Antibody stabilization for thermally accelerated deep immunostaining.

Antibodies have diverse applications due to their high reaction specificities but are sensitive to denaturation when a higher working temperature is required. We have developed a simple, highly scalable and generalizable chemical approach for stabilizing off-the-shelf antibodies against thermal and chemical denaturation. We demonstrate that the stabilized antibodies (termed SPEARs) can withstand up to 4 weeks of continuous heating at 55 °C and harsh denaturants, and apply our method to 33 tested antibodies. SPEARs enable flexible applications of thermocycling and denaturants to dynamically modulate their binding kinetics, reaction equilibrium, macromolecular diffusivity and aggregation propensity. In particular, we show that SPEARs permit the use of a thermally facilitated three-dimensional immunolabeling strategy (termed ThICK staining), achieving whole mouse brain immunolabeling within 72 h, as well as nearly fourfold deeper penetration with threefold less antibodies in human brain tissue. With faster deep-tissue immunolabeling and broad compatibility with tissue processing and clearing methods without the need for any specialized equipment, we anticipate the wide applicability of ThICK staining with SPEARs for deep immunostaining.

Exploring Intestinal Surface Receptors in Oral Nanoinsulin Delivery.

Subcutaneous and inhaled insulins are associated with needle phobia, lipohypertrophy, lipodystrophy, and cough in diabetes treatment. Oral nanoinsulin has been developed, reaping the physiologic benefits of peroral administration. This review profiles intestinal receptors exploitable in targeted delivery of oral nanoinsulin. Intestinal receptor targeting improves oral insulin bioavailability and sustains blood glucose-lowering response. Nonetheless, these studies are conducted in small animal models with no optimization of insulin dose, targeting ligand type and content, and physicochemical and molecular biologic characteristics of nanoparticles against the in vivo/clinical diabetes responses as a function of the intestinal receptor population characteristics with diabetes progression. The interactive effects between nanoinsulin and antidiabetic drugs on intestinal receptors, including their up-/downregulation, are uncertain. Sweet taste receptors upregulate SGLT-1, and both have an undefined role as new intestinal targets of nanoinsulin. Receptor targeting of oral nanoinsulin represents a viable approach that is relatively green, requiring an in-depth development of the relationship between receptors and their pathophysiological profiles with physicochemical attributes of the oral nanoinsulin. SIGNIFICANCE STATEMENT: Intestinal receptor targeting of oral nanoinsulin improves its bioavailability with sustained blood glucose-lowering response. Exploring new intestinal receptor and tailoring the design of oral nanoinsulin to the pathophysiological state of diabetic patients is imperative to raise the insulin performance to a comparable level as the injection products.

SERPINA12 promotes the tumorigenic capacity of HCC stem cells through hyperactivation of AKT/ß-catenin signaling.

BACKGROUND AND AIMS: HCC is an aggressive disease with poor clinical outcome. Understanding the mechanisms that drive cancer stemness, which we now know is the root cause of therapy failure and tumor recurrence, is fundamental for designing improved therapeutic strategies. This study aims to identify molecular players specific to CD133 + HCC to better design drugs that can precisely interfere with cancer stem cells but not normal stem cell function. APPROACH AND RESULTS: Transcriptome profiling comparison of epithelial-specific "normal" CD133 + cells isolated from fetal and regenerating liver against "HCC" CD133 + cells isolated from proto-oncogene-driven and inflammation-associated HCC revealed preferential overexpression of SERPINA12 in HCC but not fetal and regenerating liver CD133 + cells. SERPINA12 upregulation in HCC is tightly associated with aggressive clinical and stemness features, including survival, tumor stage, cirrhosis, and stemness signatures. Enrichment of SERPINA12 in HCC is mediated by promoter binding of the well-recognized ß-catenin effector TCF7L2 to drive SERPINA12 transcriptional activity. Functional characterization identified a unique and novel role of endogenous SERPINA12 in promoting self-renewal, therapy resistance, and metastatic abilities. Mechanistically, SERPINA12 functioned through binding to GRP78, resulting in a hyperactivated AKT/GSK3ß/ß-catenin signaling cascade, forming a positive feed-forward loop. Intravenous administration of rAAV8-shSERPINA12 sensitized HCC cells to sorafenib and impeded the cancer stem cell subset in an immunocompetent HCC mouse model. CONCLUSIONS: Collectively, our findings revealed that SERPINA12 is preferentially overexpressed in epithelial HCC CD133 + cells and is a key contributor to HCC initiation and progression by driving an AKT/ß-catenin feed-forward loop.

Robotic-assisted surgery for left-sided colon and rectal resections is associated with reduction in the postoperative surgical stress response and improved short-term outcomes: a cohort study.

INTRODUCTION: There is growing evidence that the use of robotic-assisted surgery (RAS) in colorectal cancer resections is associated with improved short-term outcomes when compared to laparoscopic surgery (LS) or open surgery (OS), possibly through a reduced systemic inflammatory response (SIR). Serum C-reactive protein (CRP) is a sensitive SIR biomarker and its utility in the early identification of post-operative complications has been validated in a variety of surgical procedures. There remains a paucity of studies characterising post-operative SIR in RAS. METHODS: Retrospective study of a prospectively collected database of consecutive patients undergoing OS, LS and RAS for left-sided and rectal cancer in a single high-volume unit. Patient and disease characteristics, post-operative CRP levels, and clinical outcomes were reviewed, and their relationships explored within binary logistic regression and propensity scores matched models. RESULTS: A total of 1031 patients were included (483 OS, 376 LS, and 172 RAS). RAS and LS were associated with lower CRP levels across the first 4 post-operative days (p < 0.001) as well as reduced complications and length of stay compared to OS in unadjusted analyses. In binary logistic regression models, RAS was independently associated with lower CRP levels at Day 3 post-operatively (OR 0.35, 95% CI 0.21-0.59, p < 0.001) and a reduction in the rate of all complications (OR 0.39, 95% CI 0.26-0.56, p < 0.001) and major complications (OR 0.5, 95% CI 0.26-0.95, p = 0.036). Within a propensity scores matched model comparing LS versus RAS specifically, RAS was associated with lower post-operative CRP levels in the first two post-operative days, a lower proportion of patients with a CRP ≥ 150 mg/L at Day 3 (20.9% versus 30.5%, p = 0.036) and a lower rate of all complications (34.7% versus 46.7%, p = 0.033). CONCLUSIONS: The present observational study shows that an RAS approach was associated with lower postoperative SIR, and a better postoperative complications profile.

Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS.

N-Linked glycosylation in hemagglutinin and neuraminidase glycoproteins of influenza viruses affects antigenic and receptor binding properties, and precise analyses of site-specific glycoforms in these proteins are critical in understanding the antigenic and immunogenic properties of influenza viruses. In this study, we developed a glycoproteomic approach by using a timsTOF Pro mass spectrometer (MS) to determine the abundance and heterogeneity of site-specific glycosylation for influenza glycoproteins. Compared with a Q Exactive HF MS, the timsTOF Pro MS method without the hydrophilic interaction liquid chromatography column enrichment achieved similar glycopeptide coverage and quantities but was more effective in identifying low-abundance glycopeptides. We quantified the distributions of intact site-specific glycopeptides in hemagglutinin of A/chicken/Wuxi/0405005/2013 (H7N9) and A/mute swan/Rhode Island/A00325125/2008 (H7N3). Results showed that hemagglutinin for both viruses had complex N-glycans at N22, N38, N240, and N483 but only high-mannose glycans at N411 and, however, that the type and quantities of glycans were distinct between these viruses. Collisional cross section (CCS) provided by the ion mobility spectrometry from the timsTOF Pro MS data differentiated sialylation linkages of the glycopeptides. In summary, timsTOF Pro MS method can quantify intact site-specific glycans for influenza glycoproteins without enrichment and thus facilitate influenza vaccine development and production.

Lineage tracing and single-cell analysis reveal proliferative Prom1+ tumour-propagating cells and their dynamic cellular transition during liver cancer progression.

OBJECTIVE: Hepatocellular carcinoma (HCC) has high intratumoral heterogeneity, which contributes to therapeutic resistance and tumour recurrence. We previously identified Prominin-1 (PROM1)/CD133 as an important liver cancer stem cell (CSC) marker in human HCC. The aim of this study was to investigate the heterogeneity and properties of Prom1+ cells in HCC in intact mouse models. DESIGN: We established two mouse models representing chronic fibrotic HCC and rapid steatosis-related HCC. We performed lineage tracing post-HCC induction using Prom1C-L/+; Rosa26tdTomato/+ mice, and targeted depletion using Prom1C-L/+; Rosa26DTA/+ mice. Single-cell RNA sequencing (scRNA-seq) was carried out to analyse the transcriptomic profile of traced Prom1+ cells. RESULTS: Prom1 in HCC tumours marks proliferative tumour-propagating cells with CSC-like properties. Lineage tracing demonstrated that these cells display clonal expansion in situ in primary tumours. Labelled Prom1+ cells exhibit increasing tumourigenicity in 3D culture and allotransplantation, as well as potential to form cancers of differential lineages on transplantation. Depletion of Prom1+ cells impedes tumour growth and reduces malignant cancer hallmarks in both HCC models. scRNA-seq analysis highlighted the heterogeneity of Prom1+ HCC cells, which follow a trajectory to the dedifferentiated status with high proliferation and stem cells traits. Conserved gene signature of Prom1 linage predicts poor prognosis in human HCC. The activated oxidant detoxification underlies the protective mechanism of dedifferentiated transition and lineage propagation. CONCLUSION: Our study combines in vivo lineage tracing and scRNA-seq to reveal the heterogeneity and dynamics of Prom1+ HCC cells, providing insights into the mechanistic role of malignant CSC-like cells in HCC progression.

Pomegranate bioactive constituents target multiple oncogenic and oncosuppressive signaling for cancer prevention and intervention.

Cancer remains to be the second highest cause of mortality in our society, falling just short of heart disease. Despite major advancement in cancer therapy over the past decade, momentum has been gaining for an alternative approach of using naturally-occurring and dietary agents for cancer prevention and management. Research on pomegranate (Punica granatum L.), a fruit of the Punicaceae family, has shown enormous potential for cancer prevention and intervention. In addition to a rich source of polyphenols, including flavonoids and ellagitannins, in its juice, pomegranate also houses hundreds of other phytochemicals in its pericarp, seed, flower, bark, flowers and leaves. These phytochemicals provide powerful antiproliferative, anti-inflammatory, antioxidant, anti-invasive, antimigratory, anti-angiogenic and anti-metastatic effects without significant toxicity. This makes the use of its various extracts a very attractive strategy to our current battle against cancer. This review article presents a systematic, comprehensive and critical review of research on pomegranate-derived products in both cancer prevention and intervention. It discusses the chemical constituents of pomegranate, the results of both preclinical (in vitro, ex vivo and in vivo) and clinical studies on the anticancer effect of pomegranate phytochemicals and molecular targets in numerous types of cancers, such as breast, gastrointestinal tract (oral, colon, liver and pancreas), gynecological (uterine and ovarian), hematological (lymphoma, leukemia and myeloma), lung, neurological (glioma), urogenital (bladder and prostate), bioavailability, pharmacokinetics and safety of pomegranate constituents. In order to guide the direction of future research, we have also included current limitations and challenges in the field and our post analysis recommendation.

Anticancer potential of garlic and its bioactive constituents: A systematic and comprehensive review.

Vegetables of the Allium genus, such as garlic (Allium sativum L.), onions, shallots, leaks, and chives, have been used for many years for food consumption and for medicinal purposes. Historical medical texts have indicated the therapeutic applications of garlic as an antitumor, laxative, diuretic, antibacterial and antifungal agent. Specifically, garlic's antitumor abilities have been traced back 3500 years as a chemotherapeutic agent used in Egypt. Other beneficial effects of garlic consumption include lowering blood pressure, blood cholesterol, sugar and lipids. The processing and aging of garlic result in the production of non-toxic organosulfur by-products. These sulfur-containing compounds, such as allicin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, alliin, S-allylcysteine, and S-allylmercaptocysteine, impact various stages of carcinogenesis. The anticancer mechanisms of action of these garlic-derived phytochemicals include altering mitochondrial permeability, inhibiting angiogenesis, enhancing antioxidative and proapoptotic properties, and regulating cell proliferation. All these effects of garlic's sulfur-compounds have been demonstrated in various human cancers. The intent of this literature research is to explore the potential of garlic-derived products and bioactive organosulfur compounds as cancer chemopreventive and chemotherapeutic agents. This investigation employs criteria for systematic review and critically analyzes published in vitro, in vivo and clinical studies. Concerns and limitations that have arisen in past studies regarding standards of measurement, bioavailability, and method of delivery are addressed. Overall, it is hoped that through this systematic and comprehensive review, future researchers can be acquainted with the updated data assembled on anticancer properties of garlic and its phytoconstituents.

Identification of novel biomarkers in prostate cancer diagnosis and prognosis.

Prostate cancer (PCa) is a common urinary malignancy. The lack of specific and sensitive biomarkers for the early diagnosis and prognosis of PCa makes it important to seek alternatives. R software was used to analyze the PCa expression profile from data sets in Gene Expression Omnibus. Core differential genes were identified by String and Cytoscape and further validated by Gene Expression Profiling Interactive Analysis (GEPIA) and The Human Protein Atlas (HPA). Gene Ontology analysis was done in the DIVID database and visualization analysis was conducted by Hiplot. Pathway enrichment was analyzed by IPA. To identify potential competitive endogenous RNAs (ceRNA) networks, the experimentally validated microRNA-target interactions database (miRTarBase), The Encyclopedia of RNA Interactomes (StarBase), lncBase, and GEPIA were used. The lncLocator was utilized to perform subcellular localization of long noncoding RNAs (lncRNAs). Both miRTarBase and StarBase were used to find the binding site of mRNAs-miRNAs and miRNAs-lncRNAs. Visualization of the ceRNA network was performed with Cytoscape. Nine genes closely related to the diagnosis and prognosis of PCa were obtained, including four identified biomarkers by HPA, CENPF, TPX2, TK1, and CCNB1, and five novel PCa biomarkers, RRM2, UBE2C, TOP2A, BIRC5, and ZWINT. Pathway analysis indicated that PCa carcinogenesis was highly correlated with liver fibrosis pathways, ILK signaling, and NRF2-mediated oxidative stress response. Two sets of ceRNA networks, BIRC5/hsa-miR-218-5p/NEAT1 and UBE2C/hsa-miR-483-3p/NEAT1 were found to be novel biomarkers for the identification of PCa. The quantitative real-time polymerase chain reaction results verified that UBE2C, BIRC5, and NEAT1 were upregulated and hsa-miR-218-5p and hsa-miR-483-3p were downregulated in human PCa cells compared with normal prostate epithelial cells. The novel identified biomarkers in this study would be valuable for the diagnosis and prognosis of PCa.

Effect of Chicken Egg White-Derived Peptide and Hydrolysates on Abnormal Skin Pigmentation during Wound Recovery.

Abnormal skin pigmentation commonly occurs during the wound healing process due to the overproduction of melanin. Chicken egg white (CEW) has long been used to improve skin health. Previous published works had found CEW proteins house bioactive peptides that inhibit tyrosinase, the key enzyme of melanogenesis. The current study aimed to evaluate the anti-pigmentation potential and mechanism of the CEW-derived peptide (GYSLGNWVCAAK) and hydrolysates (CEWHmono and CEWHdi), using a cell-based model. All of these peptide and hydrolysates inhibited intracellular tyrosinase activity and melanin level up to 45.39 ± 1.31 and 70.01 ± 1.00%, respectively. GYSLGNWVCAAK and CEWHdi reduced intracellular cAMP levels by 13.38 ± 3.65 and 14.55 ± 2.82%, respectively; however, CEWHmono did not affect cAMP level. Moreover, the hydrolysates downregulated the mRNA expression of melanogenesis-related genes, such as Mitf, Tyr, Trp-1 and Trp-2, but GYSLGNWVCAAK only suppressed Tyr gene expression. Downregulation of the genes may lower the catalytic activities and/or affect the structural stability of TYR, TRP-1 and TRP-2; thus, impeding melanogenesis to cause an anti-pigmentation effect in the cell. Outcomes from the current study could serve as the starting point to understand the underlying complex, multifaceted melanogenesis regulatory mechanism at the cellular level.

Qualitative and Quantitative Analysis of Edible Bird's Nest Based on Peptide Markers by LC-QTOF-MS/MS.

Edible bird's nest (EBN) is an expensive health food. There are many adulterants in the market. It remains challenging to discriminate EBN from its adulterants due to a lack of high-specificity markers. Besides, the current markers are confined to soluble fraction of EBN. Here, both soluble and insoluble fractions were analyzed by LC-QTOF-MS/MS. A total of 26 high-specificity peptides that were specific to EBN were selected as qualitative authentication markers. Among them, 10 markers can discriminate EBN from common adulterants, 13 markers discriminate white EBN from grass EBN/common adulterants, and 3 markers discriminate grass EBN from white EBN/common adulterants. Three of them, which showed high signal abundance (Peak area ≥ 106) and satisfactory linearity (R2 ≥ 0.995) with EBN references, were selected as the assay marker; and their peptide sequences were confidently identified by searching database/de novo sequencing. Based on these markers, a qualitative and quantitative analytical method was successfully developed and well-validated in terms of linearity, precision, repeatability, and accuracy. The method was subsequently applied to detect EBN products on the market. The results indicated that more than half of EBN products were not consistent with what the merchants claimed.

Qualitative and Quantitative Analysis of Ejiao-Related Animal Gelatins through Peptide Markers Using LC-QTOF-MS/MS and Scheduled Multiple Reaction Monitoring (MRM) by LC-QQQ-MS/MS.

Donkey-hide gelatin, also called Ejiao (colla corii asini), is commonly used as a food health supplement and valuable Chinese medicine. Its growing popular demand and short supply make it a target for fraud, and many other animal gelatins can be found as adulterants. Authentication remains a quality concern. Peptide markers were developed by searching the protein database. However, donkeys and horses share the same database, and there is no specific marker for donkeys. Here, solutions are sought following a database-independent strategy. The peptide profiles of authentic samples of different animal gelatins were compared using LC-QTOF-MS/MS. Fourteen specific markers, including four donkey-specific, one horse-specific, three cattle-specific, and six pig-specific peptides, were successfully found. As these donkey-specific peptides are not included in the current proteomics database, their sequences were determined by de novo sequencing. A quantitative LC-QQQ multiple reaction monitoring (MRM) method was further developed to achieve highly sensitive and selective analysis. The specificity and applicability of these markers were confirmed by testing multiple authentic samples and 110 batches of commercial Ejiao products, 57 of which were found to be unqualified. These results suggest that these markers are specific and accurate for authentication purposes.

Deficiency in Embryonic Stem Cell Marker Reduced Expression 1 Activates Mitogen-Activated Protein Kinase Kinase 6-Dependent p38 Mitogen-Activated Protein Kinase Signaling to Drive Hepatocarcinogenesis.

BACKGROUND AND AIMS: Embryonic stem-cell-related transcription factors are central to the establishment and maintenance of stemness and pluripotency, and their altered expression plays key roles in tumors, including hepatocellular carcinoma (HCC), a malignancy with no effective treatment. Here, we report on the embryonic stem cell marker, reduced expression 1 (REX1; also known as zinc finger protein 42), to be selectively down-regulated in HCC tumors. APPROACH AND RESULTS: Deficiency of REX1 in HCC was attributed to a combination of hypermethylation at its promoter as well as histone modification by methylation and acetylation. Clinically, hypermethylation of REX1 was closely associated with neoplastic transition and advanced tumor stage in humans. Functionally, silencing of REX1 potentiated the tumor-initiating and metastasis potential of HCC cell lines and xenografted tumors. Lentivirus-mediated Rex1 ablation in liver of male immunocompetent mice with HCC, induced by hydrodynamic tail vein injection of proto-oncogenes, enhanced HCC development. Transcriptome profiling studies revealed REX1 deficiency in HCC cells to be enriched with genes implicated in focal adhesion and mitogen-activated protein kinase (MAPK) signaling. From this lead, we subsequently found REX1 to bind to the promoter region of mitogen-activated protein kinase kinase 6 (MKK6), thereby obstructing its transcription, resulting in altered p38 MAPK signaling. CONCLUSIONS: Our work describes a critical repressive function of REX1 in maintenance of HCC cells by regulating MKK6 binding and p38 MAPK signaling. REX1 deficiency induced enhancement of p38 MAPK signaling, leading to F-actin reorganization and activation of nuclear factor erythroid 2-related factor 2-mediated oxidative stress response, which collectively contributed to enhanced stemness and metastatic capabilities of HCC cells.

CRAF Methylation by PRMT6 Regulates Aerobic Glycolysis-Driven Hepatocarcinogenesis via ERK-Dependent PKM2 Nuclear Relocalization and Activation.

BACKGROUND AND AIMS: Most tumor cells use aerobic glycolysis (the Warburg effect) to support anabolic growth and promote tumorigenicity and drug resistance. Intriguingly, the molecular mechanisms underlying this phenomenon are not well understood. In this work, using gain-of-function and loss-of-function in vitro studies in patient-derived organoid and cell cultures as well as in vivo positron emission tomography-magnetic resonance imaging animal models, we showed that protein arginine N-methyltransferase 6 (PRMT6) regulates aerobic glycolysis in human hepatocellular carcinoma (HCC) through nuclear relocalization of pyruvate kinase M2 isoform (PKM2), a key regulator of the Warburg effect. APPROACH AND RESULTS: We found PRMT6 to methylate CRAF at arginine 100, interfering with its RAS/RAF binding potential, and therefore altering extracellular signal-regulated kinase (ERK)-mediated PKM2 translocation into the nucleus. This altered PRMT6-ERK-PKM2 signaling axis was further confirmed in both a HCC mouse model with endogenous knockout of PRMT6 as well as in HCC clinical samples. We also identified PRMT6 as a target of hypoxia through the transcriptional repressor element 1-silencing transcription factor, linking PRMT6 with hypoxia in driving glycolytic events. Finally, we showed as a proof of concept the therapeutic potential of using 2-deoxyglucose, a glycolysis inhibitor, to reverse tumorigenicity and sorafenib resistance mediated by PRMT6 deficiency in HCC. CONCLUSIONS: Our findings indicate that the PRMT6-ERK-PKM2 regulatory axis is an important determinant of the Warburg effect in tumor cells, and provide a mechanistic link among tumorigenicity, sorafenib resistance, and glucose metabolism.

Post-transcriptional negative feedback regulation of proteostasis through the Dis3 ribonuclease and its disruption by polyQ-expanded Huntingtin.

When proteostasis is disrupted by stresses such as heat shock, the heat stress response will be stimulated, leading to up-regulation of molecular chaperones by transcriptional activation and mRNA stabilization for restoring proteostasis. Although the mechanisms for their transcriptional activation have been clearly defined, how chaperone mRNAs are stabilized remains largely unknown. Starting by exploring the coupling between the apparently unrelated RNA degradation and protein quality control (PQC) systems, we show that the Dis3 ribonuclease, catalytic subunit of the RNA exosome required for RNA degradation, suppresses PQC activity in unstressed cells by degrading mRNAs encoding the Hsp70 cofactors Sis1, Ydj1 and Fes1, as well as some other chaperones or PQC factors, thereby limiting their protein expression. Dis3 is stabilized through its binding to Sis1 and the Hsp70s Ssa1/2. Upon heat stress, loss of Sis1 and Ssa1/2 availability triggers Dis3 ubiquitination and degradation, leading to stabilization of those chaperone mRNAs originally targeted by Dis3. We further demonstrate that polyQ-expanded huntingtin delays Dis3 degradation during heat stress and thus hinders chaperone mRNA stabilization. Our findings not only reveal a post-transcriptional negative feedback loop for maintaining proteostasis, but also uncover a mechanism that contributes to the impaired heat stress response in Huntington's disease.

Polyethylene glycol-coated porous magnetic nanoparticles for targeted delivery of chemotherapeutics under magnetic hyperthermia condition.

PURPOSE: Although magnetite nanoparticles (MNPs) are promising agents for hyperthermia therapy, insufficient drug encapsulation efficacies inhibit their application as nanocarriers in the targeted drug delivery systems. In this study, porous magnetite nanoparticles (PMNPs) were synthesized and coated with a thermosensitive polymeric shell to obtain a synergistic effect of hyperthermia and chemotherapy. MATERIALS AND METHODS: PMNPs were produced using cetyltrimethyl ammonium bromide template and then coated by a polyethylene glycol layer with molecular weight of 1500 Da (PEG1500) and phase transition temperature of 48 ± 2 °C to endow a thermosensitive behavior. The profile of drug release from the nanostructure was studied at various hyperthermia conditions generated by waterbath, magnetic resonance-guided focused ultrasound (MRgFUS), and alternating magnetic field (AMF). The in vitro cytotoxicity and hyperthermia efficacy of the doxorubicin-loaded nanoparticles (DOX-PEG1500-PMNPs) were assessed using human lung adenocarcinoma (A549) cells. RESULTS: Heat treatment of DOX-PEG1500-PMNPs containing 235 ± 26 mg·g-1 DOX at 48 °C by waterbath, MRgFUS, and AMF, respectively led to 71 ± 4%, 48 ± 3%, and 74 ± 5% drug release. Hyperthermia treatment of the A549 cells using DOX-PEG1500-PMNPs led to 77% decrease in the cell viability due to the synergistic effects of magnetic hyperthermia and chemotherapy. CONCLUSION: The large pores generated in the PMNPs structure could provide a sufficient space for encapsulation of the chemotherapeutics as well as fast drug encapsulation and release kinetics, which together with thermosensitive characteristics of the PEG1500 shell, make DOX-PEG1500-PMNPs promising adjuvants to the magnetic hyperthermia modality.

In Vitro Drug Dissolution/Permeation Testing of Nanocarriers for Skin Application: a Comprehensive Review.

This review highlights in vitro drug dissolution/permeation methods available for topical and transdermal nanocarriers that have been designed to modulate the propensity of drug release, drug penetration into skin, and permeation into systemic circulation. Presently, a few of USFDA-approved in vitro dissolution/permeation methods are available for skin product testing with no specific application to nanocarriers. Researchers are largely utilizing the in-house dissolution/permeation testing methods of nanocarriers. These drug release and permeation methods are pending to be standardized. Their biorelevance with reference to in vivo plasma concentration-time profiles requires further exploration to enable translation of in vitro data for in vivo or clinical performance prediction.

Alginate-C18 Conjugate Nanoparticles Loaded in Tripolyphosphate-Cross-Linked Chitosan-Oleic Acid Conjugate-Coated Calcium Alginate Beads as Oral Insulin Carrier.

Simple alginate, alginate-stearic acid, and alginate-C18 conjugate nanoparticles and tripolyphosphate-cross-linked chitosan-oleic acid conjugate-coated calcium alginate beads as the vehicle of nanoparticles were designed. Their size, ζ potential, morphology, drug load, drug release, matrix molecular characteristics, mucus penetration, HT-29 cell line cytotoxicity and intracellular trafficking, in vivo blood glucose lowering, and insulin delivery profiles were characterized. Alginate-C18 conjugate nanoparticles were nontoxic. Among all nanoparticle variants, they had reduced size and ζ potential thus enhancing particulate mucus penetration and intracellular trafficking. Their insulin reabsorption tendency was minimized as alginate active COOH/COO- sites were preoccupied with C18. Their loading into coated beads was translated to reduced drug release in simulated gastric phase with nanoparticles being released in the intestinal phase. The combination dosage form increased the blood glucose lowering extent of insulin and blood insulin level compared with nanoparticles or beads alone. Nanoparticles in beads represented a viable approach for oral insulin delivery.

Reprogramming of central carbon metabolism in cancer stem cells.

Cancer metabolism has been studied for years and adopted in the clinic for monitoring disease progression and therapy response. Despite our growing knowledge of a distinctly altered metabolic behavior in cancer, drugs targeting cancer metabolism have remained less than promising. Recent efforts in cancer stem cell (CSC) biology suggest that a subpopulation of tumor-initiating cells within the tumor bulk represents the root of tumor recurrence and therapy resistance. In recent years, metabolic phenotype of CSCs of various tumor types has been identified. This breakthrough has shed light on the underlying mechanism by which CSCs maintain stemness, confer resistance to therapies and initiate tumor relapse. The distinct metabolic characteristics of CSCs compared to non-CSCs provide an opportunity to target CSCs more specifically and have become a major focus in cancer research in recent years with substantial efforts conducted towards discovering clinical targets. This perspective article summarizes the current knowledge of CSC metabolism in carcinogenesis and highlights the potential of targeting CSC metabolism for therapy.

Anti-Fatigue Effects of the Unique Polysaccharide Marker of Dendrobium officinale on BALB/c Mice.

Dendrobium officinale extract shows potent anti-fatigue effects; however, the active substance responsible for these effects remains undetermined. A glucomannan with a huge molecular size of 730 kDa, called DOP, was identified as the unique authentication marker of this expensive herb. DOP exhibited immunomodulating effects on macrophages and lymphocytes in our previous study. Clinical reports also showed that people with fatigue syndrome have a disturbed immune system. Because DOP is the unique and dominant component of D. officinale, we hypothesize that DOP may also have anti-fatigue activity. The present study aims to evaluate the anti-fatigue activity of DOP on BALB/c mice, with Rhodiola rosea extract as a positive control. DOP and Rhodiola rosea extract were orally administered at doses of 50 mg/kg and 100 mg/kg, respectively, for four weeks, and the anti-fatigue activity of DOP on BALB/c mice was evaluated using the weight-loaded swimming test. The contents of lactic dehydrogenase (LDH), creatine phosphokinase (CK), triglyceride (TG), blood urea nitrogen (BUN), superoxide dismutase (SOD), malondialdehyde (MDA), lactic acid (LD), and glutathione peroxidase (GSH-Px) in serum, glycogen of liver and gastrocnemius muscle were also determined. Their effects on variability of T cells and B cells were determined by using tetrazolium compound (MTS) method. The weight-loaded swimming exercise caused fatigue syndrome, mainly including the decreases of serum SOD/GSH-Px and gastrocnemius glycogen, as well as the increases of LDH, BUN, MDA, CK, TG, and LD in serum. All of these indicators of fatigue were inhibited to a certain extent by both DOP and Rhodiola rosea extract; however, the effects of DOP were much stronger than those of Rhodiola rosea extract. Compared to the positive control, mice dosed with DOP showed increases in endurance, body weight, and food intake. Furthermore, DOP-feeding mice significantly increased the cell variability of T lymphocytes and B lymphocytes, compared with that of mice in control group. This study indicates that the unique and dominant polysaccharide DOP of D. officinale has stronger anti-fatigue activity than Rhodiola rosea extract. As such, DOP has promising potential for pharmaceutical development into health products to reduce fatigue.