Drug nanocrystals, which are comprised of active pharmaceutical ingredients and only a small amount of essential stabilizers, have the ability to improve the solubility, dissolution and bioavailability of poorly water-soluble drugs; in turn, drug nanocrystal technology can be utilized to develop novel formulations of chemotherapeutic drugs. Compared with passive targeting strategy, active tumor-targeted drug delivery, typically enabled by specific targeting ligands or molecules modified onto the surface of nanomedicines, circumvents the weak and heterogeneous enhanced permeability and retention (EPR) effect in human tumors and overcomes the disadvantages of nonspecific drug distribution, high administration dosage and undesired side effects, thereby contributing to improving the efficacy and safety of conventional nanomedicines for chemotherapy. Continuous efforts have been made in the development of active tumor-targeted drug nanocrystals delivery systems in recent years, most of which are encouraging and also enlightening for further investigation and clinical translation. According to the Global Cancer Observatory (GLOBOCAN), an estimated 19.3 million newly diagnosed cancer cases and nearly 10.0 million cancer deaths occurred worldwide in 2020, and cancer is not only a leading cause of death, but also a major obstacle to increasing life expectancy in countries all over the world . NotablyCompared with conventional pharmaceutical formulations, drug NCs have the advantages of high drug loading, nominal excipient utilization and simple preparation process . Basical® Technology) and high pressure homogenization , employ high mechanical force to turn large coarse drug powders into nanosized particles [®, SmartCrystal® and Nanopure® [Top-down approaches, mainly including media milling , which is fast and repeatable to show basic information of drug NCs that directly affects saturation solubility, physical stability and dissolution/drug release rate . Zeta poSolid state form is a key factor for the delivery of poorly water-soluble drugs. Water acts as a plasticizer to stabilize drug crystallinity in the preparation of drug NCs by top-down approaches, while the amorphous form is more typical in bottom-up approaches, and the presence of thermodynamically less stable form might lead to unwanted changes in solid state form during storage, shipping and administration that alter the performance of the system solubility and dissolution rate, diminishing the therapeutic efficacy . The cryPhysical stability is usually evaluated in phosphate buffer saline (PBS) or fetal bovine serum (FBS), with particle size and surface charge measured by DLS and absorbance by enzyme labeling instrument, respectively ,27. ChemDissolution/drug release rate, which can be evaluated by paddle method, dialysis and reverse dialysis sac methods, continuous flow-through cell method and flow-nuclear magnetic resonance (NMR) spectroscopy, reflects the quality of drug NCs and is indispensable for establishing an in vitro-in vivo correlation (IVIVC) ,28,29. TDrug NCs have a smaller particle size than bulk drugs, contributing to increased solubility, dissolution rate and bioavailability and facilitating penetration of drugs through biological barriers inside human body to get to the place where the lesions occur. However, those NCs are endowed with high interfacial free energy by large specific surface area due to reduced particle size so that they are thermodynamically unstable and tend to aggregate ,23. AddiGood stability ensures efficacy as well as safety. In order to improve the stability of drug NCs, suitable stabilizers are carefully selected so as to reduce interfacial free energy and prevent NCs aggregation . StabiliGenerally, ionic (charged) surfactants offer electrostatic stabilization by enhancing repulsive Coulomb forces between particles, whereas non-ionic surfactants and polymeric stabilizers provide steric hindrance stabilization by adsorbing onto particle surfaces and forming a steric layer between particles ,57. Not ® (doxorubicin-loaded PEGylated liposomes), Genexol-PM® (paclitaxel-loaded polymeric micelles) and Abraxane®  [The rapid tumor growth requires abundant nutrients supplied through blood vessels, but the hyperpermeable tumor vasculature is completely different from normal blood vessels, which is structurally characterized by lack of basement membranes, morphological abnormalities in pericytes, and low and loose coverage of endothelial cells by pericytes as compared to the tight junction between normal vascular linings ,63. The rticles) ,12.Although the EPR effect has long been regarded as the basis for tumor-targeted drug delivery, with evidence showing that the above drug-loaded nanocarriers improve therapeutic efficacy in tumor-bearing murine models ,65,66, lActive tumor-targeted drug delivery often refers to a strategy that ligands or molecules such as proteins, nucleic acids, polysaccharides, peptides, and antibodies, with the ability to specifically target surface receptors highly expressed at targeted sites , are modified onto the surface of nanomedicines ,67. The Passive targeting facilitates accumulation of nanomedicines in tumor interstitium, but limited internalization by tumor cells is achieved, and furthermore nonspecific uptake by RES might prevent nanomedicines from targeting specific organs (except liver and spleen) or lesion sites . On the In a nutshell, active tumor-targeted drug delivery shows advantages of accurate drug biodistribution and thus reduced administration dosage, as well as biological-barrier penetration over passive targeting, leading to good efficacy and acceptable safety.Shape and size are two key factors that determine physicochemical, pharmacokinetic as well as pharmacodynamic properties of drug NCs. Benefits seem to be achieved from drug NCs with a shape of high aspect ratio such as rod/needle . 10-hydrFor liposomal drug NCs formulations, different shapes and sizes can be achieved by varying lipid bilayer compositions and drug-to-lipid ratio, leading to controllable drug release behavior ,84. PEGyThe efficacy and safety of active tumor-targeted drug NCs delivery systems have been greatly investigated and discussed both in vitro and in vivo, yielding a large number of valuable results for reference. Generally, there are two types of active targeting strategies: one is the EPR utilization and tumor cell-based active targeting evaluated in the subcutaneous tumor-bearing animal model, and the other is biological-barrier crossing- and tumor cell-based active targeting evaluated in the orthotopic tumor-bearing animal model .i.Folic acid conjugateα that is attached to cell membrane through a glycosylphosphatidylinositol (GPI) anchor is the most commonly expressed one, which is overexpressed in a variety of epithelial cancers but restrictedly expressed in normal tissues [The term folate encompasses both natural folates and folic acid , with the latter employed to signify the fully oxidized chemical compound constituting an important source of ingested folates through supplements . Folate  tissues . In addi tissues .o-quinone moieties, benefiting diverse surface functionalization [FA-conjugated PTX NCs (PTX/F127-folate) resulted in significantly higher cytotoxicity in the FR-positive human epithelial carcinoma cell line KB than unconjugated PTX NCs at the concentrations ranging from 0.08 to 10 μM . PIK-75,lization ,91,92,93lization . Lipid llization .ii.Transferrin conjugateTransferrin (Tf) is a glycoprotein that is important in iron metabolism, by which iron is safely carried in blood circulation and delivered to different tissues such as liver and spleen . Tf spec® [Compared with free docetaxel (DTX), both Tf-modified DTX NCs (Tf-DTX-NCs) and unmodified DTX NCs displayed rapid drug release behavior, but Tf-DTX-NCs showed significantly higher cellular uptake for 2-h incubation and higher toxicity for 72-h incubation than DTX NCs in human non-small cell lung cancer (NSCLC) cell line A549 . Similar® . Modulat® .iii.Hyaluronic acid conjugateN-acetylglucosamine, which can be utilized as a sustained-release carrier for drugs ascribed to its high viscoelasticity, plasticity together with nonimmunogenicity, good biocompatibility and degradability [Hyaluronic acid (HA) is a natural polysaccharide made up of glucuronic acid and dability . HA has dability ,102.HA-modified PTX-NCs (HA-PTX-NC) showed faster drug release, higher cellular uptake efficiency and stronger inhibitory effect in CD44-expressed cancer cells as well as lower toxicity in normal cells as compared to unmodified PTX-NCs . Anotheriv.Albumin conjugateAlbumin is the most abundant plasma protein involved in the transendothelial transport of nutrients or drugs with low toxicity, biocompatibility and the ability to reduce opsonization and phagocytosis, and rapidly proliferating cancer cells have an increased ability to take up albumin because of increased demand for nutrients and energy ,105. Upt®, and high cellular uptake efficiency, comparable cytotoxicity to that of Abraxane® in the SPARC-positive murine melanoma cell line B16F10. Cim-F-alb also demonstrated higher antitumor efficacy than Abraxane® in subcutaneous B16F10 tumor-bearing mice at the same dosage [®, and less interaction with serum proteins were ascribed to albumin coating [The formulation of albumin-conjugated PTX NCs crystalized in F127  had the smallest particle size and the most native albumin as compared to that in hexadecyltrimethylammonium bromide (CTAB) and pure NCs. Cim-F-alb showed better serum stability than Abraxanee dosage . Further coating . Similar coating .v.Chondroitin sulfate conjugateChondroitin sulfate (CS) is a natural polysaccharide belonging to sulfated glycosaminoglycan (GAG) that has been found in ECM, corneas, skin and cartilage, showing good compatibility, and similar to HA, receptor-mediated endocytosis can be triggered by CS specifically binding to CD44 .® (DTX injection) as evidenced by enhanced cellular uptake together with higher degree of cytotoxicity and G2-phase arrest in a time-dependent manner in MDA-MB-231 cells, mechanistically due to disrupted mitochondrial membrane potential and lysosomal membrane integrity. Furthermore, intravenously administered DTX@CSA-NCs showed higher plasma concentration in healthy SD rats and stronger tumor inhibition in subcutaneous 4T1 tumor-bearing mice [CS-conjugated DTX NCs (DTX@CSA-NCs) outperformed the unconjugated counterpart and Taxotereing mice . Increasing mice .vi.Mannuronic acid conjugateD-mannuronic acid (MA) is an important component of a natural polysaccharide alginate that can specifically bind to mannose receptor (MR) mostly expressed by macrophages, dendritic cells and endothelial cells, and MR is also found overexpressed in many cancer cells because of their higher affinity for carbohydrate molecules than that of normal cells due to higher demand for nutrients . A cytosolic co-delivery system of immunological adjuvant Poly I:C and PTX NCs coated with MA (MA-PNRplex), which was rod-shaped with particle size of about 218 nm, greatly enabled immunogenicity of cancer cells as evidenced by increased secretion of cytokines and chemokines and enhanced immune response in vivo, leading to improved antitumor efficacy. Specifically, the intracellular asynchronous release of Poly I:C and PTX amplified antitumor immune response .vii.Herceptin conjugate®  is a humanized monoclonal antibody targeting HER2, and has been approved by the U.S. Food and Drug Administration (FDA) as monotherapy or combination with chemotherapy for the treatment of early-stage HER2-positive breast cancer or metastatic breast cancer [The human epidermal growth factor receptor (HER) family are transmembrane glycoproteins associated with pathogenesis of various cancers, and HER2 has been found overexpressed in approximately 20% of all breast cancers, which is characterized by activation of downstream signaling after dimerization ,114. Hert cancer .Rod-shaped HCT-conjugated PTX NCs (PNCs-HCT) were stable for at least 1 month at 4 °C and displayed similar sustained drug release behavior to unconjugated PTX NCs. Higher binding affinity and cell-specific internalization to HER2-positive cancer cell lines  with PNCs-HCT than with unconjugated PTX NCs were evidenced by increased cellular uptake and cytotoxicity as well as enhanced cell-cycle arrest in G2/M phase . A similviii.Peptide conjugateTumor-targeting peptides, usually composed of less than 50 amino acids, are naturally existing or artificially synthesized low molecular weight ligands that are able to specifically bind to receptors expressed on the surface of cells. They have higher cell or tissue permeation ability as compared to proteins or antibodies, improved pharmacokinetic behavior with little attenuated targeting ability by suitable chemical modification, and good stability in vivo owing to resistance to protease hydrolysis .XQ-1 (HAIYPRHGGGF) is a novel tumor-targeting peptide with high affinity for TfR. Rod-shaped PDA-coated CPT NCs with surface modification by XQ-1 exhibited improved stability, dispersion property and dissolution rate, and more importantly, enhanced targeting ability to TfR-overexpressed A549 and HeLa cells and thus cytotoxicity as compared to free CPT and unmodified CPT NCs .Vβ3 is found to be overexpressed in tumor cells and neovasculature [Vβ3-targeting peptide that has been widely used to improve the antitumor efficacy of nanomedicines [Vβ3. NR1-modified inorganic silver nanoparticles (AgNP)-decorated PTX NCs showed dramatically improved cellular uptake efficiency and cytotoxicity in MDA-MB-231 cells [Integrins are large heterodimeric transmembrane glycoproteins made up of noncovalently bound α- and β-subunits, and they are critical in cell-to-ECM and cell-to-cell communications and play an active role in signaling pathways concerning cell proliferation, division, and survival . Integriculature , and RGDedicines . Based oedicines . NR1 (RG31 cells .ix.Triphenylphosphonium cation conjugate+) is a mitochondrial homing moiety with high lipophilicity. TPP+-modified PTX NCs were reported to show the strongest cytotoxicity against 2D monolayer of MCF-7 and MCF-7/ADR cells as well as deeper penetration on 3D multicellular spheroids (MCs) of MCF-7 cells and more potent growth inhibition on MCF-7 and MCF-7/ADR MCs, providing a promising therapeutic option to overcome drug-resistant breast cancer [Mitochondria are novel targets for treating cancer, especially in the setting of MDR, and triphenylphosphonium cation (TPPt cancer .x.Cell membrane-coated formulationCirculating tumor cells (CTCs) derived from local tumors are capable of escaping immunosurveillance and targeting homotypic tumors due to cell surface interactions including Thomsen–Friedenreich antigen and E-cadherin, and inspired by this self-adherence property, researchers have developed the biomimetic cancer cell membrane-coating technique for active tumor-targeted drug delivery ,68. Flake-shaped 4T1 cancer cell membrane-coated HCPT NCs sandwiched with a photosensitizer indocyanine green (NCs/ICG/CM) greatly induced effective uptake by 4T1 cells rather than HeLa cells, human umbilical vein endothelial cells (HUVEC) or murine macrophages J774A.1, and showed enhanced antitumor efficacy with little toxicity in subcutaneous 4T1 tumor-bearing mice, which provides a promising modality for utilization of hydrophobic chemotherapeutic agents in cancer treatment .i.Albumin conjugateAlbumin facilitates drug permeation across tumor vessels. Compared with current cyclodextrin-based formulation that are limited by short circulation time of carfilzomib (CFZ) due to extensive hepatic and extra-hepatic metabolism and nonspecific biodistribution, a novel albumin-coated CFZ NCs  had advantages of improved metabolic stability, enhanced cellular uptake and cytotoxicity in different breast cancer cell lines , and greater antitumor efficacy in orthotopic 4T1 tumor-bearing mice owing to the improved ability to cross the blood-tumor barrier (BTB) and then target tumor cells .ii.Peptide conjugateVβ3 [As mentioned before, constant changes in crystalline state during NCs growth will be detrimental to dosing, especially via intravenous route . It has Vβ3 .iii.Cell membrane-coated formulationIn the absence of targeting ligand modification, HCPT NCs coated with cancer cell membrane from rat glioma cell line C6 (HCPT-NS/CCM) still had the ability to penetrate BBB and BBTB, and target glioma via immune escape and homotypic binding as evidenced by enhanced BBB and BBTB permeability, increased cellular uptake by C6 cells and increased glioma accumulation in intracranial C6 tumor-bearing mice when compared to HCPT-NS .iv.Peptide-conjugated cell membrane-coated formulationDWSW (DSDYDPDGDWDSDW), a D-peptide exhibiting good proteolytic stability and barrier-penetrating ability [DWSW-CCM-(PTX)NS). DWSW-CCM-(PTX)NS could effectively penetrate BBB and specifically target glioma tissues, and compared with unmodified CCM-(PTX)NS, pure (PTX)NS and free PTX, DWSW-CCM-(PTX)NS via intravenous administration showed the strongest tumor inhibitory effect and led to longest lifespan in intracranial C6 tumor-bearing mice, suggesting the feasible application of this integrated strategy in the treatment of glioma and probably other cancers [To further the targeting ability, the strategy of tumor-targeting peptide modification and that of biomimetic cancer cell membrane coating are combined. PTX NCs were coated with C6 cancer cell membrane and then modified with  ability , to obta cancers .To overcome the limitations of conventional antitumor pharmaceutical agents, considerable efforts have been made in the delivery of poorly water-soluble drugs using the technology of drug NCs for the advantages of high drug loading, good physical stability as well as improved drug release behavior and bioavailability. Active tumor-targeted strategies such as ligand modification and cell membrane coating not only improve antitumor efficacy but also reduce toxicity ascribed to the ability endowed to penetrate biological barriers and specifically target tumor site. Drug NCs have provided a good nano-platform for the delivery of drugs via oral, intravenous, and other administration routes, and they can also be encapsulated into nanocarriers such as liposomes for better stability. Despite abundant encouraging results and significant progress, some key points should be taken into meticulous consideration, and also some problems warrant further investigation.Currently, common chemotherapeutic drugs, including classical taxanes PTX and DTX, have been selected as the model drugs in drug NCs formulations for cancer treatment. Cabazitaxel (CTX) is a novel semi-synthetic taxane with poor affinity for drug resistance-related drug efflux pump p-gp and improved BBB-penetrating ability that has shown better antitumor activity than PTX and DTX, but disadvantages such as poor water solubility, toxicity and low selectivity to tumor tissues limit its application , which mFrom the perspective of current active targeting strategies, targeted receptors should be highly expressed on tumor endothelial cells and/or tumor cells rather than normal cells so as to prevent off-target toxicity. Ligands with high affinity for targeted receptors are modified onto the surface of drug NCs, and specific ligand-receptor binding triggers receptor-mediated endocytosis for internalization of drug-loaded particles and subsequent intracellular drug release. Distinct from peripheral tumors, the physiologically existing BBB and subsequently formed BBTB are major obstacles to efficient drug delivery for glioma occurring in the brain, so tumor-targeting ligands with multiple functions, such as peptides consisting of BBB- and BBTB-penetrating molecules via a chemical linker, will boost the antitumor efficacy to the fullest extent and ensure safety simultaneously . SuitablOther active targeting strategies gaining increasing attractions might be a source of inspiration for the development of novel active tumor-targeted drug NCs delivery systems. Combination of tumor-targeting ligand modification and biomimetic cancer cell membrane coating will enhance the specificity towards tumor cells and/or tissues via ligand-receptor binding and homotypic binding. An integrated strategy of surface modification with tumor-targeting peptide and cell-penetrating peptide (CPP) conjugate will contribute to improved antitumor efficacy due to dual benefits from specific targeting at tumor site and enhanced internalization across cell membranes ,133,134.Accumulating evidence has supported the potential of drug NCs for active tumor-targeted drug delivery, but it should be aware that the subcutaneous tumor-bearing animal models, which have been broadly used in current studies, are actually not that appropriate because of the augmented EPR effect as compared to that in human tumors clinically. Therefore, more attention should be paid to the utilization and improvement of orthotopic tumor-bearing animal models that take into account efficient penetration through biological barriers such as BBB, BTB and BBTB. This is an important issue for active targeting in addition to receptor-mediated endocytosis and will lay the foundation for substantial clinical evaluation in the future to take a further step from bench to bedside. In this seemingly endless fight against cancer, we sincerely hope that more and more safe and efficacious nanomedicines, represented by active tumor-targeted drug NCs, will be developed and eventually succeed in clinical translation.


Primary liver cancer (PLC), consisting mainly of hepatocellular carcinoma and intrahepatic cholangiocarcinoma, is one of the major causes of cancer-related mortality worldwide. The curative therapy for PLC is surgical resection and liver transplantation, but most PLCs are inoperable at diagnosis. Even after surgery, there is a high rate of tumor recurrence. There is an unmet clinical need to discover more effective treatment options for advanced PLCs. Pre-clinical mouse models in PLC research have played a critical role in identifying key oncogenic drivers and signaling pathways in hepatocarcinogenesis. Furthermore, recent advances in single-cell RNA sequencing (scRNA-seq) have provided an unprecedented degree of resolution in such characterization. In this review, we will summarize the recent studies that utilized pre-clinical mouse models with the combination of scRNA-seq to provide an understanding of different aspects of PLC. We will focus particularly on the potentially actionable targets regarding the cellular and molecular components. We anticipate that the findings in mouse models could complement those in patients. With more defined etiological background, mouse models may provide valuable insights. Primary liver cancer (PLC) is the sixth most common cancer and the third leading cause of cancer-related deaths worldwide ,2. Geogrβ-catenin and AKT-mTOR-MAPK signaling [The key etiologies of HCC include chronic viral infection with hepatitis B or C virus (HBV or HCV), excessive alcohol consumption, non-alcoholic fatty liver disease (NAFLD), cirrhosis of any etiology, and exposure to carcinogens, such as aflatoxins ,11,12. Gignaling . HoweverCurrently, the most effective curative treatment for early HCC patients is surgical resection and liver transplantation . HoweverICC is the second commonest primary liver cancer. It has shown an increasing trend in the past decade . For theThe PLC tumors are of high intratumoral heterogeneity, consisting of highly admixed cell populations residing in the tumor microenvironment. To this end, single-cell sequencing strategies are ideal for carrying out relevant cellular and molecular investigations. Regarding the development of single-cell RNA sequencing (scRNA-seq), Tang et al. first reported transcriptome sequencing analysis at single-cell level in 2009 . SubsequPre-clinical mouse models are particularly useful in recapitulating the process of hepatocarcinogenesis in human and have played a critical role in revealing the key oncogenic drivers and signaling pathways of liver cancer ,27. Theyβ-oxidation in mitochondria [The positive correlation between NAFLD or nonalcoholic steatohepatitis (NASH) and the risk of HCC is well established. NAFLD is a common syndrome in people who are overweight or obese, whereas NASH is a combination of diabetes, hypertension and obesity. NAFLD is the most prevalent etiology of HCC in western countries due to the growing obesity epidemic. The incidence of HCC among patients with NASH-associated cirrhosis is approximately 2.6% per year . To mimichondria . Therefochondria . Previouchondria . A recenchondria . A comprchondria  revealed+PD1+ T cells related to cytotoxicity and effector-function was observed in the livers of mice with NASH using scRNA-seq on cells expressing T cell receptor β-chains (TCRβ). Moreover, this type of cell expressed genes that are related to cytotoxicity and effector functions (Gzmk and Gzmn), as well as some exhaustion markers (Pdcd1 and Tox). While anti-PD1 treatment is an effective therapy in non-NASH HCC to a certain degree, it did not result in significant regression of tumors in the NASH-HCC mice. Besides, they also displayed increased fibrosis and contained an increased number of CD8+PD1+ T cells, which indicates that CD8+PD1+ T cells failed to execute an effective immune surveillance role, and instead showed tissue-damaging potential. They validated their hypothesis by depleting CD8+ T cells in NASH mice without liver cancer and found that CD8+PD1+ T cells not only had defected immune surveillance but were also able to promote HCC development in a NASH background. Consistently, similar results were also found in patients with NASH.Notably, Pfister et al. utilized the scRNA-seq analysis on pre-clinical NASH-associated HCC mouse models and demonstrated that immune checkpoint blockade (ICB) therapy targeting programmed death-1 (PD1) indeed exacerbated, rather than suppressed, NASH-HCC, unlike HCC of other etiology such as HBV infection . FirstlyThe gradual accumulation of genetic alterations in the genome occurs during the onset of PLC. To this end, in order to recapitulate the processes in mice, toxins and carcinogens are administrated in mouse models. There are some highly reproducible and frequently used chemicals that can cause chronic or acute liver damage, including DEN, carbon tetrachloride (CCl4), streptozotocin (STZ) and alcohol. DEN can directly form covalent bonds with DNA leading to oncogenic mutations and immune modifications that are associated with HCC initiation and progression . A singlMiz1hep∆) or its essential transcriptional POZ domain (POZhep∆) was specifically deleted in hepatocytes, a 2-fold increase of tumor mass was observed when compared with wild-type (WT) littermates (F/F) control mice in DEN/CCl4-induced and STZ-HFD induced HCC models. ScRNA-seq was conducted in the tumor tissues of mice in the DEN/CCl4 model. While there was no significant difference in the cell populations between (POZhep∆) and Miz1F/F mice, an interesting sub-cluster (sc-2) was identified in the pool of hepatocytes using both Miz1F/F and Miz1hep∆ scRNA-seq data. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, this sub-cluster demonstrated the highest activation level of the NF-kB pathway. This sub-cluster was also only highly enriched in the Miz1hep∆ models. Moreover, such hepatocytes showed a special cell state with high expression levels of inflammatory cytokines and chemokines, such as Tnf, Cxcl2, and Ccl4. These inflammatory cytokines and chemokines were able to promote the polarization of tumor-infiltrating macrophages towards pro-inflammatory activity. The study also validated that, mechanistically, the loss of Miz1 could cause NF-kB activation. Previous papers reported that the major NF-kB transactivating subunit RelA, when bound to oncoprotein metadherin (MTDH), activated the NF-kB signaling pathway upon stimulation by tumor necrosis factor α (TNF-α) [A recent study by Zhang et al. utilized chemical-induced an HCC mouse model and scRNA-seq to uncover the tumor suppressor role of the zinc finger protein Miz1 . In this (TNF-α) ,44. NotaTo better reflect the clinical complexities due to intra- and inter-tumor heterogeneity, transgenic mice can be engineered with the expression of oncogenes and/or constitutive or conditional silencing of tumor-suppressor genes . NowadayIn PLC, phosphatase and tensin homolog (PTEN) is a well-known tumor suppressor . PTEN muInterestingly, a recent study performed sleeping beauty (SB) transposon-based screening in liver-specific PTEN-KO ICC mouse model and identified Traf3 to be the most significant trunk driver for tumor development . Next, tβ and calcium signaling, one subtype of tumor endothelial cells (TEC- NOTCH) promoted the differentiation of fibroblasts into myofibroblastic CAF (myCAF) subtype and aggravated the ICC growth. Hence, the findings revealed functionally distinct cell subpopulations within ICC and provided hints to better understand ICC initiation and progression.Some studies employed SB transposase-based HDTVi spontaneous ICC tumorigenesis model with overexpression of AKT and NICD (intracellular domain of the NOTCH1 receptor) in mouse hepatocytes to investigate the transcriptomic profiles of PLC ,52. The GKRASD12/p19 and YAP/AKT are popular combos for ICC preclinical mouse models. GKRASD12 is a common oncogenic mutation in ICC patients and is associated with overgrowth of tumor cells [Besides overexpressing more than one oncogene, the combination of overexpressing an oncogene and depleting a tumor suppressive gene is another commonly used strategy in HDTVi spontaneous tumorigenesis models. or cells . Tumor sor cells . YAP , red fluorescent protein (RFP) and firefly luciferase) are conventional markers used in reporter knock-in transgenic mouse models . When a + HCC cells increased with HCC development. Next, they tried to deplete Prom1+ HCC lineage in vivo and observed that HCC growth and malignant progression were impeded, which indicates that Prom1+ HCC cells have CSC-like features. They further performed scRNA-seq for Prom1+ and Prom1- models at different time points . Almost a total of 34,000 cells were obtained. In the Prom1+ cells, the dominant cell type was HCC cells, accounting for about 95% throughout the disease development, suggesting that the Prom1+ cells did not transdifferentiate into other lineages in this chronic HCC model and they were the expanding epithelial tumor cells during HCC progression. They detected well-known CSC markers expressed in Prom1+ cells with distinct pattern, suggesting the heterogeneity of Prom1-derived HCC subpopulations. Functional annotation analysis also indicated Prom1+ cells lost most of the normal liver functions, which may represent dedifferentiation status. More importantly, Prom1-lineage gene signature predicted poor prognosis HCC patients and the malignancy might depend on the activated oxidant detoxification system. In summary, the combination of in vivo cell linage tracing and scRNA-seq has uncovered a unique CSC-like pro-oncogenic role of Prom1+ HCC cells and this finding may shed light on the HCC cell heterogeneity in human HCC.A recent study used knock-in reporter mouse line, carcinogen-induced HCC model and scRNA-seq to conduct cell linage tracing in mouse and has revealed a subpopulation of Prom1-expressing HCC cells with CSC properties . It showPatient-derived tumor xenograft (PDX) mouse models are a commonly employed translational research tool to facilitate precision medicine. Resected clinical tumor tissues are transplanted into immunodeficient mice . PDX mouIn order to depict the primary resistance mechanism of sorafenib, the first-line kinase inhibitor drug for HCC, the study by Guan et al.  inoculatIn recent studies ,67,69,75By integrating the findings of the aforementioned papers, we have noted some key insights: one is the pivotal involvement of NF-kB signaling in PLC, and the other is the functional significance of CAF, which might be worthy for further functional characterization as therapeutic targets. Regarding NF-kB signaling, NF-kB-inducing kinase (NIK) demonstrated an inverse correlation with TRAF3 , and the targeting of TRAF3/NIK axis may provide potential therapy in ICC patients . MoreoveHowever, CAF is one of the most abundant cell types in the TME and it supports multiple aspects of tumor development by suppressing antitumor immune responses and remodeling the TME to support tumor growth. Some studies ,64 have Many studies focused on the immune components of the TME of PLC tumors. Notably, there were recent and insightful findings utilizing lineage tracing and mouse models to pinpoint the importance of CSC marker-expressing cells in the tumor initiation and progression . We beliIn summary, using animal models to pre-clinically study PLC can help deepen our understanding of the pathogenesis of PLC. The pre-clinical mouse models are extremely valuable in recapitulating the process of hepatocarcinogenesis ,82. By aRecently, more attention has been paid to utilize spatial transcriptomics (ST) in studying HCC, which can provide the spatial information of liver zonation and other applications . Compare


GaSb-based single-transverse-mode narrow ridge waveguide (RW) lasers with high power and simultaneous good beam quality have broad application prospects in the mid-infrared wavelength region. Yet its design and formation have not been investigated systematically, while the beam characteristics that affect their suitability for specific applications remain rarely analyzed and optimized. The present work addresses these issues by theoretically establishing a waveguide parameter domain that generalizes the overall possible combinations of ridge widths and etch depths that support single-transverse-mode operation for GaSb-based RW lasers. These results are applied to develop two distinct and representative waveguide designs derived from two proposed major optimization routes of model gain expansion and index-guiding enhancement. The designs were evaluated experimentally based on prototype 1-mm cavity-length RW lasers in the 1950 nm wavelength range, which were fabricated with waveguides having perpendicular ridge and smooth side-walls realized through optimized dry etching conditions. The model gain expanded RW laser design with a relatively shallow-etched (i.e., 1.55  Semiconductor diode lasers operating in the mid-infrared (mid-IR) 2–4 mentclass2pt{minim therapy . Among t therapy –5. In famentclass2pt{minimnear 2 W . NonetheThe potential for higher output power with simultaneously good beam quality makes GaSb-based single-transverse-mode narrow ridge waveguide (RW) lasers ideally suited light sources for various scientific and commercial applications, such as pumping rare-earth-doped fiber amplifiers and solid-state lasers –10, seedIn fact, various applications emphasize different aspects of the single-transverse-mode beam characteristics. For example, applications that perform beam transformations in complicated optical systems strictly require excellent beam quality, low astigmatism, and highly stable single-transverse-mode emission , 12. In tigation , 5, 17. The present work addresses these issues by establishing a waveguide parameter domain that generalizes the overall theoretical possible combinations of ridge widths and etch depths that support single-transverse-mode operation, based on the higher-order mode cutoff condition according to the analysis of the effective-index slab waveguide model that extracted from a 1950-nm GaSb-based narrow RW laser diode. The two dimensions of the waveguide structure design, including transverse mode confinement and guiding mechanism, have been demonstrated. These results are applied to develop two distinct waveguide designs A and B, representing the two major waveguide optimization routes of model gain expansion and index-guiding enhancement, adopting either shallow or deep etching  characteristics and power conversion efficiency (PCE) of the fabricated versions of Designs A and B under CW operation and different heat sink temperature are presented in Fig. The far-field characteristics measured using a rotating photoelectric detector for Designs A and B are presented in Fig. mentclass2pt{minimmentclasspt{minimag effect . The resThe beam quality factor Man shape . These dechanism .The insets of Fig. w = 5 d = 1.80 For comparison, we also fabricated an equivalently sized RW laser diode based on the conventional design with As demonstrated in the preceding section, both RW laser diode designs offer single-transverse-mode emission with excellent comprehensive performances. In addition to these, the nearly astigmatism-free characteristic and highly stable fundamental-mode operation of Design A make this design ideally suited for use in complicated optical systems that require beam transformation and propagation, while offering promising prospects as seed sources for various external cavity seeding applications. Moreover, the deeply etched RW employed in Design A makes this design compatible as well with other etched structures, such as curved waveguides or Bragg gratings, which facilitates the fabrication of SLD, DFB, DBR, and other devices. Meanwhile, the ultra-high single-transverse-mode power and brightness obtained by Design B offer promising prospects in pumping fiber amplifiers and solid-state laser systems. In addition, the reduced lateral beam divergence observed for Design B allows the lateral bulk-coupling or the use of relatively simple and low-cost optics for coupling into commercial single-mode fibers, such as SM1950 or PM1950 , 27, andw < 4 w > 7 d. However, the corresponding diminished effective index step may cause severe transverse mode instability owing to the predominant gain-guiding effect. Nonetheless, this problem may be solved by introducing structures that increase the threshold for higher-order modes and thereby stabilize the fundamental mode emission. Possible structures include leaky, corrugated, metallic, lateral absorbing, curved, flared, or highly resistive regions [More importantly, the selectively improved beam characteristics obtained via customized modifications in the waveguide structure according to the two proposed optimization routes provide substantial evidence supporting the validity and practicability of the waveguide design concepts of this work. Therefore, the waveguide parameter domain and the trends presented in Fig.  regions , 28, 29.w = 4 d = 2.05 w = 7 d = 1.55 The present work has addressed the waveguide structure design factors that affect the single-transverse-mode beam characteristics of GaSb-based narrow RW lasers. The possible combinations of ridge widths and etch depths that support single-transverse-mode operation have been established, and two optimization routes of index-guiding enhancement and model gain expansion are indicated. These results were applied to develop two distinct optimized waveguide designs, denoted as Design A (


Polymeric micelles, nanosized assemblies of amphiphilic polymers with a core–shell architecture, have been used as carriers for various therapeutic compounds. They have gained attention due to specific properties such as their capacity to solubilize poorly water-soluble drugs, biocompatibility, and the ability to accumulate in tumor via enhanced permeability and retention (EPR). Moreover, additional functionality can be provided to the micelles by a further modification. For example, micelle surface modification with targeting ligands allows a specific targeting and enhanced tumor accumulation. The introduction of stimuli-sensitive groups leads to the drug’s release in response to environment change. This review highlights the progress in the development of multifunctional polymeric micelles in the field of cancer therapy. This review will also cover some examples of multifunctional polymeric micelles that are applied for tumor imaging and theragnosis. Advancements in nanoscience and nanotechnology have proposed innovative ways for the treatment of diseases, especially cancer, that pose a significant challenge for clinicians ,2,3,4. RPreviously, specific reviews have been published on polymeric micelles for cancer therapy. For example, the review by Kaur et al., (2022) specifically focused on the clinical challenges of polymer-based micelles . SimilarTherefore, the present review focuses on the new developments on polymeric micelles, which have a core–shell nanostructure formed by self-assembly of amphiphilic polymers. Adding to the tumor-targeting property of polymeric micelles via the EPR effect, various modifications of polymeric micelles enabling active targeting and stimuli-sensitive release are discussed. Finally, we discuss multifunctional polymeric micelles, which are applied for tumor imaging and theragnosis.Before discussing multifunctional polymeric micelles, the basic property of polymeric micelles will be reviewed in this section. Polymeric micelles are nanosized constructions formed from the self-assembly of amphiphilic copolymers with both hydrophobic and hydrophilic compartments in their structure. The self-assembly above critical micelle concentration (CMC) is an entropically favored phenomenon. During the process, the hydrophobic compartment of the copolymer forms the core of polymeric micelles, whereas the hydrophilic compartment forms the shell.Various amphiphilic di- or triblock copolymers have been used to form the polymeric micelles. The widely adopted hydrophilic compartment for the copolymer is poly(ethylene glycol) (PEG). PEG is hydrophilic, nonionic, biocompatible, and inert to many organic compounds. The antifouling property of PEG, which is related to steric hindrance effects, is known to contribute to the protection of drugs from enzymatic degradation. In some cases, poly(N-isopropylacrylamide) (PNIPAAm) was used as a hydrophilic compartment in a copolymer to introduce a thermos-sensitive property to micelles . Unlike Polymeric micelles have several features suitable for drug-delivery applications. Most anticancer drugs have low solubility in water due to their polycyclic structure, and their clinical uses are limited . FurtherSome of the recent polymeric micelles and their physicochemical characteristics and applications are listed in Longer circulation allows the polymeric micelles to be accumulated in a tumor. The accumulation has been known to be preceded by an enhanced permeability and retention (EPR) effect, which is the basis of passive targeting of polymeric micelles. Growing tumors require a high amount of nutrients and oxygen, and tumors drive blood vessel growth. Tumor vasculatures grow fast, and vascular density tends to decrease as tumors grow. The resulting structural abnormality makes the tumor blood vessels highly permeable . As a re®-PM  is a polymeric micellar formulation of paclitaxel. Genexol®-PM showed greater anticancer activity and higher tumor tissue concentration compared to paclitaxel . . 65]. b-PMCC) containing hydrophilic poly(ethylene glycol) and cyano-functionalized polycarbonate. Upon NIR irradiation, these micelles were able to inhibit the tumor growth effectively in vivo ..27].It is well known that the synergy between PTT  and chemotherapy could improve overall anticancer efficacy. However, developing stable photosensitizer-loaded micelles is always challenging. A biodegradable hybrid micelles (HMs) nano system was developed for the for co-delivery of paclitaxel (PTX) and IR825. The hybrid micelle (PTX/IR825-TAT HMs) was self-assembled through hydrophobic interactions between polyethyleneimine-polycaprolactone (PEI-PCL) and TAT peptide-modified-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)5000 (TAT-PEG-DSPE). In vivo antitumor results showed that PTX/IR825-TAT HMs can selectively enter tumor tissue, enabling chemophototherapy to be effective .l-tyrosine-125I) for PTT and multifaceted imaging such as fluorescence, SPECT (ingle photon emission computed tomography), and PAI, as well [Similarly, indocyanin green (ICG) was loaded into amphiphilic diblock polymer poly(ethylene glycol)–poly have been made by click chemistry, which is catalyzed by a copper(I) reaction. In 2022, the Nobel Prize—which was shared by Carolyn R. Bertozzi , Morten Meldal , and K. Barry Sharpless  for their contributions along with bio-orthogonal chemistry ,69,70,71Sone et al. developed a targeting-clickable and tumor-cleavable polyurethane micelle for multifunctional delivery of DOX. The multifunctional polymer was prepared by the conjugation of folic acid (FA) ligand via click chemistry. In the tumor site, PEG is detached in response to tumoral pH by the cleavage of the benzoic–imine bond. Then, intracellular drug release is triggered by the disulfide bond cleavage in response to glutathione (GSH). In the flow cytometry study, FA-conjugated micelles showed an enhanced cellular uptake compared to nontargeting micelles. FA-conjugated micelles efficiently delivered DOX in the cells and DOX fluorescence was observed mainly in the cell cytoplasm. The cytotoxicity of DOX-loaded FA-conjugated micelles were compared with DOX-loaded micelles and free DOX. The study result showed that free DOX was more toxic than DOX-loaded micelles, since free DOX was internalized to the cell nucleus faster than DOX released from DOX-loaded micelles. However, FA-conjugated micelles exhibited a higher cytotoxicity compared to non-targeted micelles, which is consistent with its enhanced cellular uptake observed in flow cytometry .co-AN)), was ~43 °C. The developed micelles were used for DOX and Gd-CuS nanoparticles (Gd-CuS NPs) encapsulation for chemophotothermal treatment of HCC guided by magnetic resonance (MR)/photoacoustic (PA) dual-mode imaging. The resultant SA-PEG-p(AAm-co-AN)/DOX/Gd-CuS (SPDG) had superior potential in MR/PA dual-mode imaging-guided chemophotothermal treatment [A smart micellar multifunctional nanosystem was made for hepatocellular carcinoma (HCC) by sialic acid (SA) modification on the micelles. The upper critical solution temperature (UCST) of the micelle, i.e., -poly(acrylamide-co-acrylonitrile), SA-PEG-p(AAm-reatment .Click chemistry was used for creating self-assembled polymer-drug conjugates (PDCs). Two amphiphilic PDCs were modified with PEG for loading 5-fluorouracil and coumarin, providing a unique opportunity to develop promising codelivery carriers for synergistic cancer therapy. The micelles showed anticancer efficacy on PANC-1 and BxPC-3, confirming that both coumarin and 5-fluorouracil retain their anticancer properties after conjugation with PEG .b-PLLA block copolymer (glucose-PEO-b-PLLA-TEMPO), which can encapsulate CUDC101 and IR780 effectively. The specific core–shell rod structure formed by the designed copolymer gives TEMPO radicals excellent stability against reduction-induced magnetic resonance imaging (MRI) silence. The glucose enables tumor targeting, whereas the TEMPO can have trimodal imaging via MRI, photoacoustic imaging, and fluorescence. Efficient delivery of CUDC101 and IR780 is achieved to synergize the antitumor immune activation through IR780-mediated photodynamic therapy (PDT) and CUDC101-triggered CD47 inhibition, showing M1 phenotype polarization of tumor-associated macrophages (TAMs). More intriguingly, this study demonstrates that PDT-stimulated p53 can also re-educate TAMs, providing a combined strategy of using dual tumor microenvironment remodeling to achieve the synergistic effect in the transition from a cold immunosuppressive to a hot immunoresponsive tumor microenvironment [Inflammatory bowel diseases ((ulcerative colitis (UC)) can be treated successfully by siRNAs by oral route. However, oral administration of naked siRNA is challenging, as it can easily be damaged by the harsh gastrointestinal tract. A nanocarrier of micelles based on MPEG-PCL-CH2R4H2C was used for oral delivery of siRNA to treat a dextran sodium sulfate-induced UC model . The preRecently, there have been huge improvements in theranostic approaches using polymer-based micelles; however, there must still be careful analysis of their long-term applicability to improve cancer therapy.Even though there are many reported studies related to polymer micelles in the clinical trials, some of the recent ones are listed in max, availing a safer therapeutic window than the intact cisplatin. Laboratory experiments confirmed the safety of NC-6004 in terms of lowering the nephrotoxicity and neurotoxicity more than the intact cisplatin at an equivalent dose [The NC-6004 clinical-drug-based polymer micelle nanotechnique has been studied. This specific drug has a particle size of ~30 nm and the basic reaction chemistry is based on a polymer–metal complex between polyethylene glycol poly (glutamic acid) block copolymers (PEG-P(Glu)) . This ulent dose . SeveralIn this review, we have discussed various examples that cover a wide range of polymeric micelles applied in the field of anticancer drug delivery. Passive targeting with polymeric micelles has great potential; however, TME (tumor microenvironment) heterogeneity limits the potential. To overcome this, various functionalities were employed in polymeric micelles. The block copolymers forming micelles can be chemically modified, facilitating the employment of various functionalities. Due to this, polymeric micelles could be further developed for active targeting and triggered drug release in response to special cues provided by the tumor microenvironment. However, the design of functional polymeric micelles should consider various factors, including targeting, drug release, therapeutic efficacy, biodistribution, and systemic toxicity. In spite of rapid benchside developments, the translation of polymeric micelle to the bedside has been less impressive. Only a few micelle-based drug delivery systems have been approved by the Food and Drug Administration (FDA). The slow bench-to-bedside translation is because of several challenges that need careful research. The main reason is the lack of preclinical tissue culture systems that mimic biological conditions to predict the therapeutic activity and targeting efficiency performance of polymeric micelles. Thus, new evaluation tools should be further developed to accelerate the clinical translation of polymeric micelles. With the development of multifunctional polymeric micelles, the structure of the polymeric micelles becomes more complex, leading to difficulties in reproducible synthesis and scale-up. Impediments to clinical translation can originate from the challenge in developing a robust manufacturing process meeting the regulatory requirements.In spite of the hurdles mentioned above, more well-designed multifunctional micelles will be investigated, and there will be more accumulated cases translated to clinical trials. In addition, a micellar drug-free therapeutic area should also be well utilized with new functionalities in the coming days. The multidisciplinary field related to chemistry, medicinal science and clinical research holds the promise of delivering breakthroughs in polymeric micelle-based drug delivery, facilitating their move from concept to clinical fields. However, there is long way to go for such establishments because of the lack of detailed toxicity assessments on novel polymer micelles. Therefore, newly developed micelles should be thoroughly checked for toxicity and long-term human applications.


Immunocompromised patients can experience prolonged SARS-CoV-2 infections in the setting of a lack of protectivity immunity despite vaccination. As circulating SARS-CoV-2 strains become more heterogeneous, concomitant infection with multiple SARS-CoV-2 variants has become an increasing concern. Immunocompromised patient populations represent potential reservoirs for the emergence of novel SARS-CoV-2 variants through mutagenic change or coinfection followed by recombinatory events. Identification of SARS-CoV-2 coinfections is challenging using traditional next generation sequencing pipelines; however, targeted genotyping approaches can facilitate detection. Here we describe five COVID-19 cases caused by coinfection with different SARS-CoV-2 variants (Delta/Omicron BA.1 and Omicron BA.1/BA.2) as identified by multiplex fragment analysis. Epidemiological surveillance of SARS-CoV-2 has enabled clinical and public health interventions in response to increasing COVID-19 case numbers. These efforts have been aided by broad sequencing/genotyping initiatives, associating case trajectories with the emergence of dominant genetic lineages harboring characteristic mutations. Single nucleotide polymorphisms (SNPs) or insertion/deletion mutations (indels) characterize these divergent strains which exhibit altered cellular tropism, decreased vaccine-derived antibody protection and/or marked increases in transmissibility, morbidity, and mortality. Emergence of these SARS-CoV-2 variants of concern (VOCs) has occurred in succession, with new VOCs often displacing the previous dominant lineage amongst the global population.−4–10−6 substitutions/nucleotide/round of replication have been reported  devoid of proofreading activity. Due to the rapid speed of the RdRP, mutation rates on the order of 10reported . To mitireported . Thus, wreported , mutagenHost immunity  also drives the emergence of strains with advantageous mutations . We and Finally, intra- and interhost recombinatory events enhance coronaviral genomic diversity . RecombiThus, factors governing VOC emergence are multifactorial and influenced by a combination of genetic drift, recombination, host immunity, antiviral therapy, and duration of SARS-CoV-2 infection. In this work, we investigate SARS-CoV-2 coinfections among patients at a large referral center. Using a rapid genotyping method based on targeted RT-PCR and multiplex fragment analysis  we have A 76-year-old male who underwent bilateral lung transplantation 8 years prior due to idiopathic pulmonary fibrosis presented with fever and respiratory symptoms in November 2021 consistent with COVID-19. SARS-CoV-2 PCR was positive upon admission. Viral genotyping revealed mutations consistent with the Delta VOC which was epidemiologically dominant at the time of sampling. The patient’s immunosuppressive regimen included prednisone, tacrolimus, and monthly belatecept infusions. He had received three doses of the Pfizer/BioNTech COVID-19 vaccine prior to presentation. The patient was initially treated with Casirivimab/Imdevimab (Regeneron) followed by dexamethasone and remdesivir for 5 and 10 days, respectively . He was Forty-five days later (early January 2022), he returned with fever, worsening shortness of breath, and productive cough. Respiratory RT-PCR was again positive for SARS-CoV-2, but given the proximity to prior infection, was initially thought to be a result of persistent viral shedding and alternative etiologies were sought. The patient was treated for hospital acquired pneumonia with piperacillin-tazobactam and vancomycin, and with methylprednisolone for acute cellular rejection. However, genotyping of the second PCR-positive specimen identified SARS-CoV-2 mutational signatures consistent with the presence of two unique VOCs. Delta  and Omicron BA.1 (secondary infection) were identified . FollowiSARS-CoV-2 variant status is routinely determined for all RT-PCR-positive respiratory samples at our institution through a combination of whole genome sequencing (WGS) and genotyping by multiplex fragment analysis of RT-PCR products. Briefly, mutational hotspots of the SARS-CoV-2 genome are amplified with fluorescently labeled primers. These PCR amplicons are separated by capillary electrophoresis, and size differences determine the presence or absences of characteristic deletions. Nucleotide substitutions are determined though the competitive incorporation of primers with alternative florescent labeling .This patient’s initial SARS-CoV-2 PCR-positive specimen contained only Delta VOC sequences, while the positive specimen from the subsequent admission contained mutational signatures consistent with a mixture of both Delta and the newly emerged Omicron BA.1 VOCs. At the time of both infections, the patient had undetectable levels of nucleocapsid-specific IgG of IgM antibodies, often see among patient groups receiving immunosuppressive therapies . As partGiven the patient’s immunosuppressed and coinfected status, we wanted to determine if there was any evidence of recombination between the detected VOCs. WGS revealed mutations specific to Delta and Omicron BA.1 were found on independent reads using WGS. While a total of eight fragment analysis targets are used in genotyping, only three are highlighted here to demonstrate the difference in Omicron BA.1 and Delta VOCs. ORF1A detects the 3 amino acid deletion from ORF1A:Del3675_3677. Spike recurrently deleted region (RDR)1 and 2 detect S:Del69_70 and mutations in the 140–160 amino acid range, respectively. The Delta variant has a 2 amino acid deletion in Spike region 2 (S:Del157_158), while Omicron has a 3 amino acid deletion  were identified at the relative onset of the outbreak, indicative of rapid evolutionary change . While BRespiratory samples from three unique patients revealed multiple peaks amplified for RDR1 (WT and 6 base pair deletion), RDR2 (WT and 9 base pair deletion), RDR3-4  have been recently shown to interact with SARS-CoV-2 Nsp14 and generating a lethal phenotype of SARS-CoV-2 with effective replication and/or long-term propagation . Importa+ T-cell depletion (<20 CD4+ T-cell counts) as found in IC persons, including persons living with HIV/AIDS, has been determined to increase the risk of giving rise to SARS-CoV-2 resistance mutations , which with a CT value of 27 places the prevalence of both variants in the range of active infection and thus, less likely to be due to the presence of shedding dead virus.The principal limitation to widespread adoption of this approach is the upfront cost of some capillary electrophoresis devices. However, newer, smaller, bench-top options that are attractive and less expensive can overcome this challenge.Importantly, SARS-CoV-2 genomic recombination can be rapidly and economically screened by fragment analysis, alleviating the need for the bioinformatic expertise or financial burden associated with traditional NGS pipelines. The variant identification program, Pangolin, can be programed to distinguish recombinant lineages including the recently described XD, XE, and XF variants. Nextclade, and non-amplicon-based platforms and bioinformatic tools such as RAT  or RDP (Recombination Detection Program) may also aid in the characterization of SARS-CoV-2 recombinatory events. However, by placing targets across the genome, this fragment analysis assay may detect recombination. Most assays just target the spike gene and recombination can occur across the entire SARS-CoV-2 genome. We conclude that SARS-CoV-2 coinfections will continue and represent a source of viral evolution, which in many cases can be effectively monitored using a fragment analysis genotyping approach.


Sleep disturbance specifically insomnia, non-restorative sleep, and hypersomnia are common symptoms of major depressive disorder (MDD). As it alleviates major depressive disorder, transcranial magnetic stimulation (TMS) may improve associated sleep disturbances, and may also have inherent sedating or activating properties.To examine the impact of TMS on sleep disturbances in adults with treatment resistant depression in a clinical setting, we retrospectively reviewed de-identified data from naturalistically-treated MDD patients undergoing an initial acute course of TMS therapy at St.Louis Park MinCEP Clinic.Adults with treatment-resistant depression received daily TMS treatments. 9-item Patient Health Questionnaire (PHQ-9) total scores were used to calculate % change at endpoint (relative to pretreatment baseline); response on both measures was defined as 50% reduction in scores, with remission defined as a final total score 4 on the PHQ-9. Insomnia was measured with a 3-item subscale of the Inventory of Depressive Symptomatology Self Report (IDS-SR). Hypersomnia was measured with a single IDS-SR item. Pairwise comparisons were performed using Student’s T-test. Categorical variables were compared using Fisher’s Exact test. Continuous outcome measures were tested with an analysis of covariance, using baseline PHQ-9 score as a fixed effect covariate.TMS appears to have differential modulatory effects on insomnia and hypersomnia in adults with treatment resistant depression.These results may provide the basis for further investigation into therapeutic applications of TMS in addressing sleep disturbances in treatment-resistant depression. Measures that separate hypersomnia and insomnia should be implemented in future work addressing effects of TMS in treatment-resistant depression.No significant relationships.


Nigella sativa (NS), demonstrates very promising biomedical anti-inflammatory, antioxidant, antimicrobial and anticancer properties. Several investigations have inspected the modulative activities of TQ on different stem/progenitor cell types, but its possible role in the regulation of gingival mesenchymal stem/progenitor cells (G-MSCs) has not yet been characterized. For the first time, this study investigates the effects of TQ on G-MSCs’ stemness and Toll-like receptor expression profiles. G-MSCs (n = 5) were isolated, sorted via anti-STRO-1 antibodies and then disseminated on cell culture dishes to create colony-forming units (CFUs), and their stem/progenitor cell attributes were characterized. TQ stimulation of the G-MSCs was performed, followed by an examination of the expression of pluripotency-related factors using RT-PCR and the expression profiles of TLRs 1–10 using flowcytometry, and they were compared to a non-stimulated control group. The G-MSCs presented all the predefined stem/progenitor cells’ features. The TQ-activated G-MSCs displayed significantly higher expressions of TLR3 and NANOG with a significantly reduced expression of TLR1 . TQ-mediated stimulation preserves G-MSCs’ pluripotency and facilitates a cellular shift into an immunocompetent-differentiating phenotype through increased TLR3 expression. This characteristic modulation might impact the potential therapeutic applications of G-MSCs.Thymoquinone (TQ), the key active component of  In periodontal disease and during the subsequent healing process, gingival mesenchymal stem/progenitor cells (G-MSCs) represent the vital cellular components involved in the repair of damaged tissues ,2. RecenNigella sativa, a “herb of blessing” established in Islamic medicine and other traditional therapies, also known as black cumin, black seed, or habbatul barakah, is among the most effective evidence-based herbal remedies with various medicinal assets . Increasing the sample size might have an effect on the performed statistical evaluation and increase the number of obtained significances .Within these limitations, the outcome of the current study provides important insight into the several effects of TQ on G-MSCs. TQ priming of G-MSCs during periodontal treatment might positively affect the cells’ therapeutic properties through their biomodulation. Nevertheless, this topic needs further elaboration and expansion, especially on a clinical level, to confirm the suggested therapeutic value.