Gold Nanourchins Improve Virus Targeting and Plasmonic Coupling for Virus Diagnosis on a Smartphone Platform.

Point-of-care detection of pathogens is critical to monitor and combat viral infections. The plasmonic coupling assay (PCA) is a homogeneous assay and allows rapid, one-step, and colorimetric detection of intact viruses. However, PCA lacks sufficient sensitivity, necessitating further mechanistic studies to improve the detection performance of PCA. Here, we demonstrate that gold nanourchins (AuNUs) provide significantly improved colorimetric detection of viruses in PCA. Using respiratory syncytial virus (RSV) as a target, we demonstrate that the AuNU-based PCA achieves a detection limit of 1400 PFU/mL, or 17 genome equivalent copies/µL. Mechanistic studies suggest that the improved detection sensitivity arises from the higher virus-binding capability and stronger plasmonic coupling at long distances (∼10 nm) by AuNU probes. Furthermore, we demonstrate the virus detection with a portable smartphone-based spectrometer using RSV-spiked nasal swab clinical samples. Our study uncovers important mechanisms for the sensitive detection of intact viruses in PCA and provides a potential toolkit at the point of care.

Digital plasmonic nanobubble detection for rapid and ultrasensitive virus diagnostics.

Rapid and sensitive diagnostics of infectious diseases is an urgent and unmet need as evidenced by the COVID-19 pandemic. Here, we report a strategy, based on DIgitAl plasMONic nanobubble Detection (DIAMOND), to address this need. Plasmonic nanobubbles are transient vapor bubbles generated by laser heating of plasmonic nanoparticles (NPs) and allow single-NP detection. Using gold NPs as labels and an optofluidic setup, we demonstrate that DIAMOND achieves compartment-free digital counting and works on homogeneous immunoassays without separation and amplification steps. DIAMOND allows specific detection of respiratory syncytial virus spiked in nasal swab samples and achieves a detection limit of ~100 PFU/mL (equivalent to 1 RNA copy/µL), which is competitive with digital isothermal amplification for virus detection. Therefore, DIAMOND has the advantages including one-step and single-NP detection, direct sensing of intact viruses at room temperature, and no complex liquid handling, and is a platform technology for rapid and ultrasensitive diagnostics.

mTOR kinase is a therapeutic target for respiratory syncytial virus and coronaviruses.

Therapeutic interventions targeting viral infections remain a significant challenge for both the medical and scientific communities. While specific antiviral agents have shown success as therapeutics, viral resistance inevitably develops, making many of these approaches ineffective. This inescapable obstacle warrants alternative approaches, such as the targeting of host cellular factors. Respiratory syncytial virus (RSV), the major respiratory pathogen of infants and children worldwide, causes respiratory tract infection ranging from mild upper respiratory tract symptoms to severe life-threatening lower respiratory tract disease. Despite the fact that the molecular biology of the virus, which was originally discovered in 1956, is well described, there is no vaccine or effective antiviral treatment against RSV infection. Here, we demonstrate that targeting host factors, specifically, mTOR signaling, reduces RSV protein production and generation of infectious progeny virus. Further, we show that this approach can be generalizable as inhibition of mTOR kinases reduces coronavirus gene expression, mRNA transcription and protein production. Overall, defining virus replication-dependent host functions may be an effective means to combat viral infections, particularly in the absence of antiviral drugs.

Single-Particle Counting Based on Digital Plasmonic Nanobubble Detection for Rapid and Ultrasensitive Diagnostics.

Rapid and sensitive diagnostics of infectious diseases is an urgent and unmet need as evidenced by the COVID-19 pandemic. Here we report a novel strategy, based on DIgitAl plasMONic nanobubble Detection (DIAMOND), to address these gaps. Plasmonic nanobubbles are transient vapor bubbles generated by laser heating of plasmonic nanoparticles and allow single-particle detection. Using gold nanoparticles labels and an optofluidic setup, we demonstrate that DIAMOND achieves a compartment-free digital counting and works on homogeneous assays without separation and amplification steps. When applied to the respiratory syncytial virus diagnostics, DIAMOND is 150 times more sensitive than commercial lateral flow assays and completes measurements within 2 minutes. Our method opens new possibilities to develop single-particle digital detection methods and facilitate rapid and ultrasensitive diagnostics. ONE SENTENCE SUMMARY: Single-particle digital plasmonic nanobubble detection allows rapid and ultrasensitive detection of viruses in a one-step homogeneous assay.

Retraction Note: miR-34a blocks osteoporosis and bone metastasis by inhibiting osteoclastogenesis and Tgif2.

A Retraction to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: miR-34a blocks osteoporosis and bone metastasis by inhibiting osteoclastogenesis and Tgif2.

Change history: In this Letter, the citation to 'Fig. 4e, f' in the main text should be 'Fig. 3e, f'. This has not been corrected online.

mTORC1 impedes osteoclast differentiation via calcineurin and NFATc1.

Rapamycins are immunosuppressant and anti-cancer drugs that inhibit the kinase mTOR. Clinically, they often cause bone pain, bone necrosis, and high bone turnover, yet the mechanisms are unclear. Here we show that mTORC1 activity is high in osteoclast precursors but downregulated upon RANKL treatment. Loss-of-function genetic models reveal that while early Raptor deletion in hematopoietic stem cells blunts osteoclastogenesis due to compromised proliferation/survival, late Raptor deletion in osteoclast precursors instead augments osteoclastogenesis. Gain-of-function genetic models by TSC1 deletion in HSCs or osteoclast precursors cause constitutive mTORC1 activation, impairing osteoclastogenesis. Pharmacologically, rapamycin treatment at low but clinically relevant doses exacerbates osteoclast differentiation and bone resorption, leading to bone loss. Mechanistically, RANKL inactivates mTORC1 via calcineurin-mediated mTORC1 dephosphorylation, consequently activating NFATc1 by reducing mTORC1-mediated NFATc1 phosphorylation. These findings uncover biphasic roles of mTORC1 in osteoclastogenesis, dosage-dependent effects of rapamycin on bone, and a previously unrecognized calcineurin-mTORC1-NFATc1 phosphorylation-regulatory signaling cascade.

Milk lipid regulation at the maternal-offspring interface.

Milk lipids provide a large proportion of energy, nutrients, essential fatty acids, and signaling molecules for the newborns, the synthesis of which is a tightly controlled process. Dysregulated milk lipid production and composition may be detrimental to the growth, development, health and survival of the newborns. Many genetically modified animal models have contributed to our understanding of milk lipid regulation in the lactating mammary gland. In this review, we discuss recent advances in our knowledge of the mechanisms that control milk lipid biosynthesis and secretion during lactation, and how maternal genetic and dietary defects impact milk lipid composition and consequently offspring traits.

mTOR Inhibition Subdues Milk Disorder Caused by Maternal VLDLR Loss.

It is unknown whether and how very-low density lipoprotein receptors (VLDLRs) impact skeletal homeostasis. Here, we report that maternal and offspring VLDLRs play opposite roles in osteoclastogenesis and bone resorption. VLDLR deletion in the offspring augments osteoclast differentiation by enhancing RANKL signaling, leading to osteoporosis. In contrast, VLDLR deletion in the mother alters milk metabolism, which inhibits osteoclast differentiation and causes osteopetrosis in the offspring. The maternal effects are dominant. VLDLR-null lactating mammary gland exhibits higher mTORC1 signaling and cholesterol biosynthesis. Pharmacological probing reveals that rapamycin, but not statin, treatment of the VLDLR-null mother can prevent both the low bone resorption and our previously described inflammatory fur loss in their offspring. Genetic rescue reveals that maternal mTORC1 attenuation in adipocytes, but not in myeloid cells, prevents offspring osteopetrosis and fur loss. Our studies uncover functions of VLDLR and mTORC1 in lactation and osteoclastogenesis, illuminating key mechanisms and therapeutic insights for bone and metabolic diseases.

Macrophage PPARγ inhibits Gpr132 to mediate the anti-tumor effects of rosiglitazone.

Tumor-associated macrophage (TAM) significantly contributes to cancer progression. Human cancer is enhanced by PPARγ loss-of-function mutations, but inhibited by PPARγ agonists such as TZD diabetes drugs including rosiglitazone. However, it remains enigmatic whether and how macrophage contributes to PPARγ tumor-suppressive functions. Here we report that macrophage PPARγ deletion in mice not only exacerbates mammary tumor development but also impairs the anti-tumor effects of rosiglitazone. Mechanistically, we identify Gpr132 as a novel direct PPARγ target in macrophage whose expression is enhanced by PPARγ loss but repressed by PPARγ activation. Functionally, macrophage Gpr132 is pro-inflammatory and pro-tumor. Genetic Gpr132 deletion not only retards inflammation and cancer growth but also abrogates the anti-tumor effects of PPARγ and rosiglitazone. Pharmacological Gpr132 inhibition significantly impedes mammary tumor malignancy. These findings uncover macrophage PPARγ and Gpr132 as critical TAM modulators, new cancer therapeutic targets, and essential mediators of TZD anti-cancer effects.

Gata2 Is a Rheostat for Mesenchymal Stem Cell Fate in Male Mice.

Gata2 is a zinc finger transcription factor that is important in hematopoiesis and neuronal development. However, the roles of Gata2 in the mesenchymal lineages are poorly understood. In vitro studies suggest that Gata2 modulates adipocyte differentiation and mesenchymal stem cell (MSC) proliferation. To systematically determine the in vivo functions of Gata2 in the MSC lineage commitment and development, we have generated three mouse models in which Gata2 is specifically deleted in MSCs, adipocytes, or osteoblasts. During the MSC expansion stage, Gata2 promotes proliferation and attenuates differentiation; thereby Gata2 loss in MSCs results in enhanced differentiation of both adipocytes and osteoblasts. During the differentiation stage, Gata2 also plays MSC-independent roles to impede lineage commitment; hence, Gata2 loss in adipocyte or osteoblast lineages also augments adipogenesis and osteoblastogenesis, respectively. These findings reveal Gata2 as a crucial rheostat of MSC fate to control osteoblast and adipocyte lineage development.

Nur77 prevents excessive osteoclastogenesis by inducing ubiquitin ligase Cbl-b to mediate NFATc1 self-limitation.

Osteoclasts are bone-resorbing cells essential for skeletal remodeling. However, over-active osteoclasts can cause bone-degenerative disorders. Therefore, the level of NFATc1, the master transcription factor of osteoclast, must be tightly controlled. Although the activation and amplification of NFATc1 have been extensively studied, how NFATc1 signaling is eventually resolved is unclear. Here, we uncover a novel and critical role of the orphan nuclear receptor Nur77 in mediating an NFATc1 self-limiting regulatory loop to prevent excessive osteoclastogenesis. Nur77 deletion leads to low bone mass owing to augmented osteoclast differentiation and bone resorption. Mechanistically, NFATc1 induces Nur77 expression at late stage of osteoclast differentiation; in turn, Nur77 transcriptionally up-regulates E3 ubiquitin ligase Cbl-b, which triggers NFATc1 protein degradation. These findings not only identify Nur77 as a key player in osteoprotection and a new therapeutic target for bone diseases, but also elucidate a previously unrecognized NFATc1→Nur77→Cblb-•NFATc1 feedback mechanism that confers NFATc1 signaling autoresolution.

HDAC9 Inhibits Osteoclastogenesis via Mutual Suppression of PPARγ/RANKL Signaling.

Recent studies suggest that the class II histone deacetylase (HDAC)9 plays important roles in physiology such as metabolism and immunity. Here, we report that HDAC9 also controls bone turnover by suppressing osteoclast differentiation and bone resorption. HDAC9 expression is down-regulated during osteoclastogenesis. Ex vivo osteoclast differentiation is accelerated by HDAC9 deletion but diminished by HDAC9 overexpression. HDAC9 knockout mice exhibit elevated bone resorption and lower bone mass. Bone marrow transplantation reveal that the osteoclastogenic defects are intrinsic to the hematopoietic lineage, because the excessive bone resorption phenotype can be conferred in wild-type (WT) mice receiving HDAC9-null bone marrow, and rescued in HDAC9-null mice receiving WT bone marrow. Mechanistically, HDAC9 forms a negative regulatory loop with peroxisome proliferator-activated receptor gamma (PPARg) and receptor activator of nuclear factor kappa-B ligand (RANKL) signaling. On one hand, PPARγ and nuclear factor κB suppress HDAC9 expression, on the other hand, HDAC9 inhibits PPARγ activity in synergy with silencing mediator of retinoic acid and thyroid hormone receptors (SMRT)/NCoR corepressors. These findings identify HDAC9 as a novel, important and physiologically relevant modulator of bone remodeling and skeletal homeostasis.

miR-34a blocks osteoporosis and bone metastasis by inhibiting osteoclastogenesis and Tgif2.

Bone-resorbing osteoclasts significantly contribute to osteoporosis and bone metastases of cancer. MicroRNAs play important roles in physiology and disease, and present tremendous therapeutic potential. Nonetheless, how microRNAs regulate skeletal biology is underexplored. Here we identify miR-34a as a novel and critical suppressor of osteoclastogenesis, bone resorption and the bone metastatic niche. miR-34a is downregulated during osteoclast differentiation. Osteoclastic miR-34a-overexpressing transgenic mice exhibit lower bone resorption and higher bone mass. Conversely, miR-34a knockout and heterozygous mice exhibit elevated bone resorption and reduced bone mass. Consequently, ovariectomy-induced osteoporosis, as well as bone metastasis of breast and skin cancers, are diminished in osteoclastic miR-34a transgenic mice, and can be effectively attenuated by miR-34a nanoparticle treatment. Mechanistically, we identify transforming growth factor-ß-induced factor 2 (Tgif2) as an essential direct miR-34a target that is pro-osteoclastogenic. Tgif2 deletion reduces bone resorption and abolishes miR-34a regulation. Together, using mouse genetic, pharmacological and disease models, we reveal miR-34a as a key osteoclast suppressor and a potential therapeutic strategy to confer skeletal protection and ameliorate bone metastasis of cancers.

Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development.

How environmental cues regulate adult stem cell and cancer cell activity through surface receptors is poorly understood. Angiopoietin-like proteins (ANGPTLs), a family of seven secreted glycoproteins, are known to support the activity of haematopoietic stem cells (HSCs) in vitro and in vivo. ANGPTLs also have important roles in lipid metabolism, angiogenesis and inflammation, but were considered 'orphan ligands' because no receptors were identified. Here we show that the immune-inhibitory receptor human leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse orthologue paired immunoglobulin-like receptor (PIRB) are receptors for several ANGPTLs. LILRB2 and PIRB are expressed on human and mouse HSCs, respectively, and the binding of ANGPTLs to these receptors supported ex vivo expansion of HSCs. In mouse transplantation acute myeloid leukaemia models, a deficiency in intracellular signalling of PIRB resulted in increased differentiation of leukaemia cells, revealing that PIRB supports leukaemia development. Our study indicates an unexpected functional significance of classical immune-inhibitory receptors in maintenance of stemness of normal adult stem cells and in support of cancer development.

Ex vivo expanded hematopoietic stem cells overcome the MHC barrier in allogeneic transplantation.

The lack of understanding of the interplay between hematopoietic stem cells (HSCs) and the immune system has severely hampered the stem cell research and practice of transplantation. Major problems for allogeneic transplantation include low levels of donor engraftment and high risks of graft-versus-host disease (GVHD). Transplantation of purified allogeneic HSCs diminishes the risk of GVHD but results in decreased engraftment. Here we show that ex vivo expanded mouse HSCs efficiently overcame the major histocompatibility complex barrier and repopulated allogeneic-recipient mice. An 8-day expansion culture led to a 40-fold increase of the allograft ability of HSCs. Both increased numbers of HSCs and culture-induced elevation of expression of the immune inhibitor CD274 (B7-H1 or PD-L1) on the surface of HSCs contributed to the enhancement. Our study indicates the great potential of utilizing ex vivo expanded HSCs for allogeneic transplantation and suggests that the immune privilege of HSCs can be modulated.

IGF binding protein 2 supports the survival and cycling of hematopoietic stem cells.

The role of IGF binding protein 2 (IGFBP2) in cell growth is intriguing and largely undefined. Previously we identified IGFBP2 as an extrinsic factor that supports ex vivo expansion of hematopoietic stem cells (HSCs). Here we showed that IGFBP2-null mice have fewer HSCs than wild-type mice. While IGFBP2 has little cell-autonomous effect on HSC function, we found decreased in vivo repopulation of HSCs in primary and secondary transplanted IGFBP2-null recipients. Importantly, bone marrow stromal cells that are deficient for IGFBP2 have significantly decreased ability to support the expansion of repopulating HSCs. To investigate the mechanism by which IGFBP2 supports HSC activity, we demonstrated that HSCs in IGFBP2-null mice had decreased survival and cycling, down-regulated expression of antiapoptotic factor Bcl-2, and up-regulated expression of cell cycle inhibitors p21, p16, p19, p57, and PTEN. Moreover, we found that the C-terminus, but not the RGD domain, of extrinsic IGFBP2 was essential for support of HSC activity. Defective signaling of the IGF type I receptor did not rescue the decreased repopulation of HSCs in IGFBP2-null recipients, suggesting that the environmental effect of IGFBP2 on HSCs is independent of IGF-IR mediated signaling. Therefore, as an environmental factor, IGFBP2 supports the survival and cycling of HSCs.

Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair.

The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte-specific V(D)J (variable [diversity] joining) recombination. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)-dependent protein kinase catalytic subunit (DNA-PKcs)-null mice, knockin mice with the DNA-PKcs(3A/3A) allele, which codes for three alanine substitutions at the mouse Thr2605 phosphorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA-PKcs(3A/3A) HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs(3A/3A) cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphorylation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice.

Components of the hematopoietic compartments in tumor stroma and tumor-bearing mice.

Solid tumors are composed of cancerous cells and non-cancerous stroma. A better understanding of the tumor stroma could lead to new therapeutic applications. However, the exact compositions and functions of the tumor stroma are still largely unknown. Here, using a Lewis lung carcinoma implantation mouse model, we examined the hematopoietic compartments in tumor stroma and tumor-bearing mice. Different lineages of differentiated hematopoietic cells existed in tumor stroma with the percentage of myeloid cells increasing and the percentage of lymphoid and erythroid cells decreasing over time. Using bone marrow reconstitution analysis, we showed that the tumor stroma also contained functional hematopoietic stem cells. All hematopoietic cells in the tumor stroma originated from bone marrow. In the bone marrow and peripheral blood of tumor-bearing mice, myeloid populations increased and lymphoid and erythroid populations decreased and numbers of hematopoietic stem cells markedly increased with time. To investigate the function of hematopoietic cells in tumor stroma, we co-implanted various types of hematopoietic cells with cancer cells. We found that total hematopoietic cells in the tumor stroma promoted tumor development. Furthermore, the growth of the primary implanted Lewis lung carcinomas and their metastasis were significantly decreased in mice reconstituted with IGF type I receptor-deficient hematopoietic stem cells, indicating that IGF signaling in the hematopoietic tumor stroma supports tumor outgrowth. These results reveal that hematopoietic cells in the tumor stroma regulate tumor development and that tumor progression significantly alters the host hematopoietic compartment.

Angiopoietin-like protein 3 supports the activity of hematopoietic stem cells in the bone marrow niche.

The physiologic roles of angiopoietin-like proteins (Angptls) in the hematopoietic system remain unknown. Here we show that hematopoietic stem cells (HSCs) in Angptl3-null mice are decreased in number and quiescence. HSCs transplanted into Angptl3-null recipient mice exhibited impaired repopulation. Bone marrow sinusoidal endothelial cells express high levels of Angptl3 and are adjacent to HSCs. Importantly, bone marrow stromal cells or endothelium deficient in Angptl3 have a significantly decreased ability to support the expansion of repopulating HSCs. Angptl3 represses the expression of the transcription factor Ikaros, whose unregulated overexpression diminishes the repopulation activity of HSCs. Angptl3, as an extrinsic factor, thus supports the stemness of HSCs in the bone marrow niche.