contrast, TAC-seq uses thermostable Thermus aquaticus (Taq) DNA ligase27 that enables specific hybridization and ligation at temperature above 45 °C. Based on this property of Taq DNA ligase, we have carried out first specific probe-target hybridization at 60 °C and thereafter introduced ligase to join the proximity annealed strands at the same temperature.TAC-seq detects mRNA biomarkers through oligo-T primed cDNA synthesis (poly-A selection) that reflects the analysis of active transcriptome. It differs from TempO-SeqAs sequencing contributes to the majority of the NGS cost, it is critical to apply library preparation that supports low-coverage sequencing in routine NGS clinical applications. Cost-effectiveness of TAC-seq is ensured by off-the-shelf reagents and the usage of common instruments in genomic laboratory, such as standard thermocycler and benchtop NGS sequencer. The running cost of TAC-seq is only a fraction of the cost for commonly used NGS applications like whole-genome sequencing for NIPT or RNA-seq for mRNA and miRNA analysis. Set-up cost of TAC-seq depends on number of studied loci due to the need of specific detector oligonucleotides  without restrictions in usage and described in Supplementary Methods) that automates probe design procedure and provides highly specific oligonucleotide sequences with common motifs that are ready for synthesize. Probe design for miRNA molecules is even more straightforward and does not require special software  in cfDNA-like material. TAC-seq is automation-compatible method that is designed to overcome ligation- and NGS-based limitations in genetic testing laboratory. Although all presented applications need careful clinical validations before they can be utilized, the described method is the base for further specialization and optimization to provide advanced DNA and RNA biomarker analysis tools and thereby improve reach and quality of corresponding research and healthcare applications.This study was approved by the Research Ethics Committee of the University of Tartu (246/T-21 and 221/M-31). Endometrial biopsies for RNA assays were collected from healthy volunteers and written informed consent was obtained from all participants. Genomic DNAs from GM01359  and GM04616  cell lines  were extracted using the DNeasy Blood and Tissue kit (Qiagen). To mimic cfDNA, genomic DNA was fragmented to 150–200 bp using Covaris M220 Focused-ultrasonicator (Thermo Fisher).Detail description of biomarker selection, TAC-seq probe design, library preparation protocols, and TAC-seq sequencing are described in Supplementary Methods.ERCC spike-in reads were trimmed to construct length of 88-bp and demultiplexed by barcodes (6-bp) allowing 1 mismatch. Demultiplexed reads were further trimmed to length of 62-bp and 4-bp of UMI at the end of the read was inserted after 4-bp of the UMI at the start of the read. Reads with UMI that contained unallocated nucleotides, were discarded. Resulting reads per sample were demultiplexed again using target regions of the genes (54-bp) allowing up to five mismatches. Total read counts, unique molecule counts and Pearson and Spearman correlations were calculated at different UMI thresholds. Due to the fact that Pearson method assumes linear correlation and, therefore, resulted in insensitive correlations at high UMI thresholds, we chose Spearman correlation as it proved to work correctly in case of very high and very low molecule concentrations.Gene expression reads were processed as described above. To reduce potential sequencing error accumulating at UMI motif, only reads appearing at least twice were counted as unique molecules (UMI = 2). Each sample was normalized to CPM using edgeR (version 3.18.1) package in R (version 3.4.1) and log-transformed using the log10 (CPM + 1) transformation to reduce skewness. In addition to biomarker genes, both high- and low-coverage TAC-seq libraries included spike-in molecules. Furthermore, the low-coverage library included eight housekeeping genes, which were taken into account in CPM normalization. The normalization procedure was based on the published formula that was further adjusted for read-count data.Genomic DNA sequencing data quality control and pre-processing were performed as described above. Loci that were 1.5 interquartile ranges (IQRs) below the first quartile or above the third quartile were called as outliers and removed. As we constantly detected slightly higher molecule counts in chr2 compared to chr21 in euploid samples, chromosome-specific molecule counts were applied. For that, mean molecule counts of chr2 and chr21 (~1.081 at UMI threshold 2) using the euploid samples were calculated and used for normalization. User-friendly software was developed to enable TAC-seq data processing in end-user’s personal computer or in Linux environment.https://github.com/cchtEE/TAC-seq-data analysis and shown schematically in Supplementary Fig. Open-source software with installation instructions are available at Targeted assay set-up needs two specific probe oligonucleotides for mRNA and cfDNA analysis. The price we got was 27 EUR per detector pair, resulting in 800 EUR in total for 30 loci analysis, for example (Supplementary Fig. Supplementary information


RM) cells. Both tissue-resident memory ILCs and TRM cells share common characteristics in terms of dynamics, phenotype, and molecular regulation. The emergence of ILC memory expands the basic biology of ILCs and prompts us to re-examine their functions in disease progression. This review discusses the evidence supporting tissue-resident memory NK cells and other memory ILC subsets, compares them with TRM cells, and highlights key unsolved questions in this emerging field.Innate lymphoid cells (ILCs) are defined as lymphocytes that lack RAG recombinase and do not express diverse antigen receptors; however, recent studies have revealed the adaptive features of ILCs. Mouse cytomegalovirus (MCMV)- and cytokine-induced memory natural killer (NK) cells circulate in the blood and are referred to as conventional memory NK cells. In contrast, virus- and hapten-induced memory NK cells, hapten-induced memory ILC1s, and cytokine-induced memory-like ILC2s exhibit long-term residency in the liver or lung, and are referred to as tissue-resident memory ILCs. Considering their similar migration patterns and memory potential, tissue-resident memory ILCs could be regarded as innate counterparts of resident memory T (T However, in the last decade, adaptive features of NK cells have been observed by several independent laboratories in different experimental systems, including MCMV m157 protein-, cytokine- and hapten-induced NK cell recall responses , whereas the latter accounts for nearly 50%  patients  cells  in a CXCR3-dependent fashion  and selectively reside in the liver via CXCR6-CXCL16 interaction  to support the long-term survival of TRM cells  is a common occupational skin disease caused by allergens, such as nickel and house dust mite allergens, which accounts for 20% of the entire work-related health burden   repertoire, whereas NK cells and other ILCs only express germline-encoded recognition receptors. cNK cells that remember specific antigens are well characterized in the CMV infection model, including murine Ly49H recognition of MCMV m157 protein   chain cytokines, IL-7 and IL-15, are well established as homeostatic factors for the longevity of TC1s Fig. ; thus, BbrightNKG2C+ NK cells are trained in the uterus by repeated pregnancies. IL-7Rα+ ILC1s acquire memory potential against haptens and selectively reside in the liver for long-term survival. Lung-resident ILC2s show recall capacities to the cytokine IL-33. It is becoming increasingly clear that tissue-resident memory ILCs may be innate counterparts of TRM cells. They share common features, for example, similar location and dynamics, expression of resident markers, inflammatory factors driving memory cell formation, and IL-7 and IL-15 supporting their longevity. Along with a deeper understanding of memory ILCs, more detailed comparisons should be summarized.Over the past 10 years, many studies about ILCs have been focused on their innate functions in pathogenic infection, inflammation, and tumor surveillance. However, accumulating evidence has revealed the adaptive features of ILCs and supports the concept of ILC memory. Two types of memory ILCs, including circulating memory NK cells and tissue-resident memory ILCs, have been confirmed by independent laboratories using different experimental systems in both mice and humans. Apart from CMV- and cytokine-induced conventional memory NK cells, resident memory NK cells and other ILCs exhibit long-term residency in the liver, uterus, and lung. Liver-resident NK cells can confer memory responses to viruses and haptens. Human adaptive CD56In the emerging field of tissue-resident memory ILCs, several open questions remain unsolved. For example, besides the uterus, do resident memory NK cells and other ILCs exist in other human tissues? Do murine and human ILC3s have antigen-specific or non-specific memory potential? Do tissue-resident memory ILCs and T cells co-operate in the same cases? How do NK cells and ILC1s recognize different viruses and haptens? Which molecules are responsible for the tissue-residency of memory ILCs? How can the memory features of ILCs be used as powerful tools in clinical therapies, such as vaccine development? Despite many unsolved puzzles, the emergence of tissue-resident memory ILCs prompts us to re-examine the functions of ILCs in disease progression. Such new knowledge will deepen our understanding about the biology of ILCs and contribute to novel therapies in human disease.