Dear Editor,Here, we report a case of a clinically manifest human herpesvirus-6 (HHV-6) encephalitis in a neutropenic patient with acute myeloid leukemia (AML) in a non-transplant setting while on antimicrobial prophylaxis including aciclovir.A 38-year-old female patient was admitted to the hospital with newly diagnosed AML. Following second consolidation chemotherapy containing high-dose cytarabin, the patient showed signs of forgetfulness, myoclonia in both arms, and eventually suffered from a generalized tonic-clonic seizure. Electroencephalography showed generalized slowing but no epileptiform activity. The MRI showed restricted diffusion bilateral within the frontal striatum, as well as increased contrast enhancement in the lenticulostriate arteries, indicative of inflammatory lesions Fig. . PolymerWith regard to the AML therapy, indication for allogeneic hematopoietic stem cell transplantation  from an HLA-matched sibling was met due to ongoing detection of NPM1. Before allo-HSCT PCR for HHV-6 in CSF as well as blood was negative, and MRI scan showed few residual inflammatory signs within the basal ganglia Fig. . Allo-HSHHV-6 displays latency after the primary infection. End-organ disease usually occurs in an immunocompromised host and is most likely due to reactivation. HHV-6 is the most frequent cause of encephalitis in an allo-HSCT setting and typically takes a subacute course with slow beginning of confusion, amnesia, and change of personality . Post alTo our knowledge, this is the first report of clinical manifest HHV-6 encephalitis in a leukemic patient prior to allo-HSCT without any predisposing factors contributing to an immunocompromised state before the diagnosis of AML was made. Since MRI scan may be negative at the beginning of the disease, close clinical and diagnostic monitoring is necessary if a patient presents with acute-onset altered mental status , short-term memory loss, or seizures. This case indicates the significance of observing clinical signs and symptoms of incipient encephalopathy and rapid diagnostic and therapeutic measures to prevent residual damage.


Mesenchymal stromal cells (MSCs) are the most commonly tested adult progenitor cells in regenerative medicine. They stimulate tissue repair primarily through the secretion of immune-regulatory and pro-regenerative factors. There is increasing evidence that most of these factors are carried on extracellular vesicles (EVs) that are released by MSCs, either spontaneously or after activation. Exosomes and microvesicles are the most investigated types of EVs that act through uptake by target cells and cargo release inside the cytoplasm or through interactions with receptors expressed on target cells to stimulate downstream intracellular pathways. They convey different types of molecules, including proteins, lipids and acid nucleics among which, miRNAs are the most widely studied. The cargo of EVs can be impacted by the culture or environmental conditions that MSCs encounter and by changes in the energy metabolism that regulate the functional properties of MSCs. On the other hand, MSC-derived EVs are also reported to impact the metabolism of target cells. In the present review, we discuss the role of MSC-EVs in the regulation of the energy metabolism and oxidative stress of target cells and tissues with a focus on the role of miRNAs. Mitochondria are complex organelles that play a central role in energy metabolism, biosynthetic processes and control of stress responses. Mitochondrial function or ability to generate energy through OXPHOS (oxidative phosphorylation) is vital for cell homeostasis. Mitochondrial dysfunctions are a hallmark of many diseases including metabolic disorders, cardiomyopathies, neurodegenerative diseases and cancer, tightly associated with programmed cell death (apoptosis). The regulation of mitochondrial metabolism is therefore essential to maintain tissue homeostasis .+, CD73+, CD90+, and CD45-, CD34-, CD14-, or CD11b-, CD79a- or CD19-, HLA-DR-, and iii) their capacity to differentiate into osteoblasts, adipocytes and chondrocytes  have been shown to impact mitochondrial function . MSCs adrocytes . MSCs ardrocytes , 9. Theydrocytes , 11. Thedrocytes .via the delivery of their cargo either by fusion with the plasma membrane of target cells or by endocytosis and release into the cytosol  released from the endosomal compartment, microvesicles or microparticles (>150 nm) produced by budding from the plasma membrane and apoptotic bodies originating from disassembling apoptotic cells . EVs are cytosol . EVs con0 nm rele cytosol ]. miRNA Mitochondria are found in most eukaryotic cells and are the cell energy-producing organelles. There are up to 2,000 per cell (according to cell type), and are preferentially located in adenosine triphosphate (ATP)-consuming cellular areas. They are small organelles (0.5 to 1 µM) surrounded by a double membrane; each membrane being composed of a phospholipid bilayer . The twoMitochondria contribute to the cellular respiration processes through ATP production. ATP is the ubiquitous energy molecule used in a very large number of cell metabolic reactions . Apbc1 complex (complex III), cytochrome c and cytochrome c oxidase  and ATP synthase (complex V). ETC generates a proton gradient, which drives the synthesis of ATP. During OXPHOS process, mitochondria generate reactive oxygen species (ROS), which participate to cell homeostasis, primarily by the genesis of hypoxia-inducible factors (HIF)  or Krebs cycle Figure 1rs (HIF) –24. Howede novo lipid synthesis.A part from glycolysis, acetyl-CoA can be produced by the breakdown of lipids (β-oxidation) or proteins (amino acid metabolism) . Fatty aSeveral pathways of the amino acid metabolism are located in mitochondria. Amino acids are either ketogenic (lysine and leucine), glucogenic , or both . Depending on their carbon skeleton, ketogenic amino acids are converted into acetyl-CoA or acetoacetyl-CoA, while glucogenic amino acids are converted into glucose, pyruvate or a TCA cycle intermediate. In this pathway, glutaminase converts glutamine to glutamate, which is further catabolized in TCA cycle .2+ homeostasis, ROS generation and apoptosis, has been shown  and optic atrophy 1 (Opa1)] protecting cells against environmental damages by improving respiration parameters , downregulate pro-apoptotic proteins  and CYTC and finally minimize mitochondria injury  plays an important role in this process. Through SIRT3 activation and upregulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), mitochondrial biogenesis increases and ROS production decreases , 47. MSCMitochondrial transfer mechanisms between MSCs and recipient cells are based on cell fusion, EV secretion, gap junctions or tunnelling nanotubes . In the MicroRNAs (miRNAs) are non-coding endogenous ˜22nt (18 – 25nt) RNAs, which form a large family of post-transcriptional regulators. Within the nucleus, miRNA genes are transcribed by RNA polymerases as long precursor pri-miRNAs (> 500 bases), which harbour stem-loop structures . Maturatvia PGC-1 activation   .MiRNAs can target OXPHOS either directly, by targeting mRNAs of essential ETC factors and/or indirectly, by targeting the biosynthesis pathways of essential cofactors . MiR-338MiRNAs can target central biochemical reactions, shared by many pathways. For example, the citrate synthase from the TCA cycle is targeted by a set of miRNAs: miR-152, miR-148a, miR-148b, miR-299-5p, miR-19b, miR-122a, miR-421, miR-494, and miR-19a . As a reMany miRNAs, including miR-23a/b , miR-137Several miRNAs have been described to regulate lipid metabolism. MiR-370, miR-148a, and miR-33 target CPT1A, which is responsible for the translocation of fatty acids from the cytosol to mitochondrial matrix, thereby reducing fatty acid oxidation –90. MiR-The regulation of mitochondrial proteins by miRNAs can therefore modulate mitochondrial function suggesting that targeting miRNAs might provide a new therapeutic approach for the treatment of mitochondria-related diseases.Various studies have shown that EVs from different cell types can release miRNAs in the cytoplasm of recipient cells where they target mitochondrial metabolism pathways . ExposurAcute kidney injury (AKI) results from severe renal ischemia reperfusion injury (IRI), a common clinical situation during transplantation. Renal reperfusion induces oxidative stress, characterized by increased ROS and/or reactive nitrogen species (RNS) . MSC-EVsIn a rat model of AKI, IRI caused a lower expression of miR-30-b, miR-30-c, and miR-30-d in renal cells. MSC-EVs were shown to protect kidney from IRI by inhibiting mitochondrial fission through miR-30 transfer . TransfeIn vivo, MSC-EVs were shown to significantly increase the expression of the antioxidant NRF2 and SOD2, both at the protein and mRNA level, while the pro-oxidant KEAP1 was decreased. By investigating the miRNA profile of injured kidney cells, miR-200a-3p was significantly increased after MSC-EV injection. Importantly, EVs isolated from antagomiR-200a-3p-treated MSCs failed to preserve in vitro the structural integrity of mitochondria in TECs under oxidative damage. The authors concluded that miR-200a-3p was shuttled by MSC-EVs to target the KEAP1-NRF2 signaling pathway.MSC-EVs have been reported to stimulate ATP production and the antioxidant defense of tubular epithelia cells (TECs) through activation of KEAP1-NRF2 signaling pathway . In vivoIn diabetes, hyperglycemia (HG) leads to the down-regulation of COXIV in the ETC complex, leading to mitochondrial disorders and renal tissue alterations. Transfer of miR-222 from MSC-EVs to mesangial cells reduced STAT5A expression, leading to miR-21 down-regulation . A previCardiac stem cells (CSCs) have been reported to be largely involved in cardiac regeneration and repair after injury . CSCs hPrevious studies indicated that miR-214 was up-regulated by hypoxic stress to protect cardiac myocytes from damage . In a hy2O2-treated MSCs and showed to contain higher levels of miR-21 than EVs from non-treated MSCs. Actually, miR-21 up-regulation in H2o2-treated MSC-EVs was shown to regulate the apoptosis of CSCs through PTEN down-regulation and PI3K/AKT activation.Mir-21 is another miRNA whose cardioprotective role has been demonstrated . To mimiIn the 1920s, Otto Warburg described that cancer cells use higher levels of glucose than normal cells, thereby leading to increased lactate production . Since, MiR-1180 was the most abundant miRNA detected in the conditioned medium of MSCs that were able to stimulate both glycolysis of ovarian cancer cells and chemoresistance to cisplatin treatment , 111. OvPrevious studies have demonstrated that MSCs can reduce inflammatory responses, hepatocyte apoptosis, liver fibrosis and, enhance liver regeneration and functionality . This tMiR-122 has a critical role in liver function as being involved in cholesterol biosynthesis, fatty acid synthesis and β-oxidation . In normIn the current study, we described various miRNAs that are expressed by MSCs and packaged in EVs and that directly or indirectly regulate critical functions of mitochondria. MSC-derived miRNAs are promising actors in regenerative medicine and in therapeutics as they can modulate several pathways that are altered in mitochondrial dysfunction-related diseases. Data are still sparse but encouraging results have been reported. Manipulating miRNA expression using miRNA mimics and inhibitors may serve as potential therapeutic approach for diverse diseases. A better understanding of miRNA biogenesis, import and function is needed but should provide new insights on the feasibility of this novel strategy for mitochondria-associated disorders.All authors contributed to the article and approved the submitted version.We gratefully acknowledge the Agence Nationale pour la Recherche for support of the national infrastructure: “ECELLFRANCE: Development of a national adult mesenchymal stem cell based therapy platform” (ANR-11-INSB-005). The study was also supported by the European Union Horizon 2020 Programme . The materials presented and views expressed here are the responsibility of the authors only. The EU Commission takes no responsibility for any use made of the information set out. Funding for staff exchange was received by Programme Ulysses 2018 (project #: P41059WK).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.


BRCA mutations and/or homologous recombination deficiency (HRD) is critical for selecting patients for PARPi treatment. However, the currently available HRD assays are not perfect predictors of the clinical response to PARPis in EOC patients. In this review, we introduce the concept of synthetic lethality, the rationale of using PARPi when HRD is present in tumor cells, the clinical trials of PARPi incorporating the HRD assays for EOC, the current HRD assays, and other HRD assays in development.Epithelial ovarian cancer (EOC) patients are generally diagnosed at an advanced stage, usually relapse after initial treatments, which include debulking surgery and adjuvant platinum-based chemotherapy, and eventually have poor 5-year survival of less than 50%. In recent years, promising survival benefits from maintenance therapy with poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) has changed the management of EOC in newly diagnosed and recurrent disease. Identification of  BRCA mutations and/or HRD status in clinical specimens is critical to selecting EOC patients for PARPi treatment, which has been evaluated in several clinical trials  proficient. However, the currently available HRD assays are not perfect predictors of the PARPi response because, in previous trials, HRD-positive and HRD-negative patients defined by the current assays both benefited from PARPi. In this review, we introduce the concept of synthetic lethality, the rationale of using PARPis when HRD is present in tumor cells, the clinical trials of PARPis incorporating the HRD assays for EOC, the current HRD assays, and other HRD assays in development  is a major cause of death in women worldwide –5. Due tl trials –15. The l trials , 17. Thel trials . The HRDl trials Table 1.DNA damage in cells may result from exogenous or endogenous sources, such as oxidative damage, radiation, ultraviolet light, cytotoxic materials, and replication errors, among others . AccumulPARP plays multiple roles in repairing both SSBs and DSBs, such as binding to DNA breaks, recruiting repair proteins, and resolving collapsed replication forks –30. The BRCA gene mutation tests and/or HRD assays were investigated using a primary study design or retrospective exploratory analysis to determine the predictive value of these assays in stratifying EOC patients that benefit from PARPi therapy. DDR pathways are a potential target for cancer therapy based on the concept of synthetic lethality, with the aim of specifically killing cancer cells dependent on a compensatory DNA repair pathway for survival . In receBRCA1/2 (gBRCA1/2)-mutated advanced ovarian cancer who had received ≥3 prior lines of chemotherapy was 34%  and median duration of the response (DoR) was 7.9 months . The median DoR for platinum-sensitive and platinum-resistant disease was 8.2 months  and 8.0 months , respectively. These findings suggest that olaparib monotherapy has antitumor activity in patients with gBRCA1/2-mutated advanced ovarian cancer following more than three prior lines of chemotherapy.In the phase II study 42 trial , 37, theBRCA-mutated subgroup, 5.7 months in the LOH-high subgroup, and 5.2 months in the LOH-low subgroup. PFS was significantly longer in the BRCA-mutated  and LOH-high  subgroups compared with the LOH-low subgroup. Thus, ARIEL2 (Part 1) concluded that rucaparib provides significant PFS benefits in platinum-sensitive relapsed EOC patients with BRCA-mutated or BRCA wild-type LOH-high. The findings suggest that tumor LOH can be used to identify platinum-sensitive EOC patients with BRCA wild-type who might benefit from rucaparib. The LOH threshold was adjusted to ≥16% in a post-hoc analysis to improve the selection of patients who will benefit from rucaparib (In the phase II ARIEL2 (Part 1) trial , the staucaparib .BRCA-mutated patients, niraparib was more active in patients who were platinum sensitive to last line platinum-based chemotherapy (39%) compared with platinum-resistant (33%) and platinum-refractory (19%) patients. In the BRCA wild-type HRD-positive cohort, 20% of platinum-sensitive patients achieved a response, but only 2.4% of platinum-resistant and refractory patients had a response similar to the HRD-negative or unknown cohort (3%)  –42.vs. 51.4%) and PFS (13.4 vs. 9.2 months) compared with nonplatinum chemotherapy in patients with gBRCA-mutated platinum-sensitive recurrent EOC patients with at least two prior lines of platinum-based chemotherapy.In the phase III SOLO3 trial , monotheBRCA-mutated cohort  and BRCA wild-type cohort . Study 19 concluded that platinum-sensitive recurrent serous ovarian cancer patients with a BRCA mutation receive the greatest benefits from olaparib maintenance therapy.In the phase II study 19 trial , the medBRCA mutation or myChoice assay with a genomic instability score (GIS) ≥42. The median PFS was significantly longer in the niraparib group than in the placebo group among the gBRCA-mutated cohort , the non-gBRCA-mutated HRD-positive cohort , and the non-gBRCA-mutated cohort . NOVA concluded that the median PFS was significantly longer among the platinum-sensitive recurrent EOC patients receiving niraparib maintenance therapy regardless of the gBRCA mutation or HRD status.In the phase III NOVA trial , the staBRCA-mutated cohort    . The med= 0.054) . SOLO2 cBRCA-mutated cohort , the HRD cohort , and the intention-to-treat population . The benefit was greatest in BRCA-mutated patients, followed by the HRD-positive (defined as BRCA mutated or LOH-high), HRD-positive (defined as BRCA wild-type and LOH-high), and HRD-negative (defined as BRCA wild-type and LOH-low) cohorts. ARIEL3 concluded that maintenance rucaparib significantly improves PFS in platinum-sensitive relapsed EOC patients following a complete or partial response to second-line or later platinum-based chemotherapy.In the phase III ARIEL 3 trial , the staBRCA mutations receiving olaparib maintenance therapy had a significant improvement in PFS than the placebo group . In a 5-year follow-up, the median PFS was significantly longer in patients who received olaparib than those who received placebo   .BRCA mutation or myChoice assay with GIS ≥42. The median PFS was significantly longer in the niraparib group than in the placebo group among the HRD cohort  and the intention-to-treat population . Analyses of GIS in the BRCA wild-type cohort was a preplanned exploratory analysis. The results showed a benefit in all BRCA wild-type patients regardless of the GIS, though it was higher in the GIS-high subgroup compared with the GIS-low subgroup . PRIMA concluded that niraparib maintenance therapy significantly prolonged the PFS in newly diagnosed advanced stage EOC patients who responded to platinum after primary treatment regardless of homologous recombination status , the HRD-positive cohort , and the overall population . VELIA concluded that a regimen of carboplatin, paclitaxel, and veliparib induction therapy followed by veliparib maintenance therapy leads to significantly longer PFS than carboplatin plus paclitaxel induction therapy alone. The PFS benefit was less pronounced in VELIA than in the other frontline studies, possibly because the HRD cohort contained more false positives, with a higher percentage of patients classified as having HRD (55%) despite a lower percentage of BRCA-mutated patients (26%).In the VELIA trial , the staBRCA mutations or myChoice assay with GIS ≥42. The median PFS was significantly longer in the olaparib plus bevacizumab group than in the placebo plus bevacizumab group among the overall population , the BRCA-mutated cohort , the BRCA wild-type cohort , the BRCA-mutated HRD-positive cohort , and the BRCA wild-type HRD-positive cohort . PAOLA-1 concluded that maintenance olaparib provides a significant PFS benefit in advanced EOC patients receiving first-line standard chemotherapy with bevacizumab, especially in patients with HRD-positive tumors, including those without a BRCA mutation. In the HRP cohort, the addition of olaparib to bevacizumab did not improve the PFS . The findings indicate that the GIS has the potential to identify HRD-negative EOC patients who do not derive a benefit from olaparib in combination with bevacizumab  second-line maintenance therapy in recurrent platinum-sensitive disease regardless of BRCA mutation or HRD status, and (3) first-line maintenance therapy for newly diagnosed advanced stage platinum-sensitive disease with/without BRCA mutation or HRD-positive status  in the management of EOC patients: (1) salvage monotherapy in e status Table 3.In general, HRD assays are of three main categories: germline or somatic mutations in genes in the HRR pathway, genomic scars or mutational signatures representing patterns of genomic instability, and checking the function of RAD51 localization to sites of DNA damage , 48–50.BRCA1/2 mutation tests are well known in the clinical management of EOC patients. The gBRCA tests with genetic counseling should be performed near the time of diagnosis for all patients with newly diagnosed EOC     in the VELIA trial  , NOVA (nctively) , and ARIctively) . For newIA trial . However disease –15. ThisBRCA1/2 genes, non-BRCA genes involved in the HRR pathway include ATM, BRIP1, NBN, PALB2, RAD51B, RAD51C, and RAD51D  (BRCA HRR gene mutations were noted in 20 patients (10%), including ATM, BRIP1, CHEK2, FANCA, FANCI, FANCM, NBN, RAD51B, RAD51C, and RAD54L. The sensitivity of non-BRCA HR gene mutations in discriminating the rucaparib response was only 11%. Furthermore, how many non-BRCA HRR genes should be included in the testing list is still unclear. A major challenge for HRR gene testing is the annotation of variants of uncertain significance (VUS) (BRCA VUS (3%–50%), the protocols for detection, and the strategies for management between individual laboratories  . In ARIEce (VUS) –70. In tce (VUS) . There iratories . CurrentDefects in the HRR pathway generate genetic variations, chromosome structural abnormalities, and genomic instability. These so-called genomic scars or mutational signatures are permanent even if the function of the HRR pathway is restored, which represents a record of DNA damage repair through different pathways in response to harmful exposure in cells . GeneticBRCA LOH (Foundation Medicine) are currently the most common commercial assays (BRCA mutation test and GIS. The Foundation Focus CDx BRCA LOH (Foundation Medicine) includes a tumor BRCA mutation test and genomic LOH. Both assays use next-generation sequencing (NGS) platforms to identify SNVs, indels, and large rearrangements in genes of tumor tissue  and Foundation Focus CDx l assays , 79. Ther tissue , 78, ando assays . In the L2 trial . In the BRCA mutation tests are routinely recommended to identify EOC patients who should receive PARPi therapy. A validated HRD assay is reasonable to stratify BRCA wild-type EOC patients who would benefit from PARPi therapy  subgroup constituted approximately 50% of all participants in these trials , 86% in a validation breast cancer cohort (n = 80), and nearly 100% in validation ovarian cancer (n = 73) and pancreatic cancer (n = 96) cohorts. However, the ability of HRDetect to predict the PARPi response in EOC has not been confirmed  performed better than non-BRCA HRR gene mutations  or promotor hypermethylation  in predicting a response to rucaparib. Some technical problems still need to be resolved, including sample purity, quantitative methylation assays, and gene copy number changes  samples of breast cancer, and positive RAD51 foci correlated with resistance to platinum or PARPi . Samples were defined as HR-low (<10%–20% RAD51-positive cells), HR-intermediate (10%–20% to 35%–50% RAD51-positive cells), or HR-proficient (>35%–50% RAD51-positive cells). However, the correlation of the clinical response with the organoid RAD51 foci by immunohistochemistry needs to be determined , tissue resources , methods of inducing DNA damage , methods of detection (immunofluorescence or immunohistochemistry), measurement of RAD51 foci (numbers or percentages), intratumor or interobserver variability, and the definition of HRD in correlation with the clinical response .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Flavivirus genus (Flaviviridae family) pose an ongoing threat to global public health. For example, dengue, Japanese encephalitis, West Nile, yellow fever, and Zika viruses are transmitted by infected mosquitoes and cause severe and fatal diseases in humans. The means by which mosquito-borne flaviviruses establish persistent infection in mosquitoes and cause disease in humans are complex and depend upon a myriad of virus-host interactions, such as those of the innate immune system, which are the main focus of our review. This review also covers the different strategies utilized by mosquito-borne flaviviruses to antagonize the innate immune response in humans and mosquitoes. Given the lack of antiviral therapeutics for mosquito-borne flaviviruses, improving our understanding of these virus-immune interactions could lead to new antiviral therapies and strategies for developing refractory vectors incapable of transmitting these viruses, and can also provide insights into determinants of viral tropism that influence virus emergence into new species.Mosquito-borne viruses of the  Flavivirus genus (family Flaviviridae) are principally transmitted by mosquitoes and ticks, with some flaviviruses having no known vector or being insect-specific viruses with no vertebrate host [Arthropod-borne viruses (arboviruses) of the ate host . Mosquitate host . Furtherate host ,4.Flaviviruses are enveloped positive-sense single-stranded RNA viruses that encode a single open reading frame flanked by 5′ and 3′ untranslated regions (UTRs) . The strDrosophila melanogaster and are highly conserved with those of humans and mosquitoes such as Aedes aegypti, which are classified in the order Diptera (true flies) along with D. melanogaster [The innate immune response is one of the early defense mechanisms against viral infection in vertebrates  and is anogaster ,12. Mosqnogaster . MoreoveThis review highlights similarities and differences in the major innate immune responses to mosquito-borne flaviviruses in the vertebrate host and invertebrate vector. We also focus on the known molecular mechanisms by which mosquito-borne flaviviruses antagonize the antiviral innate immune responses that are conserved between their mosquito vectors and the human host. Besides encoding immune antagonists, several other strategies are utilized by mosquito-borne flaviviruses to evade the innate immune response in humans and/or mosquitoes ,18,19,20Following the detection of mosquito-borne flaviviruses in human cells, the innate immune signaling response is activated, leading eventually to the expression of proinflammatory cytokines and type I IFN . In mosqNY99) or the ectopic expression of WNVNY99 NS1 [Mosquito-borne flaviviruses block the activation of different signaling molecules including those of TLR signaling in humans . For exaNY99 NS1 ,38. MoreN-linked glycosylation of mosquito-borne flaviviruses has a great impact on viral virulence and immune evasion as protein glycosylation is divergent between the mammalian and insect cells, particularly in terms of N-linked glycans [ glycans ,40. For  glycans . The dip glycans .When viral RNA is sensed by RIG-I in human cells, RIG-I is activated through a polyubiquitination step, an activity mediated by the E3 ligase tripartite motif 25 (TRIM25) . The sfRIn addition to PRR antagonism, mosquito-borne flaviviruses can antagonize the downstream adaptor and signaling molecules in human cells . The 14-Mosquito-borne flaviviruses block the downstream signaling cascades at different levels . For exaNS2B of DENV-2 and DENV-4 is able to antagonize the cGAS-STING pathway by promoting cGAS degradation in an autophagy-lysosome-dependent manner . MoreoveFew studies have focused on the identification of innate immune antagonists of mosquito-borne flaviviruses in mosquitoes . DENV-2 IFNs are a family of cytokines that play an important role in controlling viral infection in humans, and they are classified into type I, II, and III IFNs . AlthougOnce a successful viral replication cycle is achieved in humans, IFN treatment does not restrict the replication of mosquito-borne flaviviruses such as DENV, WNV, and ZIKV ,94,95, wFollowing the activation of JAK-STAT signaling in human cells, mosquito-borne flaviviruses block STAT2 function through various conserved and virus-specific mechanisms A. STAT2 In addition to NS5, DENV-2 encodes additional STAT antagonists, as the collective activity of NS4B, NS4A and NS2A has been found to abrogate STAT1 phosphorylation in response to IFN-β stimulation . InteresCulex quinquefasciatus STAT by upregulating the E3 ubiquitin ligase cullin 4 [Aedes albopictus) cells [Mosquito-borne flaviviruses such as WNV and JEV have been shown to antagonize STAT proteins in mosquitoes B, althoucullin 4 . In addis) cells .RNAi is a highly conserved evolutionary pathway across species from different kingdoms  in which small interfering RNAs (siRNAs), microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs) regulate endogenous gene expression and/or provide antiviral immunity ,113,114.It is under debate whether mosquito-borne flaviviruses encode viral suppressors of RNAi (VSRs) that function in human cells. However, mosquito-borne flaviviruses such as ZIKV can infect the placenta and fetal brain by targeting neural progenitor cells, impairing their differentiation and proliferation ,123. ZIKOn the other hand, some studies have reported RNAi pathway suppression by mosquito-borne flaviviruses in differentiated human cells A. For exA. aegypti when expressed recombinantly by Sindbis virus [A. aegypti) cells [KUNV blocks the RNAi pathway by interacting with Ago2 and Dicer-2, which in turn, inhibits dsRNA cleavage in the RNAi-competent mosquito  cells and C. quinquefascitus mosquitoes [Culex pipiens mosquitoes [A. aegypti-derived Aag2 cells lacking Dicer-2 (but not cells lacking Ago2) [A. albopictus-derived C6/36 cell line [Mosquito-borne flaviviruses encode VSRs that antagonize vsiRNAs production in mosquitoes B 129,13. For exais virus . Similari) cells . The sfRsquitoes ,128. Howsquitoes . ZIKV sfsquitoes . ZIKV sfsquitoes . Howeverng Ago2) , no diffell line .D. melanogaster. Furthermore, insect NF-κB and Jak-STAT responses are poorly understood in terms of how they may detect and control viral infection relative to the RNAi pathway’s antiviral functions, and in this respect studying flaviviral immune antagonists may also help provide much needed new insights into basic immune functions.The innate immune response against mosquito-borne flaviviruses poses an important barrier that limits the replication and spread of viral infection, as well as influencing the host tropism of flaviviruses. In contrast to the human innate immune response, which is normally able to eliminate and clear viral infection, mosquitoes only limit viral dissemination and transmission without clearing the viral infection. Thus, revealing the intricate interplay between flavivirus-encoded immune antagonists and antiviral host effector molecules is crucially important to improve our understanding of viral pathogenicity and disease severity in humans and viral persistence and fitness costs in mosquitoes. While the molecular immune antagonism mechanisms of mosquito-borne flaviviruses are well studied in humans, these studies are less well developed in mosquitoes, particularly for the innate immune signaling cascades. An interesting question is to investigate whether the flavivirus-encoded immune antagonists of mosquito-borne flaviviruses display similar antagonistic activity against the evolutionarily conserved signaling pathways in human and mosquito cells. Another key question is to determine whether these antagonistic activities are mediated by distinct protein domains from the separate roles of these viral proteins in viral replication and maturation. Particularly in the vector, flaviviral antagonists of the immune system could prove to be useful tools for elucidating the molecular events contributing to innate immune function, which remain incompletely characterized in mosquitoes compared to the distantly related model organism Interestingly, while some flavivirus-encoded immune antagonists are conserved in their ability to antagonize innate immunity across different flaviviruses, other flavivirus-encoded immune antagonists function differently in different viral species. Elucidating how these molecular differences are driven by the evolutionary arms race between virus and host might provide insight into viral emergence and could be exploited for rational vaccine design. On a practical level, it may be possible to develop improved small animal models for flaviviral diseases by modifying their innate immune system to be better antagonized by flaviviruses . Such models would be transformative tools to facilitate the development of much-needed antiviral drugs and vaccines. Meanwhile, modifying the vector’s immune system is a potential strategy for developing refractory mosquitoes that cannot transmit flaviviruses. Overall, future developments in this area have the potential to help reduce the increasing global public health burden of flaviviral diseases while improving the preparedness for the potential future emergence of new flaviviral pathogens.


MDR1-gene are known to show adverse reactions to certain drugs, yet metabolic imbalance exacerbated by physiologically immature organs and other contributing pathologies require consideration before arriving at a diagnosis. This study analysed the brains of two male, 5-week-old Australian Shepherd siblings that died after displaying severe neurological symptoms upon administration of MilproVet® to treat severe intestinal helminth infection. Despite the initial symptoms being similar, their case histories varied in terms of the symptom duration, access to supportive therapy and post-mortem interval. Histopathology and immunohistochemistry were used to obtain more information about the phase of the pathological processes in the brain, employing protein markers associated with acute hypoxic damage (β-amyloid precursor protein/APP) and apoptosis . The results seem to reflect the course of the animals’ clinical deterioration, implicating that the hypoxic damage to the brains was incompatible with life, and suggesting the usefulness of the mentioned immunohistochemical markers in clarifying the cause of death in animals with acute neurological deficits.Seizures in puppies often present a diagnostic challenge in terms of identifying and treating the underlying cause. Dog breeds with mutations of the  MDR1 status may contribute significantly to arising neurological deficits [Puppies with seizures are challenging veterinary patients, with ongoing post-natal organ maturation predisposing them to metabolic crises such as hypoglycemia  and inabdeficits . This frdeficits . In thisdeficits  in seizun = 8) of 5-week-old Australian Shepherd puppies presented severe neurological symptoms (videos thereof may be found in the ® each (containing 2.5 mg milbemycin oxime/25 mg praziquantel): owners refer seizures, tremors and ataxia with manifestation of severe abdominal pain 12–24 h after its administration. One male (Puppy-1) died before arrival to the Veterinary Teaching Hospital of the University of Padova. A second male (Puppy-2) succumbed about an hour after initiation of supportive therapy. Their littermates recovered within 1.5 h of flow-by oxygen administration, intravenous ringer-lactate (4 mL/kg/h) and Intralipid® 20%  infusion. Puppy-3 suffered two convulsive crises 3 h upon initiation of supportive care, recovering after repeated diazepam administration. A litter . A board-certified pathologist (R.V.) checked all the slides to assess histological and IHC findings.MDR1-gene mutation via allelic discrimination [Primary polyclonal antibodies used for IHC included anti-amyloid precursor protein , rabbit anti-diacylglycerolkinase-ζ , anti-apoptotic protease activating factor-1 , and anti-B-cell lymphoma related protein . The whole litter of Australian Shepherd puppies was tested for Gross findings in the Australian Shepherd puppies were scant, with both livers appearing mildly enlarged, with fragile consistency, light-red coloration and rounded edges. Additionally, Puppy-2 displayed multifocal to coalescing pulmonary consolidation.MDR1-gene mutations were present only in Puppy-1 and Puppy-4—the rest presented the wild-type phenotype.Histopathology confirmed the mild to moderate edema and congestion observed in all examined internal organs, and further revealed multifocal cortical laminar necrosis, superficial spongiosis and meningeal congestion of mild and mild to moderate severity in Puppy-2 and Puppy-1, respectively. Multifocal Purkinje cell degeneration was observed in the cerebellum, with perivascular edema of the molecular and Purkinje cell layers and the white matter (WM). These changes were mild and multifocal in Puppy-2, but moderate and multifocal to coalescing in Puppy-1, with additional Purkinje single cell necrosis. Both animals had mild to moderate, diffuse, micro- and macro-vesicular hepatic steatosis, and mild multifocal subacute lymphohistiocytic enteritis with reactive GALT. Both lungs displayed mild to moderate, multifocal, subacute, histiocytic interstitial pneumonia. Multifocal atelectasis, severe perivascular edema, interstitial hemorrhages, and a focal eosinophilic granuloma were observed in Puppy-2, while Puppy-1 presented mild emphysema at the lobe tip, multifocal interstitial and alveolar hemorrhages, and agonal edema. Heterozygous IHC results for the brain and the markers used are summarized in Control brain tissues were included for comparison to a dog that did not have any neurological symptoms at time of death , likely having endured only mild agonal hypoxia. Two control puppies presenting neonatal immaturity and additional inflammatory symptoms in the lungs  and metabolic imbalance as evidenced by perihepatic mineralizations  were included to assess potential changes of IHC-signal deriving from post-mortem protein degradation.APP-reactivity of Control-1 was limited to a very mild, arborized or granular signal around single cortical neurons. Controls-2 and -3 both presented a background in the neuropil that was lighter than in both Australian Shepherd puppies, with multifocal cytoplasmic and synaptic neuronal immunoreactivity (+/++), with a mildly enhanced signal in the capillary vessel walls and meninges of Control-2. Control-2 and Control-3 were negative for Bcl-2, while Control-1, being mostly negative, presented findings more subtle, but reminiscent of those in Puppy-2, with a few foci of mild, cytoplasmic immunoreactivity (+) in neurons and neuropil. DGK-ζ was detected only in neuronal nuclei of Control-1 (+/++), while alternating between nuclear and cytoplasmic compartments in Control-2 (++/+++) and appearing exclusively in the cytoplasm in Control-3 (+). Apaf1-signal was most prominent as a radiating pattern from the superficial cortical layers and choroid plexus-adjoining regions in Control-2, and as a multifocal to coalescing immunoreactivity of endothelial cells and meninges (+++) in Control-3. No Apaf1 signal was observed in Control-1.Despite few macroscopic changes, histopathology revealed that the puppies were compromised under several aspects. Severe intestinal parasitic infection could have led to deficits in energy uptake and protein absorption in a challenging period such as weaning, inflicting initial damage to a physiologically immature liver . Large bLesions caused by hypoxia were evident in the brains of both Australian Shepherd puppies. Cerebellar Purkinje cells are notoriously sensitive to acute hypoxic stress , and theAPP is a synaptic membrane protein that plays a role in various physiological functions like axonal outgrowth and neuronal adhesion. It is expected in moderate concentrations in the developing brain . Under pThe DGK-ζ isoform physiologically localizes in the nucleus of neurons, but has been shown to acutely and irreversibly translocate into the cytoplasm of hippocampal neurons following ischemic injury, before gradually disappearing . Its absBcl-2 is an anti-apoptotic protein , yet accApaf1 reflects intrinsic apoptosis upstream of executioner caspase-activating caspase 9 . MultifoRelevant IHC-findings in Puppy-1 include widespread APP-, faded DGK-ζ- and multifocal Bcl-2-immunoreactivity in cortical neurons simultaneous to necrotic shrinkage, diffuse cerebellar early-apoptotic Apaf1-reactivity and severe necrosis as evidenced by complete multifocal disintegration of Purkinje-neurons. This pattern matches advanced necrotic and progressive apoptotic changes. Puppy-2’s central nervous system rather reflects acute necrosis and pre- to early apoptosis with intense cortical DGK-ζ-, multifocal cerebellar Apaf1- and lack of Bcl-2-expression, and multifocally shrunken, but mostly intact neurons. Though apoptosis is more prominent in immature brains if compared to adults , necrotiMDR1-gene mutations responding to macrocyclic lactones with mild depression and ataxia, or more severe, transient (<48 h) neurotoxicosis preceding full recovery [Single reports describe dogs with heterozygous recovery . Furtherrecovery . Compromrecovery ,30, may MDR1 mutant, responded well to the supportive care. Meanwhile, MDR1-wild-type Puppy-3 suffered further seizures but responded well to diazepam, unlike dogs with moxidectin toxicity [Puppy-4, also being a heterozygous toxicity . These fThis study is based on diagnostic cases, and therefore contains inevitable limitations. In particular, the selection of control animals without neurological changes is problematic, thus our knowledge on a healthy profile for the dog brain is limited. Ideally, controls would be healthy experimental, age-, sex-, and breed-matched dogs that had little to no hypoxic changes within the brain tissues at the time of death. In reality, every animal, whether dying of natural causes or euthanasia, experiences some extent of agonal hypoxia , therefoIn this respect, consistency between clinical state and IHC findings, as well as similar IHC-patterns seen in animals with a PMI of up to about 72 h, render it unlikely that the longer post-mortem interval of Puppy-1 significantly compromised IHC results. The robustness of some protein markers in human post-mortem neuronal tissue is postulated to be conserved at a PMI of up to 50 h, though this varies by marker . ChangesConsidered together, the anamnestic, clinical, histological and immunohistochemical findings point to a multifactorial hypoxic syndrome as the cause of acute neuronal degeneration, contributing significantly to the disruption of vital functions and resulting in the puppies’ deaths. Direct drug toxicity is unlikely but cannot be excluded, since single-gene approaches to pharmacogenetics leave considerable individual variation to be explored with pharmacogenomics . The rec


Earlier studies investigating the pathogenesis of chronic vascular inflammation associated with atherosclerosis described pro-inflammatory and vascular barrier disruptive effects of lipid oxidation products accumulated in the sites of vascular lesion and atherosclerotic plaque. However, accumulating evidence including studies from our group suggests potent barrier protective and anti-inflammatory properties of certain oxidized phospholipids (OxPLs) in the lung vascular endothelium. Among these OxPLs, oxidized 1-palmitoyl-2-arachdonyl-sn-glycero-3-phosphocholine (OxPAPC) causes sustained enhancement of lung endothelial cell (EC) basal barrier properties and protects against vascular permeability induced by a wide variety of agonists ranging from bacterial pathogens and their cell wall components, endotoxins, thrombin, mechanical insults, and inflammatory cytokines. On the other hand, truncated OxPLs cause acute endothelial barrier disruption and potentiate inflammation. It appears that multiple signaling mechanisms triggering cytoskeletal remodeling are involved in OxPLs-mediated regulation of EC barrier. The promising vascular barrier protective and anti-inflammatory properties exhibited by OxPAPC and its particular components that have been established in the cellular and animal models of sepsis and acute lung injury has prompted consideration of OxPAPC as a prototype therapeutic molecule. In this review, we will summarize signaling and cytoskeletal mechanisms involved in OxPLs-mediated damage, rescue, and restoration of endothelial barrier in various pathophysiological settings and discuss a future potential of OxPAPC in treating lung disorders associated with endothelial barrier dysfunction. These bioactive OxPLs possess profound biological activities and exert both beneficial and harmful effects on human body governed by their biochemical and biophysical properties. It has been long recognized that OxPLs accumulate in atherosclerotic lesions –3, and loperties . On the operties . Multiplsn-2 position. PUFAs are highly prone to oxidative modifications by a group of specific enzymes such as cyclooxygenases (COX) and lipoxygenases (LOX) or reactive oxygen species (ROS)-mediated non-enzymatic lipid oxidation (in vitro and LPS-induced lung injury in vivo (A diverse spectrum of OxPLs are generated from the oxidation of phospholipids that contain polyunsaturated fatty acids (PUFA) at their xidation , 12, 13.xidation , 15. Thexidation . PGE2, P in vivo . Similar in vivo –19. Furt in vivo –23. Both in vivo .sn -glycero-3-phsphocholine , among others. Conversely, fragmented OxPAPC products are oxidatively truncated at the sn-2 position. 1-Palmitoyl-2--sn-glycero-phosphatidylchonine (POVPC), 1-palmitoyl-2glutaroyl sn-glycero-phosphocholine (PGPC), 1--2-(5-keto-6-octene-dioyl)phosphatidylcholine (KOdiA-PC) represent examples of such products -sn-glycero-3-phosphatidyl choline (PEIPC) and 1-palmitoyl-2- products . Besidesproducts . Furtherproducts .A continuous monolayer of EC covers vascular lumen and provides a highly selective semi-permeable barrier between the circulation and underlying tissues. EC barrier controls the passage of fluids, solutes, and cells across the vascular endothelium. Various injurious stimuli disrupt the endothelial barrier integrity leading to an increased endothelial permeability for macromolecules and immune cells that is a hallmark of numerous disorders such as pulmonary edema, ARDS, sepsis, and other pathologies –29. BioaA role of OxPLs in various cardiopulmonary disorders including ALI , ARDS 33, pulmonIn contrast to these pathological roles of OxPLs, multiple studies have demonstrated potent anti-inflammatory and endothelial barrier protective functions of OxPLs in host defense against bacterial pathogens. Initial studies demonstrated a protective role of OxPAPC against endothelial dysfunction caused by bacterial wall lipopolysaccharide (LPS) in cultured EC as well as in murine models of ALI , 38, 39.Endothelial barrier is a dynamic structure that constantly undergoes remodeling in response to mechanical forces and various agonists that positively or negatively regulate barrier function. Altered expression of cell junction proteins, the assembly-disassembly dynamics of adherens junction  and tight junction  protein complexes and reorganization of endothelial cytoskeleton in response to chemical and mechanical stimulation are the major determinants of endothelial barrier integrity. While barrier-disruptive insults (edemagenic agonists or high magnitude cyclic stretch) stimulate stress fiber formation, actomyosin contraction, disassembly of cell junction complexes leading to cell retraction and formation of inter-cellular gaps, barrier-enhancing agonists  or physiologic mechanical forces  stimulate enhancement of cortical cytoskeleton, assembly and cooperation of AJ and TJ protein complexes , 44. Remvia interference with toll-like receptors (TLRs) inflammatory signaling cascade activated by LPS  in vivo  and Rap1 are critical regulators of endothelial barrier in physiological and pathological conditions . These G in vivo . Later s in vivo . This no in vivo . In addi in vivo . An impo in vivo . Our stu in vivo . In this in vivo . Consistcadherin . The dircadherin Figure 2via activation of PKA, PKC, and MAP kinase pathways , protein kinase C (PKC), Raf-MEK MAP kinase cascade, and JNK MAP kinase . Furtherpathways , and viativation  but its f OxPAPC . Figuref OxPAPC . It willAs discussed above, truncated products of phospholipids oxidation possess endothelial barrier disruptive properties. Moreover, even OxPAPC at higher concentration (≥ 50 µg/ml) causes barrier disruption as opposed to its barrier enhancing function at lower concentrations (5-20 µg/ml) , 68. Thevia VEGFR2 activation  as a prototype synthetic phospholipid compound with barrier-protective and anti-inflammatory properties  techniques along with the use of sophisticated bioinformatics analysis has now made possible to detect minor modifications in OxPLs structure and its possible impact on biological activities. These advanced analysis and detection techniques combined with omics analysis approaches including lipidomics, metabolomic, transcriptomic, and proteomics will reveal the signaling cascades associated with OxPLs that will assist in identifying potential therapeutic targets for OxPLs-derived diseases. Furthermore, oxidative phospholipidomics analysis may serve as a valuable tool for identification of biomarkers as suggested by a recent study characterizing oxygenated cardiolipins and phosphatidylethanolamines as predictive biomarkers of apoptotic and ferroptotic cell death, respectively . Future It is now widely appreciated that the complexity of contrasting biological effects of OxPLs largely relies on their structural heterogeneity with full-length oxidized products exerting beneficial effects and truncated species acting as pathogenic factor in various cardiopulmonary disorders. The studies so far have established that OxPAPC itself or signaling intermediates that mediate its functions on endothelium, barrier-disruptive OxPLs targeting enzymes such as PAFAH2, and structure-based synthetic analogues of barrier-protective OxPLs could be developed into therapeutics against acute endothelial dysfunction associated with injury, infection, or sepsis. The utilization of LC-MS advanced detection techniques in combination with next generation omics analytical tools has enabled to precisely monitor the lipid modifications and possibly use these modifications as biomarkers for various pathologies. Thus, the dual biological nature of OxPLs presents the opportunity to consider both “good” and “bad” oxidized products of circulating or membrane phospholipids for therapeutic interventions.PK and KB outlined the review. PK drafted the manuscript and KB finalized. All authors contributed to the article and approved the submitted version.The studies in authors laboratory have been supported by RO1HL076259, R01HL146829, and RO1HL087823 grants from National Heart, Lung, and Blood Institute and RO1GM122940 from National Institute of General Medical Sciences to KB.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Among single-cell analysis technologies, single-cell RNA-seq (scRNA-seq) has been one of the front runners in technical inventions. Since its induction, scRNA-seq has been well received and undergone many fast-paced technical improvements in cDNA synthesis and amplification, processing and alignment of next generation sequencing reads, differentially expressed gene calling, cell clustering, subpopulation identification, and developmental trajectory prediction. scRNA-seq has been exponentially applied to study global transcriptional profiles in all cell types in humans and animal models, healthy or with diseases, including cancer. Accumulative novel subtypes and rare subpopulations have been discovered as potential underlying mechanisms of stochasticity, differentiation, proliferation, tumorigenesis, and aging. scRNA-seq has gradually revealed the uncharted territory of cellular heterogeneity in transcriptomes and developed novel therapeutic approaches for biomedical applications. This review of the advancement of scRNA-seq methods provides an exploratory guide of the quickly evolving technical landscape and insights of focused features and strengths in each prominent area of progress. New data show that cells arising from the same tissue origins are far more heterogeneous than they seemingly appear . Ev,11–16. Cellular transcriptomic heterogeneity helped to establish a new paradigm of cellular heterogeneity with the invention of scRNA-seq–19. The cellular transcriptomic heterogeneity arises from stochasticity, differentiation, environmental stimuli, diseases, aging, and other factors,11,20–23. The development of single-cell analysis was overshadowed by traditional bulk cell approaches and technically limited by the absence of high throughput single-cell isolation and minute initiation materials (picogram DNA and mRNA per cell). Combined technological advances in cell isolation, high throughput multiplexing, amplification, and next generation sequencing facilitated scRNA-seq and uncovered cellular heterogeneity. Mapping transcriptomic changes at single-cell level has since revealed global gene expression profiles and exposed stochasticity, differentiation, cell fate plasticity, and diseases. In this review, we highlight the novel scRNA-seq platforms and conduct a comparative analysis of each technology and their future applications in translational science.In the new paradigm, the diverse properties of cells are mainly reflected in the heterogeneous gene expression, genomic alterations, epigenomic modifications, and proteomic fluctuationsCell purity is paramount for scRNA-seq and other single-cell analysis methods. Tissues, organoids, and 2D and 3D cultured cells are multi-cellular, and the first step to dissociate aggregated cells into individual cells risks potential contamination with cell doublets, DNA, and RNA by incomplete enzymatic digestion and cell lysis. The impurity of single cells distorts the scRNA-seq data and leads to false interpretations. To ensure the purity and integrity of single cells, several instrumental technologies have been adopted to overcome these technical challenges.. The instrumental efficiency of picking individual cells exceeds old-fashioned mouth-pipetting. Specifically, cells are maintained in suspension and manually isolated by capillary pipettes connected with a microinjector. Cellular integrity is maintained for further analysis and is particularly workable with rare cells. However, high operator skills are required through training and practice. Additionally, the throughput is relatively low compared with the other methods.Manual cell picking presents a simple and cost-effective method for single-cell isolation. This technique involves an inverted fluorescent microscope, manipulator, and microinjector for precise cell location and picking after cells are labeled with markers,28. With advanced fluorochrome and microscope techniques, 18 fluorescent, inorganic semiconductor nanocrystals (Quantum Dots) are used to label antigens on cells, which increases the specificity and sensitivity of single-cell isolation from a bulk sample,30. However, greater than 10,000 cells are required for this method and signal overlap may affect the purity of the target cells. Moreover, this method cannot perform single-cell analysis with rare cells.This high throughput method relies on antibody affinity to cell surface markers and has become the most common strategy for single-cell isolation. Cells are labeled with fluorescent or conjugated antibodies and run through flow cytometry, sensed by laser detectors or a magnetic field, and sorted with surface specific markers. In droplet-based microfluidics, each single cell is embedded in a hydrophilic droplet which suspends hydrophobic channels. The advantages of this approach are high throughput and yield, making it feasible to isolate rare cell types. Besides, genetic barcodes can be added within the cell droplet that record the cell origin, allowing profiling of cells from simultaneous preparation of thousands of single-cell libraries,34. In channel-based microfluidics, single cell is controlled and confined by pneumatic membrane valves according to the biological requirements. This selection approach increases the accuracy of cell isolation and the flexibility of experimental design. However, it is limited by the low throughput compared with droplet-based microfluidics. Hydrodynamic traps such as Fluidigm C1 passively isolate and trap single cells based on cell size,32. Both channel-based microfluidics and hydrodynamic traps enable long-term cell culture and high-resolution observation for further biological experiments such as drug treatment or cDNA library preparation.This technology for single-cell isolation can be divided into three main approaches: droplet-based microfluidics, channel-based microfluidics, and hydrodynamic traps. These methods rely on cell adhesion, hydrodynamics, physical characteristics , cellular density, and elasticity. Microfluidic technology platforms can actively or passively recognize and sort single cells from a heterogenic population,37. Laser capture microdissection is a rapid and precise isolation technology that maintains versatility for further analysis including scRNA-seq,39.This microscopy-based technology carries out isolation of specific single cells on a microscope slide without cell dissociation from solid samples. Tissue sections are either top-covered by or laid on a thermoplastic polymer film, which is heat-activated by infrared or ultraviolet laser. The boundaries of a single cell on a tissue section are recognized and severed precisely by laser, and the dissected cell is captured,41. The protocols of scRNA-seq have fruitfully advanced in a decade [,25,42–44. The technical variations have strengths and weaknesses in linear amplification, length coverage, low copy RNA species detection, multiplexing, high throughputs, and cost reduction. Tang’s protocol was the first scRNA-seq modality and was based on single-cell RNA amplification from RNA microarray assays,46. Tang’s protocol uses poly(T) primers to generate full-length cDNA of transcripts less than 3 kb and can detect ~13,000 genes, 65% of microarray genes. Two years later, Single-cell Tagged Reverse Transcription sequencing (STRT-seq) introduced template switching to incorporate bead-linked barcoded primers for strand-specific amplification of 3’ ends and high throughput 96-cell multiplexing with 2000–4000 genes detected in individual cells,47,48. In 2012, a significant advancement for full-length cDNA synthesis of 40% transcripts was made with Smart-seq, and it was updated with the Smart-seq2 in 2013–51. Smart-seq has laid the foundation for future scRNA-seq methods, employing more stable template switching ribo(guanosine)3 oligos and having more unique mapping reads, higher recovery rates of low expression genes, and a two-fold increase in spliced forms discovered. At about the same time, Cell Expression by Linear amplification and sequencing (CEL-seq) utilized linear strand-specific in vitro transcript amplification mainly at 3’ ends, and an improved CEL-seq2 version reduced mRNA molecule counting biases with the introduction of unique molecular identifiers (UMIs)–55. Single Cell RNA Barcoding and sequencing (SCRB-seq) is a protocol for high throughput of 12,000 cells at a low cost and one of the first scRNA-seq protocols to include UMIs. Previous scRNA-seq platforms utilized relative measures such as reads per kilobase per million reads (RPKM), which masked differences in total mRNA content. As an example, a gene may be “upregulated” in terms of RPKM and have a decrease in absolute expression level. UMIs are short unique sequences integrated in cDNAs before PCR amplification to allow for unique identification of amplified DNAs carrying the same UMI sharing the same mRNA/RNA molecule origin and reduce nonlinear PCR amplification bias. For full length transcript coverage and analysis of noncoding RNA, Multiple Annealing and dC-Tailing-based Quantitative single-cell RNA-seq (MATQ-seq) and Random Displacement Amplification sequencing (RamDA-seq) can be employed, which allow for poly(A)+ and non-poly(A) scRNA-seq, useful for characterization of lncRNA or circRNA–59. RamDA-seq also detects enhancer RNAs differentially expressed in a cell-type specific manner. Quartz-Seq builds upon both CEL-seq2 and STRT-seq to perform 3’ coverage scRNA-seq, vastly improving poly(A) tailing and initial read UMI conversion and augmenting sequencing depth and accuracy.The uniform and full coverage of cDNA synthesis from single-cell mRNA/RNA is a crucial step for the success of scRNA-seq because the limited starting materials are as little as 5–30 pg and need to be amplified for next generation sequencing. The cDNA synthesis from single cells has been attempted for qRT-PCR and microarrays, and the technical predecessors were adopted and modified for scRNA-seqa decade [19,25,42. Drop-seq and InDrop are two similar methods that employ droplet capture microfluidic methods to isolate cells. The main differences are that Drop-seq uses reagent containing beads, while InDrop uses reagent carrying hydrogel microspheres. Both platforms can quickly process tens of thousands of cells daily,34. Chromium is similar to both previous methods, employing a gel bead in emulsion microfluidic capture method, but has the advantage of being able to process eight samples at once or a single sample more quickly due to its eight-channel microfluidic chip. Seq-Well is a low-cost alternative, not requiring any expensive microfluidic devices and instead utilizing semi-permeable membranes on a picowell plate with wells that contain one barcoded capture bead and space for one single cell per well. Seq-Well plates have ~86,000 wells, but the actual capture efficiency varies. Similar to the other platforms , CytoSeq employs a microfluidic method automating high throughput cell settling in 1/10 of 100,000-well plate by gravity. It employs a similar plate system to Seq-Well with 30-μm well sizes only allowing one cell per well and one magnetic bead with a universal primer plus 106 diverse UMIs created by a split-pool synthesis process. It can easily reach up to 10,000 cells with detection of ~100 genes per cell.Many methods employ cDNA amplification strategies, as mentioned above, but they each have unique methods of single-cell sorting and significant high throughput improvement. MAssively parallel RNA Single-cell sequencing (MARS-seq) uses fluorescence-activated cell sorting sorting to separate and sort 100–1000 cells into individual wells. sci-RNA-seq performs the analysis of single cells or nuclei isolated from methanol-fixed whole organisms , with 3’ coverage and high depth sequencing employing double UMI barcoding. Split-Pool Ligation-based Transcriptome sequencing (SPLiT-seq) is an extremely high throughput 3’ coverage method distinct from other methods, offering scRNA-seq analysis without single-cell isolation from 1.33% formaldehyde-fixed tissues. It employs combinatorial indexing to identify single cells without isolation, by performing three successive barcoding steps through in situ reverse transcription on groups of cells and mixing after each time, leaving each cell with a unique identifier totaling up to 21 million for downstream data analysis. This invention posits potential cost reduction per cell and time effectiveness.For harder to work with tissues, such as frozen samples, DroNC-seq is able to salvage samples and produce high quality data, employing single nucleus RNA-seq with 3’ coverage. The protocol requires knowledge of the intended target’s genome prior to use. SC3-seq provides 3’ coverage method for only 3’ end characterization of mRNA, allowing for higher reproducibility and reduced noise, thus it is useful for projects not requiring deep sequencing with a low budget,70.Not all methods employ UMIs. Designed Primer-based RNA-sequencing (DP-seq) is a 3’ coverage method useful for small samples, analyzes at least 50 pg of RNA, and employs random hexamer-based amplification and sequencing. Six commonly used methods - CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq, and Smart-seq2 - were performed side by side using mouse embryonic stem cells. Overall, Smart-seq2 detected the most genes per cell, while the other methods displayed less amplification noise due to the use of UMIs. Power analysis showed that Drop-seq provides a less costly option for a larger number of cells analyzed, whereas MARS-seq, SCRB-seq, and Smart-seq2 were suitable for fewer cells. To evaluate sensitivity and accuracy as performance metrices for 15 different methods, in silico power analysis of External RNA Controls Consortium spike-in standards was performed in those scRNA-seq studies. The vast majority of methods displayed high accuracy. The exceptions were CEL-seq and MARS-seq data, which show more variations among cells. Better sensitivity appeared in SMARTer (C1), CEL-Seq2 (C1), STRT-Seq, and InDrop-seq that could detect digital copies of spike-ins; however, the sensitivity was sequencing depth-dependent. For the droplet-based high-throughput platforms , a thorough comparative study revealed insights regarding their efficacy and limits. The 10X Genomics Chromium protocol was maturely developed with a higher cost and delivered high degree of sensitivity and accuracy with less technical noise. Drop-seq provided similar data quality with fewer cells, but a more affordable cost. InDrop is also less expensive with high compatibility with other protocols, such as Smart-seq2. In a large-scale validation of 13 protocols by multi-centered collaboration effort using mixed human and murine cells, CEL-seq2, Quartz-seq2, SMAR-seq2, and Chromium platforms were superior in producing high-resolution transcriptomic profiles.Attempts have been made to evaluate diverse scRNA-seq protocols using systematic comparisons,75. However, major efforts in the development of advanced algorithms and computational strategies as well as adaptations of existing workflows have shown great promise for comprehensive and detailed analysis of scRNA-seq data [,76. Seurat,78 and Single Cell ANalysis in PYthon are the two most comprehensive packages that can, respectively, integrate scRNA-seq data with other single-cell data and enable scaling-up to simultaneously analyze millions of cells at the same time. Of note, the core analysis pipelines show higher resemblance with the bulk RNA-seq and can be broadly categorized in the following: (1) quality control; (2) read alignment and generation of counts; (3) removal of confounding factors; and (4) normalization and annotation of cell types and cellular states. The quality of individual single-cell libraries needs to be carefully assessed to abolish the underlying noise as downstream interpretation relies heavily on the preprocessing steps. Generic quality control (QC) metrics including FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), High-Throughput Quality Control, or Kraken provide insights into overall quality of raw sequence files. Characterization of heterogeneity is one of the primary purposes of performing single-cell analysis, however not all outliers contribute to unique cell populations. Single-cell specific quality evaluation tools such as SinQC, SCell, and Celloline enable identification of technical artifacts that interfere with gene expression patterns. Mapping sequencing reads to a reference genome or transcriptome allows identification of the specific location from which the transcripts originate and subsequent quantification. Although dedicated mappers for scRNA-seq are not available, existing aligners such as TopHat, STAR, and Hierarchical Indexing for Spliced Alignment of Transcripts have shown considerable precision and accuracy. Recently, two pseudo-alignment tools, Kallisto and Salmon, have been proposed which pseudo-align splicing isoforms to a reference transcriptome and overcome the requirement of significant amount of computational power and time to process the reads. An improved Salmon with Selective Alignment and expanded decoy sequences was introduced recently and significantly reduced false mappings. However, careful consideration should be made while implementing pseudo-alignment with scRNA-seq data since the data themselves can have lower depth in the first place and 3’ coverage bias. Another important step in the analysis pipeline is normalization of the expression data, which is particularly important in single-cell analysis as many technical parameters including cell capture efficiency, drop out events, read depth, and coverage bias can induce variation,92. Tagging individual RNA molecules using UMIs enables absolute quantification of the transcripts from each cell. In the cases without the use of UMIs, external spike-in RNAs  can be used as internal controls. Additionally, several single-cell specific normalization approaches including SAMstrt, Bayesian Analysis of Single-Cell Sequencing (BASiCS), Gamma Regression Model, sctransform, Scran, SCnorm, and Linnorm can be utilized, of which the last three do not require incorporation of additional spike-ins. Eight commonly applied normalization methods  were subject to benchmarking. Data show that scRNA-seq normalization methods outperformed bulk RNA-seq counterparts. However, a bulk RNA-seq normalization method using Differentially Expressed Genes Elimination Strategy is competitive with scRNA-seq normalization methods,104. Three scRNA-seq imputation methods, namely K-Nearest Neighbor smoothing (kNN-smoothing), DrImpute, and Single-cell Analysis Via Expression Recovery (SAVER), were evaluated for their capacity to tackle the zero-inflation issue either derived from a technical contribution or a normal distribution,108,109 [Synthesized and amplified cDNAs are subsequently subject to library preparation and NGS to generate massive sequencing short reads, as depicted in seq data . Several,108,109 . Both kN,111. Batch correcting algorithms such as Mutual Nearest Neighbors Correct, Seurat 3, Harmony, scGen, and scMerge, among many others, can compensate the discrepancy. In unsynchronized cells, cell-cycle variation can also mask other important physiological variations, which can be overcome by eliminating cell-cycle factors using packages such as single-cell Latent Variable Model (scLVM) and ccRemover. Drop-out, high number of zero counts, sparsity, and multimodality are some of the unique events encountered in single-cell expression analysis which demand more sophisticated algorithms for identifying differentially expressed genes (DEGs).Single-cell RNA isolation at different time points or in different laboratories can induce systemic variations and batch effects which may compromise biologically meaningful interpretation of signals–120.Algorithms for scRNA-seq data analysis have been developed recently in different computer languages, such as R program or Python, and few are designed as a website interface or software package . scRNA-s,44,121. Recently, many software algorithms have been developed to achieve cell clustering and cell type identification [,122. Based on an unsupervised clustering method, such as Seurat or Monocle 3, novel cell types or populations might be revealed with scRNA-seq data,78,123. Recently, cell type identification of scRNA-seq data has been exponentially applied to studies in developmental biology, neurology, cancer biology, and immunology and can provide the type, quantity, and gene signature of different cell populations–129.Cell clustering and cell type identification are critical features of scRNA-seq and, unlike bulk cell RNA-seq, can reveal heterogeneous cell types using entire transcriptomes from an enormous quantity of cellsfication  through et al. identified a group of cell-cycle genes upregulated in a subpopulation which had an attenuated androgen response using Single Cell Differential Expression (SCDE) algorithm in R program. DEGs can also be identified among different cell types in a tissue or organ based on unsupervised algorithms, such as Seurat or Monocle 3. Wang et al. identified single-cell transcriptome profiling of cardiopharyngeal lineages and characterized their cell fate using Seurat package in R program. SCDE, PAthway and Gene set OverDispersion Analysis (PAGODA), Model-based Analysis of Single-cell Transcriptomics, Monocle, and SigEMD algorithms and SINgle CEll RNA-seq profiling Analysis workflows have addressed some common challenges to some extent and improved sensitivity in calling DEGs. Since each single cell has the potential to behave as a unique entity, oftentimes the curse of high dimensionality can impose restrictions in clustering and data visualization. UMAP, Zero Inflated Factor Analysis, Single-cell Interpretation via Multikernel LeaRning, and scvis belong to recent dimensionality reduction techniques that can address the underlying confounding factors and enable proper visualization of diverse expression patterns over conventional PCA analysis,136,137. With the advent of automated advanced tools and packages including SingleR and scMatch, cell-type annotations have significantly improved, leading to the identification of rare events or specific cell populations with the ability to scale-up–140. Cell BLAST is a cell type query algorithm for the analysis of new scRNA-seq data. It utilizes a neural network-based generative model to extract low-dimensional cell-to-cell relationships from high-dimensional transcriptomic data and predicts cell types via batch correction with a large-scale curated reference cell type database.Differential expression analysis can reveal significant DEGs to identify novel pathways or biological functions in different cell types or treatments. Identification of DEGs can be performed by comparing gene expression between two predetermined groups or treatments. For example, Horning ,143. Based on transcriptome information, some algorithms provide a pseudotime scale and cell fate branches within all the cells to reveal potential progression or direction of cell types based on cell phenotypic clusters,144. Cell fate trajectory analysis provides an opportunity to investigate the dynamic processes of large-scale cells in developmental processes, cellular differentiation, or drug responses,146. Several software packages can perform trajectory inference. Monocle 3, Diffusion PseudoTime, and Single-cell Trajectories Reconstruction, Exploration And Mapping are well-developed algorithms to perform cell fate trajectory prediction but require a mastery of computer programming skills. Tools for Single Cell ANalysis (TSCAN) provides a friendly webpage interface to access and perform cell fate trajectory.Following cell type identification, cell fate trajectory is the next step to uncover how different cell types coordinate in many aspects of biology including the developmental process or cancer progression,78, Slingshot, Monocle 2, Waterfall, Single-cell Clustering Using Bifurcation Analysis, and TSCAN can allow construction of pseudotime trajectory and assessment of expression kinetics that can provide novel insights into cellular differentiation of stem cells as well as oncogenic progression during tumor development. Tian et al. evaluated five trajectory analysis methods in a thorough combination of normalization and imputation of four independent scRNA-seq datasets and found that Slingshot and Monocle 2 led to more robust results. Integration of single-cell transcriptome profiling with other single-cell or bulk analysis and spatial measurements can significantly enhance our understanding of molecular basis of cellular heterogeneity and crosstalk among cellular populations in in-vivo studies,153.Of note, transcriptional dynamics represent an important feature of single-cell analysis which enables the analysis of gene expression in given time series such that the output can generate biological signals inferring potential cellular lineages. Seurat. The differentiational modulation from the inner cell mass to blastocysts and ESCs was not fully understood based on bulk cell studies, but they provided a good initial model for scRNA-seq analysis,154. DEGs showed self-renewal and pluripotency signals with high gene expression variations, particularly for genes with medium expression levels. While epigenetic repressor expression was increased, a suppressive transcription became apparent during the development. A group of miRNAs targeting early differentiation genes and pluripotency genes plays a role in transcriptional alterations. Meanwhile, many spliced forms were discovered for the first time. The same approach was also applied to profile the transcriptional dynamics of earlier embryonic stages, preimplantation human embryos, using 124 cells from oocyte to blastocyst stage. Previous bulk studies have shown that the expressions of ~1900 genes were mainly transcriptionally suppressed during the stages. In this study, about 2,495 and 2,675 genes were significantly up- and downregulated between the four- and eight-cell stages. Splicing isoforms of 4,822 transcripts were enriched in different stages and 20% of transcripts displayed more than two splicing variants. FOXP1 with exon 18b transcripts, ESC-specific splicing species, are 25-fold more abundant than those with exon 18a in undifferentiated ESCs. From the 90 single embryonic cells analyzed, 64% of the total known human lncRNAs  were found to be expressed. Another study using Smart-seq method with deep sequencing analyzed the cell fate determination between two- and four-cell embryos and later stage blastomeres. However, this scRNA-seq study discovered that dozens of protein-encoding genes, including Gadd45a, showed significant differential bimodal expression between blastomeres at two- and four-cell embryonic stages. Differential monoallelic expression in 24% genes was clearly observed to be independently regulated in early embryonic development using scRNA-seq. In later embryonic developmental stages within 5–7 days, X-chromosome dose compensation was found in single-cell transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. The cell lineage expression patterns were concurrent as an intermediate status before the establishment of the trophectoderm, epiblast, and primitive endoderm lineages that are contemporary with blastocyst formation. Linnarson’s group studied the differential expression between mouse embryonic stem cells and fibroblasts with a high throughput scRNA-seq method. Nematode embryogenesis between two- and eight-cell stage was dissected with CEL-seq with RNA linear amplification by scoring single-cell transcriptomes. Seventeen genes had significant two-fold mean difference between AB and P1 cells. EMS had more genes expressed compared to P2 cells, while P3 had fewer new genes expressed than C lineage. Taken together, the single-cell transcriptome data map the cell fates in early embryonic differentiation and ESC pluripotency establishment.In the last decade, cumulative efforts have been undertaken to explore the uncharted territory of cellular heterogeneity in different species, organs, tissues, developmental stages, and microenvironments. The first attempt was to interrogate the transitional transcription profiles in the formation of pluripotent embryonic stem cells (ESCs) in early embryogenesis. Dendric cells (DCs) are a group of highly heterogeneous antigen-presenting cells and important for pathogen recognition and immune defense. The bulk RNA-seq of marker-sorted subpopulations did not sufficiently capture their complex functions and led to great controversy. An unbiased global transcriptomic mapping of 18 bone-marrow-derived DCs exposed to lipopolysacharrides (LPS) using Smart-seq revealed hundreds of genes expressed in high variability and unique bimodal profiles that were similarly observed during early mammalian embryogenesis,156. Among them, 137 genes are anti-virus genes. The spleen is the largest lymphatic organ in the human body. The heterogeneity of 1,536 splenic cells was explored using massively parallel MART-seq with low-depth RNA sampling. From them, the method coupled with a probabilistic mixture model demonstrated sensitive cell classification for distinct identification of B cells, natural killer cells, macrophages, monocytes, and plasmacytoid DCs. In DCs, four subpopulations were found either significantly linked or supported by internal combinatorial marker gene expressions. After exposure to LPS, 1,536 spleen cells’ scRNA-seq displayed the heterogeneity in DCs with enriched CD11c expression and their response to LPS. In the adaptive immune system, differentiation of naïve T cells into T helper 2 (Th2) cells is a feedback loop to restrain immune overreaction. From 91 single Th2 cells acquired post infection of naïve T cells, scRNA-seq revealed unique subpopulations with transcriptional profiles and changes in transcription factors, cytokines, surface receptors, and other pathways,164.With multiplexing and high throughputs improvements, scRNA-seq has served as a molecular scalpel directed at the heterogeneity of cells in much more complex tissues, systems, and organisms. The immune system is a complex of bone-marrow-derived differentiated cells. More and more scRNA-seq technologies are adopted for exploring transcriptomes and functional relevance in this biological systemCaenorhabditis elegans) with more than 50-fold somatic cellular coverage at the L2 larval stage. From the data, consensus expression profiles for 27 cell types were defined and rare neuronal cell types with one or two cells were sensitively recovered. The global view of regulatory networks for human skeletal muscle myoblast differentiation has been masked by the low resolution of bulk genomic data. ScRNA-seq coupled with a nonlinear MONOCLE pseudotime trajectory prediction model discovered dynamic expression in 1,061 genes that clustered in gene regulatory groups responsible for activation and suppression at three time points after differentiation initiation. During Embryonic Days 16.5–18.5, murine lung cell lineages at respiratory airway tips are developed from columnar epithelial progenitor cells into flat alveolar type 1 (AT1) or cuboidal type 2 (AT2) cells for gas exchange or surfactant secretion, respectively. A few markers have been identified for four cell types but the global transcriptomic dynamics during the transition is unknown. Microfluidic scRNA-seq of 196 cells have delineated transcriptional signatures for an intermediate bipotential progenitor cells that precede AT1 and AT2 cells, in addition to Clara and ciliated cells. CEL-seq of 238 randomly selected cells from intestinal organoids composed of major intestinal cell lineages brought a better understanding of diversity in intestinal differentiation. Hierarchical clustering of gene expression correlation and rare cell identification method identified the major intestinal cell lineages and 10 clusters as novel diverse subtypes of cells. Spermatogenesis in testes is a complicated and highly orchestrated process including the differentiation of diploid spermatogonia into haploid sperm. The whole picture of spermatogenesis is still far from complete. Two research groups have run scRNA-seq on thousands of dissociated cells from testis samples using Drop-seq and STAR,171. A conserved continuous temporal trajectory of transcriptional dynamics was consistent in both murine and monkey reproductive models. Novel subpopulations were identified in several time points of differentiation and displayed unique transcriptional regulators and signatures. Based on CEL-seq2 data of pancreatic islet cells from four deceased patients, cell clusters by t-distributed Stochastic Neighbor Embedding (t-SNE) analysis showed the classical pancreatic cell types with marker genes and additional novel markers that have not been reported previously.Mining efforts on heterogeneity of other tissues are ongoing in muscle, lung, intestine, testis, pancreas, and the nervous system. The sci-RNA-seq was applied to profile nearly 50,000 cells from nematodes (. Single-cell transcripts of ~3000 cells from mouse somatosensory S1 cortex and hippocampus Cornu Ammonis (CA) were analyzed by STRT/C1. Cell type classification identified nine major classes and 47 molecularly distinct subclasses. scRNA-seq of 30,000 nuclei from mouse and human archived brain tissues from hippocampus and prefrontal cortex was carried out by DroNc-seq. With fewer genes detected, cell clustering analysis still identified novel cell types along with well-known cell types.The central nervous system is composed of large amounts of neuronal and glial cells with numerous types, and the classical methods to identify them with some molecular markers were limited and not definitive,175. Ribosomes And Intact Single Nucleus (RAISIN) RNA-seq and MIning RAre Cells sequencingMIRACL-seq processed transcriptomes of thousands of neurons in mouse and human enteric nervous system for species-specific transcription signatures and dozens of neuronal subtypes. From 44,808 mouse retinal cells, 39 transcriptionally distinct cell populations were identified, creating an atlas of gene expression for the classification of retinal cells and novel rare subtypes.In other independent studies, there were more than 100 subclasses of cells found in mouse brain and spinal cord,7,177. ScRNA-seq is a powerful tool to address the tumoral heterogeneity, particularly for rare cells and previously unrecognizable subpopulations. Smart-seq was applied to stratify heterogenous cell subpopulations in 672 cells from five glioblastoma tumors. Despite apparent cell-to-cell variability, unbiased cell hierarchical clustering showed four meta-signatures comprised of cell-cycle, hypoxia, complement/immune response, and oligodendritic function. Gene expression profiling of 4,347 cells from six Isocitrate dehydrogenase 1(IDH1) or IDH2 mutant human oligodendrogliomas displayed distinct expression signatures. With bulk exome sequencing and copy number variation estimation, a hierarchical cell lineage map with variant stem/progenitor cell components was delineated in each tumor. Noncanonical WNT activation signaling was noted in retrospective analysis of 77 circulating tumor cells from 13 prostate cancer (PCa) patients following tumor progression compared with stable counterparts undergoing androgen deprivation therapy. This study indicated a potential novel therapeutic target and predictive biomarker for PCa. From multicellular ecosystem of metastatic melanoma, 4,645 single cells isolated from 19 patients were subject to analysis for profiling malignant, immune, stromal, and endothelial cells. The principle component analysis of scRNA-seq data showed that the transcriptomic expression could discern malignant cells from tumor and nonmalignant cells  independent of biopsy sites. The transcriptional signatures for malignant cells consist of a core set of cell-cycle genes and a set of immediate early-activation transcription factors that displayed spatial difference. Meanwhile, a drug-resistant subpopulation with high AXL or MITF signals was present in treatment-naive tumors. Treatment-naïve tumors are usually sensitive to initial therapy and generally respond to first-line therapy. However, most advanced tumors acquire drug resistance and lead to poor survival outcomes. Androgen deprivation therapy is effective for the majority of PCa but biochemical recurrence occurs in 30% of patients subject to treatment, and there is a limited understanding of the underlying mechanisms. From 144 cells treated or untreated with androgen, subpopulations of heterogeneous LNCaP cells were revealed and exhibited high levels of ten cell-cycle-related genes using Smart-seq2 analysis. The subpopulations of cells showed cancer stemness phenotype and became resistant to cell-cycle targeting agents. ScRNA-seq and imaging found transcriptional variation and a pre-adapted subpopulation that exhibited resistance to endocrine therapy. ScRNA-seq identified a stem-like subpopulation of PCa cells from monolayer and organoid culture.The transcriptomic heterogeneity of tumors evolves temporospatially during tumor progression with genetic, epigenetic, and tumor immune microenvironmental fluctuations. The scRNA-seq data of 23,261 cells from 49 samples show high-power resolution of high cellular heterogeneity and that residual disease tumors have fewer proliferative markers and increased alveolar cell markers. In TKI-resistant tumors, the upregulated genes were related to oncogenesis and inflammation. Moreover, progressive disease had increased infiltration of immune cells, predominant MF2 macrophages, and suppressive T cells in tumor microenvironments.Smart-seq2 was deployed to sequence single cells derived from treatment naïve, residual disease, and progressive disease following tyrosine kinase inhibitor (TKI)-based therapies in tumor derived from non-small cell lung cancer patients for mapping transcriptional alterations unique to drug-sensitive and drug-resistant tumor cell populations. The three temporal subpopulations of stromal cells displayed unique functional signatures. The lymphocytes from lymph nodes underwent activation and clonal expansion in tumors. To map the heterogeneity in the immune cells within hepatocellular carcinoma tumors, scRNA-seq methods were used to study CD45+ cells isolated from tumors and four immune-relevant sites of 16 treatment-naïve liver cancer patients. it was found that LAMP3+ dendric cells contain unique transcriptional features affecting other immune cell types and show the ability to migrate to lymph nodes. Exhibiting distinct transcriptional states, tumor-associated macrophages were associated with poor prognosis. The inflammatory roles of SLC40A1 and GPNMB were clearly demonstrated in these cells.Melanoma-associated immune and stromal cells were isolated and analyzed by Smart-seq2 at three time points during tumor developmentCell heterogeneity has been more appreciated under the light of a new paradigm due to the advances of scRNA-seq and other single-cell analysis technologies. Since its induction, scRNA-seq has been well received and undergone fast-paced technical advances in uniform cDNA amplification, length coverage, rare copy detection, multiplexing, high throughput, processing of metadata, DEG calling, cell clustering, subpopulation identification, and cell fate trajectory predictions. Along with the new technology progress with higher sensitivity and accuracy, our understanding about the extent of cellular heterogeneity has been swiftly updated and repeatedly brought to another level. The discovery of new cell subpopulations and rare cell types with transcriptomic signatures posit new mechanisms for cell functions and defects that lead to novel biomedical applications and rising therapeutic venues.