A single-cell transcriptome atlas of the adult human retina.

The retina is a specialized neural tissue that senses light and initiates image processing. Although the functional organization of specific retina cells has been well studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile the human retina, we performed single-cell RNA sequencing on 20,009 cells from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct cell populations representing all known neural retinal cells: rod photoreceptors, cone photoreceptors, Müller glia, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, astrocytes, and microglia. Our data captured molecular profiles for healthy and putative early degenerating rod photoreceptors, and revealed the loss of MALAT1 expression with longer post-mortem time, which potentially suggested a novel role of MALAT1 in rod photoreceptor degeneration. We have demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to understanding retinal biology and disease.

Disruption of Iron Homeostasis to Induce Ferroptosis with Albumin-Encapsulated Pt(IV) Nanodrug for the Treatment of Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer , accounting for approximately 85% of lung cancers. For more than 40 years, platinum (Pt)-based drugs are still one of the most widely used anticancer drugs even in the era of precision medicine and immunotherapy. However, the clinical limitations of Pt-based drugs, such as serious side effects and drug resistance, have not been well solved. This study constructs a new albumin-encapsulated Pt(IV) nanodrug (HSA@Pt(IV)) based on the Pt(IV) drug and nanodelivery system. The characterization of nanodrug and biological experiments demonstrate its excellent drug delivery and antitumor effects. The multi-omics analysis of the transcriptome and the ionome reveals that nanodrug can activate ferroptosis by affecting intracellular iron homeostasis in NSCLC. This study provides experimental evidence to suggest the potential of HSA@Pt(IV) as a nanodrug with clinical application.

TAK1 blockade as a therapy for retinal neovascularization.

Retinal neovascularization, or pathological angiogenesis in the retina, is a leading cause of blindness in developed countries. Transforming growth factor-ß-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) activated by TGF-ß1 and other proinflammatory cytokines. TAK1 is also a key mediator of proinflammatory signals and plays an important role in maintaining vascular integrity upon proinflammatory cytokine stimulation such as TNFα. However, its role in pathological angiogenesis, particularly in retinal neovascularization, remains unclear. Here, we investigate the regulatory role of TAK1 in human endothelial cells responding to inflammatory stimuli and in a rat model of oxygen-induced retinopathy (OIR) featured retinal neovascularization. Using TAK1 knockout human endothelial cells that subjected to inflammatory stimuli, transcriptome analysis revealed that TAK1 is required for activation of NFκB signaling and mediates its downstream gene expression related to endothelial activation and angiogenesis. Moreover, pharmacological inhibition of TAK1 by 5Z-7-oxozeaenol attenuated angiogenic activities of endothelial cells. Transcriptome analysis also revealed enrichment of TAK1-mediated NFκB signaling pathway in the retina of OIR rats and retinal neovascular membrane from patients with proliferative diabetic retinopathy. Intravitreal injection of 5Z-7-oxozeaenol significantly reduced hypoxia-induced inflammation and microglial activation, thus attenuating aberrant retinal angiogenesis in OIR rats. Our data suggest that inhibition of TAK1 may have therapeutic potential for the treatment of retinal neovascular pathologies.

Nintedanib Alleviates Chronic Pancreatitis by Inhibiting the Activation of Pancreatic Stellate Cells via the JAK/STAT3 and ERK1/2 Pathways.

BACKGROUND: Nintedanib (Ninte) has been approved for the treatment of pulmonary fibrosis, and whether it can ameliorate chronic pancreatitis (CP) is unknown. AIMS: This study was conducted to investigate the effect and molecular mechanism of Ninte on pancreatic fibrosis and inflammation in vivo and in vitro. METHODS: The caerulein-induced CP model of murine was applied, and Ninte was orally administered. Pathological changes in pancreas were evaluated using hematoxylin & eosin, Sirius Red, Masson's trichrome, and anti-Ki-67 staining. For in vitro studies, the effects of Ninte on cell viability, apoptosis, and migration of pancreatic stellate cells (PSCs) were determined by CCK-8, flow cytometry, and wound healing assays, respectively. The potential molecular mechanisms of the effects of Ninte on PSCs were analyzed by RNA-Seq and verified at the gene expression and protein activity levels by qRT-PCR and Western Blot. RESULTS: Ninte significantly alleviated the weight loss in mice with caerulein-induced CP and simultaneously attenuated the pancreatic damage, as evidenced by reduced acinar atrophy, collagen deposition, infiltration of inflammatory cells, and inhibited cell proliferation/regeneration. Besides, Ninte markedly suppressed the transcription of fibrogenic and proinflammatory genes in pancreatic tissues. Further in vitro studies showed that Ninte significantly inhibited the transcription and protein expression of genes corresponding to fibrogenesis and proliferation in PSCs. The results of RNA-Seq analysis and subsequent verification assays indicated that Ninte inhibited the activation and proliferation of PSCs via the JAK/STAT3 and ERK1/2 pathways. CONCLUSIONS: These findings indicate that Ninte may be a potential anti-inflammatory and anti-fibrotic therapeutic agent for CP.

Broadening prime editing toolkits using RNA-Pol-II-driven engineered pegRNA.

The prime editor is a versatile tool for targeted precise editing to generate point mutations, small insertions, or small deletions in eukaryotes. However, canonical PE3 system is less efficient, notably in primary cells or pluripotent stem cells. Here, we employed RNA polymerase II promoter instead of RNA polymerase III promoter, whose application is limited by specific DNA contexts, to produce Csy4-processed intronic prime editing guide RNAs (pegRNAs) and, together with other optimizations, achieved efficient targeting with poly(T)-containing pegRNAs, as well as combinatorial and conditional genetic editing. We also found simultaneous suppression of both DNA mismatch repair and DNA damage response could achieve efficient and accurate editing in human embryonic stem cells. These findings relieve the restrictions of RNA polymerase III (RNA-Pol-III)-based base editors and broadened the applications of prime editing.

Computational and in vitro validation of cardiogenic induction of quercetin on adipose-derived mesenchymal stromal cells through the inhibition of Wnt and non-Smad-dependent TGF-ß pathways.

Exploiting human mesenchymal stem cells (hMSCs) was proposed as a promising therapeutic approach for cardiovascular disease due to their capacity to differentiate into cardiac cells. Though modulation of the intracellular signaling pathways dominantly WNT/ß catenin and transforming growth factor-ß (TGF-ß) have been reported to promote differentiation of hMSCs into cardiomyocytes in the prevailing literature, a safe and reproducible system for their clinical application has not yet turned into reality. In the present study, the molecular docking-based strategy was first applied for evaluating the potency of some natural phenolic compounds in the modulation of Wnt and TGF-ß signaling pathways using a vital class of crystallographic protein structures of WNT signaling regulators such as Frizzled, Disheveled, GSK3-ß, ß-catenin, LRP 5/6 extracellular domain, Tankyrase and their variety of active pockets. Then, the impacts of plant-derived chemical compounds on the regulation of the relevant signals for the differentiation of hMSCs into the definitive mesoderm lineage and cardiac progenitors were assessed in vitro. Data obtained revealed the synergistic activity of Wnt and TGF-ß superfamily to direct cardiac differentiation in human cardiogenesis by comparing cardiac gene expression in the presence and absence of the TGF-ß inhibitors. We found that the inhibitory effect of canonical Wnt/ß-catenin is sufficient to cause proper cardiomyocyte differentiation, but the TGF-ß pathway plays a vital role in enhancing the expression of the cardiomyocyte-specific marker (cTnT). It was found that quercetin, a p38MAPK inhibitor with the high energy dock to the active pocket of Wnt receptors, promotes cardiac differentiation via the inhibition of both Wnt and non-Smad TGF-ß pathways. Altogether, data presented here can contribute to the development of a feasible and efficient cardiac differentiation protocol as an "off-the-shelf" therapeutic source using novel natural agents for cardiac repair or regeneration.

The adipokine orosomucoid alleviates adipose tissue fibrosis via the AMPK pathway.

The excess deposition of underlying extracellular matrix (ECM) in adipose tissue is defined as adipose tissue fibrosis that is a major contributor to metabolic disorder such as obesity and type 2 diabetes. Anti-fibrosis therapy has received much attention in the treatment of metabolic disorders. Orosomucoid (ORM) is an acute-phase protein mainly produced by liver, which is also an adipokine. In this study, we investigated the effects of ORM on adipose tissue fibrosis and the potential mechanisms. We showed that ORM1-deficient mice exhibited an obese phenotype, manifested by excessive collagen deposition in adipose tissues and elevated expression of ECM regulators such as metalloproteinases (MMP-2, MMP-13, MMP-14) and tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, TIMP-3). Administration of exogenous ORM (50 mg· kg-1· d-1, ip) for 7 consecutive days in high-fat diet (HFD)-fed mice and leptin receptor (LepR)-deficient db/db mice attenuated these abnormal expressions. Meanwhile, ORM administration stimulated AMP-activated protein kinase (AMPK) phosphorylation and decreased transforming growth factor-ß1 (TGF-ß1) level in adipose tissues of the mice. In TGF-ß1-treated 3T3-L1 fibroblasts, ORM (10 µg/mL) improved the impaired expression profiles of fibrosis-related genes, whereas a selective AMPK inhibitor dorsomorphin (1 µmol/mL) abolished these effects. Together, our results suggest that ORM exerts a direct anti-fibrosis effect in adipose tissue via AMPK activation. ORM is expected to become a novel target for the treatment of adipose tissue fibrosis.

Approach for in vivo delivery of CRISPR/Cas system: a recent update and future prospect.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system provides a groundbreaking genetic technology that allows scientists to modify genes by targeting specific genomic sites. Due to the relative simplicity and versatility of the CRISPR/Cas system, it has been extensively applied in human genetic research as well as in agricultural applications, such as improving crops. Since the gene editing activity of the CRISPR/Cas system largely depends on the efficiency of introducing the system into cells or tissues, an efficient and specific delivery system is critical for applying CRISPR/Cas technology. However, there are still some hurdles remaining for the translatability of CRISPR/Cas system. In this review, we summarized the approaches used for the delivery of the CRISPR/Cas system in mammals, plants, and aquacultures. We further discussed the aspects of delivery that can be improved to elevate the potential for CRISPR/Cas translatability.

Gene Therapy Intervention in Neovascular Eye Disease: A Recent Update.

Aberrant growth of blood vessels (neovascularization) is a key feature of severe eye diseases that can cause legal blindness, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). The development of anti-vascular endothelial growth factor (VEGF) agents has revolutionized the treatment of ocular neovascularization. Novel proangiogenic targets, such as angiopoietin and platelet-derived growth factor (PDGF), are under development for patients who respond poorly to anti-VEGF therapy and to reduce adverse effects from long-term VEGF inhibition. A rapidly advancing area is gene therapy, which may provide significant therapeutic benefits. Viral vector-mediated transgene delivery provides the potential for continuous production of antiangiogenic proteins, which would avoid the need for repeated anti-VEGF injections. Gene silencing with RNA interference to target ocular angiogenesis has been investigated in clinical trials. Proof-of-concept gene therapy studies using gene-editing tools such as CRISPR-Cas have already been shown to be effective in suppressing neovascularization in animal models, highlighting the therapeutic potential of the system for treatment of aberrant ocular angiogenesis. This review provides updates on the development of anti-VEGF agents and novel antiangiogenic targets. We also summarize current gene therapy strategies already in clinical trials and those with the latest approaches utilizing CRISPR-Cas gene editing against aberrant ocular neovascularization.

Albumin-stabilized layered double hydroxide nanoparticles synergized combination chemotherapy for colorectal cancer treatment.

Combination chemotherapy with two or more complimentary drugs has been widely used for clinical cancer treatment. However, the efficacy and side effects of combination chemotherapy still remain a challenge. Here, we constructed an albumin-stabilized layered double hydroxide nanoparticle (BLDH) system to simultaneously load and deliver two widely used anti-tumor drugs, i.e. 5-fluorouracil (5FU) and albumin-bound PTX (Abraxane, ABX) for colorectal cancer treatment. The cellular uptake test has revealed that 5FU-ABX encapsulated BLDH (BLDH/5FU-ABX) nanoparticles were efficiently internalized by the colorectal cancer cell (HCT-116), synergistically inducing apoptosis of colon cancer cells. The in vivo test has demonstrated that BLDH/5FU-ABX nanomedicine significantly inhibited the tumor growth after three intravenous injections, without any detectable side effects. The enhanced therapeutic effectiveness is attributed to efficient accumulation of BLDH/5FU-ABX at tumor sites and acid-sensitive release of co-loaded drugs. Thus, combination chemotherapy based on BLDH/5FU-ABX nanomedicine would be a new strategy for colorectal cancer treatment.

Role of acute-phase protein ORM in a mice model of ischemic stroke.

The only Food and Drug Administration-approved treatment for acute ischemic stroke is tissue plasminogen activator, and the discovery of novel therapeutic targets is critical. Here, we found orosomucoid (ORM), an acute-phase protein mainly produced by the liver, might act as a treatment candidate for an ischemic stroke. The results showed that ORM2 is the dominant subtype in mice normal brain tissue. After middle cerebral artery occlusion (MCAO), the level of ORM2 is significantly increased in the ischemic penumbra compared with the contralateral normal brain tissue, whereas ORM1 knockout did not affect the infarct size. Exogenous ORM could significantly decrease infarct size and neurological deficit score. Inspiringly, the best administration time point was at 4.5 and 6 hr after MCAO. ORM could markedly decrease the Evans blue extravasation, and improve blood-brain barrier-associated proteins expression in the ischemic penumbra of MACO mice and oxygen-glucose deprivation (OGD)-treated bEnd3 cells. Meanwhile, ORM could significantly alleviate inflammation by inhibiting the production of interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α), reduce oxidative stress by improving the balance of malondialdehyde (MDA) and superoxide dismutase (SOD), inhibit apoptosis by decreasing caspase-3 activity in ischemic penumbra of MCAO mice and OGD-treated bEnd.3 cells. Because of its protective role at multiple levels, ORM might be a promising therapeutic target for ischemic stroke.

Controlled Attachment of Pseudomonas aeruginosa with Binary Colloidal Crystal-Based Topographies.

Micro- and nanotopographies can interfere with bacteria attachment, however, the interplay existing between surface chemistry and topography remains unclear. Here, self-assembled spherical micrometer- silica and nanometer poly(methyl methacrylate) (PMMA)-sized particles are used to make binary colloidal crystal (BCC) topographical patterns to study bacterial attachment. A uniform surface chemistry of allylamine plasma polymer (AAMpp) is coated on the top of the BCCs to study only the topography effects. The uncoated and coated BCCs are exposed to Pseudomonas aeruginosa, and the surfaces and bacteria are characterized using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and fluorescence microscopy. It is found that bacteria attachment to the uncoated BCCs is delayed and individual cells are attracted to the small particle regions of the patterns. Surprisingly, this phenomenon is also observed for the AAMpp-coated BCCs, with bacteria attaching to the small particle regions of the pattern, in stark contrast to uniform flat films of AAMpp that are highly adhesive toward P. aeruginosa. Also, the overall levels of bacterial attachment are significantly reduced by the BCC patterns compared to controls. Thus, there is a trade-off that exists between chemistry and topography that can be exploited to delay the onset of P. aeruginosa biofilm formation on surfaces.

Numerical investigation of nematic liquid crystals in the THz band based on EIT sensor.

This paper introduces the concept of electromagnetically induced transparency (EIT) into the permittivity extraction of an anisotropic material-nematic liquid crystal (NLC). A novel two-step strategy is presented to extract the complex permittivity of the NLC at the THz band, which evaluates the relative permittivity tensor from the resonant frequencies and then determines the loss tangent from the quality factor Q of the EIT sensor. The proposed method features high accuracy due to the sharp resonance of the EIT sensor and also high robustness to the thickness of the NLC layer because only amplitude rather than phase information of the transmission coefficients is required. The NLC filled EIT sensor shows a sensitivity of 56.8 µm/RIU (the resonance wavelength shift over the refractive index change unit (RIU)) and Figure of Merit (FoM) of 6.92. The uncertainty of the proposed technique in the relative permittivity and loss tangent is 3% and 8.2%, respectively.

New Combination/Application of Polymer-Based Nanoparticles for Biomedical Engineering.

Polymer-based nanoparticles (PNPs) are attractive in part due to their ultra-small size, versatility and target specificity. Therefore, PNPs have been increasingly used in a variety of biomedical applications including diagnoses and therapeutic treatment. In this chapter, we focus on the recent studies (within 5 years) with some new ideas/agent's application in biomedical field and roughly divide applications of PNPs into four categories: (1) Delivery, (2) In vivo imaging, (3) Therapies, and (4) Other applications. First, we introduce how PNPs can enhance the treatment and delivery efficiency of therapeutic agent. Second, how PNPs can be used to help in vivo imaging system for disease tracking and monitor. Then, we reveal some novel PNPs which is able to function as an agent in photodynamic, photothermal, sonodynamic and neuron capture therapy. Furthermore, we also mention some interesting applications of PNPs for biomedical field in individual form or cluster employment, such as immunoswitch particles, surface fabrication. Finally, the challenges and future development of PNPs are also discussed. In delivery section, we focus on how polymer "can be used" as vehicles in delivery application. But, in the section of imaging and therapies, we carried on how polymer as an "adjuvant" for functional enhancement. The biodegradable property of PNPs is the feature that they can be controllable for itself degradation and drug release as a chief actor. Besides, in imaging and therapies application, PNPs can be the support role for helping contrast agent or photo/sonosensitizer to enlarge their imaging or therapeutic effect.

Ocular Drug Delivery: Role of Degradable Polymeric Nanocarriers for Ophthalmic Application.

Ocular drug delivery has been a major challenge for clinical pharmacologists and biomaterial scientists due to intricate and unique anatomical and physiological barriers in the eye. The critical requirement varies from anterior and posterior ocular segments from a drug delivery perspective. Recently, many new drugs with special formulations have been introduced for targeted delivery with modified methods and routes of drug administration to improve drug delivery efficacy. Current developments in nanoformulations of drug carrier systems have become a promising attribute to enhance drug retention/permeation and prolong drug release in ocular tissue. Biodegradable polymers have been explored as the base polymers to prepare nanocarriers for encasing existing drugs to enhance the therapeutic effect with better tissue adherence, prolonged drug action, improved bioavailability, decreased toxicity, and targeted delivery in eye. In this review, we summarized recent studies on sustained ocular drug/gene delivery and emphasized on the nanocarriers made by biodegradable polymers such as liposome, poly lactic-co-glycolic acid (PLGA), chitosan, and gelatin. Moreover, we discussed the bio-distribution of these nanocarriers in the ocular tissue and their therapeutic applications in various ocular diseases.

c-Jun enhances intestinal epithelial restitution after wounding by increasing phospholipase C-γ1 transcription.

c-Jun is an activating protein 1 (AP-1) transcription factor and implicated in many aspects of cellular functions, but its exact role in the regulation of early intestinal epithelial restitution after injury remains largely unknown. Phospholipase C-γ1 (PLCγ1) catalyzes hydrolysis of phosphatidylinositol 4,5 biphosphate into the second messenger diacylglycerol and inositol 1,4,5 triphosphate, coordinates Ca2+ store mobilization, and regulates cell migration and proliferation in response to stress. Here we reported that c-Jun upregulates PLCγ1 expression and enhances PLCγ1-induced Ca2+ signaling, thus promoting intestinal epithelial restitution after wounding. Ectopically expressed c-Jun increased PLCγ1 expression at the transcription level, and this stimulation is mediated by directly interacting with AP-1 and CCAAT-enhancer-binding protein (C/EBP) binding sites that are located at the proximal region of the rat PLCγ1 promoter. Increased levels of PLCγ1 by c-Jun elevated cytosolic free Ca2+ concentration and stimulated intestinal epithelial cell migration over the denuded area after wounding. The c-Jun-mediated PLCγ1/Ca2+ signal also plays an important role in polyamine-induced cell migration after wounding because increased c-Jun rescued Ca2+ influx and cell migration in polyamine-deficient cells. These findings indicate that c-Jun induces PLCγ1 expression transcriptionally and enhances rapid epithelial restitution after injury by activating Ca2+ signal.

Modulation of human mesenchymal and pluripotent stem cell behavior using biophysical and biochemical cues: A review.

In vitro manipulation of human stem cells is a critical process in regenerative medicine and cellular therapies. Strategies and methods to maintain stem cells and direct them into specific lineages are ongoing challenges in these fields. To date, a number of studies have reported that besides biochemical stimulation, biophysical cues in the form of surface patterning and external stimulation also influence stem cell attachment, proliferation, and differentiation, and can be used in cell reprogramming and the maintenance of pluripotency. While biochemical cues are generally effective and easy to deliver, biophysical cues have many other advantages for scalability as they are cost efficient, have a longer lifetime, and can be easily defined. However, different protocols and cell sources utilized in a variety of studies have led to difficulties in obtaining clear conclusions about the effects of the biophysical environment on stem cells. In addition, the examination of different types of external stimulation is time consuming and limited by available fabrication techniques, resulting in a delay in commercialization and clinical applications. In this review, we aim to summarize the most important biophysical cues and methods for the culture of human stem cells, including mesenchymal and pluripotent stem cells, to facilitate their adoption in stem cell biology. The standard classical protocols of using biochemical cues will also be discussed for comparison. We believe that combining biochemical and biophysical stimulation has the greatest potential to generate functionally mature cells at a scalable and inexpensive rate for diverse applications in regenerative medicine and cell therapy. Biotechnol. Bioeng. 2017;114: 260-280. © 2016 Wiley Periodicals, Inc.

Rapid Self-Assembly of Shaped Microtiles into Large, Close-Packed Crystalline Monolayers on Solid Surfaces.

The rapid self-assembly of photolithographic microtiles into large crystalline monolayers is achieved. Crystalline monolayers get trapped at the liquid-liquid interface and re-emerge at the air-liquid interface by mixing a cosolvent, which then deposits on the solid surface in seconds. This method has the potential to assemble different shapes and sizes of microtiles into complex architectures.

MicroRNA-124 negatively regulates LPS-induced TNF-α production in mouse macrophages by decreasing protein stability.

AIM: MicroRNAs play pivotal roles in regulation of both innate and adaptive immune responses. In the present study, we investigated the effects of microRNA-124 (miR-124) on production of the pro-inflammatory cytokine TNF-α in lipopolysaccharide (LPS)-treated mouse macrophages. METHODS: Mouse macrophage cell line RAW264.7 was stimulated with LPS (100 ng/mL). The levels of miR-124 and TNF-α mRNA were evaluated using q-PCR. ELISA and Western blotting were used to detect TNF-α protein level in cell supernatants and cells, respectively. 3'-UTR luciferase reporter assays were used to analyze the targets of miR-124. For in vivo experiments, mice were injected with LPS (30 mg/kg, ip). RESULTS: LPS stimulation significantly increased the mRNA level of miR-124 in RAW264.7 macrophages in vitro and mice in vivo. In RAW264.7 macrophages, knockdown of miR-124 with miR-124 inhibitor dose-dependently increased LPS-stimulated production of TNF-α protein and prolonged the half-life of TNF-α protein, but did not change TNF-α mRNA levels, whereas overexpression of miR-124 with miR-124 mimic produced the opposite effects. Furthermore, miR-124 was found to directly target two components of deubiquitinating enzymes: ubiquitin-specific proteases (USP) 2 and 14. Knockdown of USP2 or USP14 accelerated protein degradation of TNF-α, and abolished the effect of miR-124 on TNF-α protein stability. CONCLUSION: miR-124, targeting USP2 and USP14, negatively regulates LPS-induced TNF-α production in mouse macrophages, suggesting miR-124 as a new therapeutic target in inflammation-related diseases.