A microfluidic gradient device for drug screening with human iPSC-derived motoneurons.

We developed a microfluidic gradient device to utilize as a drug screening system with human induced pluripotent stem cell (hiPSC)-derived motoneurons. The microfluidic channel was asymmetrically designed to generate the concentration gradients and a micropillar array was used to trap and culture the motoneuron spheroids containing motoneurons for 9 days. We optimized the concentration gradients in the microfluidic device using a computational fluid dynamics (CFD) model. We also observed that the motoneuron spheroid-derived neurite network was generated in response to the concentration gradients of riluzole in the microfluidic device. Therefore, this microfluidic gradient device could be useful for screening of various drugs for neurological disease applications.

Liposomal Texaphyrin Theranostics for Metastatic Liver Cancer.

Reported here is a new theranostic agent, 1, which consists of a Gd3+-texaphyrin core conjugated to a doxorubicin prodrug via a disulfide bond. Conjugate 1 was designed to undergo cleavage in the presence of glutathione (GSH), a species typically upregulated in cancer cells. As prepared, conjugate 1 displays no appreciable fluorescence. However, when exposed to excess GSH an increase in the fluorescence intensity at 592 nm is observed that is ascribed to release of free doxorubicin. To improve the solubility and enhance the tumor targeting of 1, it was loaded into folate-receptor-targeted liposomes to produce FL-1 (for folate liposome loaded with 1). As inferred from both fluorescence turn on studies and independent HPLC analyses, FL-1 was found to undergo selective uptake and cleavage to release free Dox in the KB and CT26 cell lines, which express folate receptors on the cell surface, relative to the HepG2 and NIH3T3 cell lines, which show low expression of those receptors. FL-1 was found to produce a greater antiproliferative effect in the case of the KB and CT26 cell lines as compared to that in the HepG2 and NIH3T3 cell lines. FL-1 was also found to provide enhanced magnetic resonance imaging in vivo under conditions of T1 contrast in the early stage of metastatic cancer progression. Finally, time-dependent tumor regrowth studies involving both subcutaneous and metastatic liver cancer mouse models revealed that FL-1 is capable of reducing the tumor burden in vivo.

An activatable prodrug for the treatment of metastatic tumors.

Metastatic cancers have historically been difficult to treat. However, metastatic tumors have been found to have high levels of reactive oxygen species such as hydrogen peroxide (H2O2), supporting the hypothesis that a prodrug could be activated by intracellular H2O2 and lead to a potential antimetastatic therapy. In this study, prodrug 7 was designed to be activated by H2O2-mediated boronate oxidation, resulting in activation of the fluorophore for detection and release of the therapeutic agent, SN-38. Drug release from prodrug 7 was investigated by monitoring fluorescence after addition of H2O2 to the cancer cells. Prodrug 7 activated by H2O2, selectively inhibited tumor cell growth. Furthermore, intratracheally administered prodrug 7 showed effective antitumor activity in a mouse model of metastatic lung disease. Thus, this H2O2-responsive prodrug has therapeutic potential as a novel treatment for metastatic cancer via cellular imaging with fluorescence as well as selective release of the anticancer drug, SN-38.

Association of protein expression in isolated milk epithelial cells and cis-9, trans-11 conjugated linoleic acid proportions in milk from dairy cows.

BACKGROUND: The effects of the individual variation among dairy cows on the synthesis of cis-9, trans-11 conjugated linoleic acid (CLA) are still not well characterised. Therefore, the protein expression profiles of isolated milk epithelial cells (MECs) were detected by two-dimensional electrophoresis and their correlation with the various proportion of cis-9, trans-11 CLA were evaluated. RESULTS: Although animals were offered the same diet, the proportion of cis-9, trans-11 CLA in group High (1.02 ± 0.10%) was twice as high as that in group Low (0.59 ± 0.14%) (P < 0.05). MECs with the characteristics of native epithelial cells were successfully isolated from the milk and these cells had no obvious RNA degradation or were hardly contaminated with leucocytes or blood red cells. Moreover, the protein expression pattern of cathelicidin 5 in isolated MECs was positive, whereas annexin I (confirmed by real-time polymerase chain reaction), ZW10 interactor and κ-casein were negatively related to the proportion of cis-9, trans-11 CLA in the milk fat. CONCLUSION: The varied individual content of cis-9, trans-11 CLA in cows may be associated with annexin I. These findings may provide some theoretical basis for studies concerning the effects of the individual variation among dairy cows of the synthesis of cis-9, trans-11 CLA. © 2013 Society of Chemical Industry.

An activatable theranostic for targeted cancer therapy and imaging.

A new theranostic strategy is described. It is based on the use of an "all in one" prodrug, namely the biotinylated piperazine-rhodol conjugate 4 a. This conjugate, which incorporates the anticancer drug SN-38, undergoes self-immolative cleavage when exposed to biological thiols. This leads to the tumor-targeted release of the active SN-38 payload along with fluorophore 1 a. This release is made selective as the result of the biotin functionality. Fluorophore 1 a is 32-fold more fluorescent than prodrug 4 a. It permits the delivery and release of the SN-38 payload to be monitored easily in vitro and in vivo, as inferred from cell studies and ex vivo analyses of mice xenografts derived from HeLa cells, respectively. Prodrug 4 a also displays anticancer activity in the HeLa cell murine xenograft tumor model. On the basis of these findings we suggest that the present strategy, which combines within a single agent the key functions of targeting, release, imaging, and treatment, may have a role to play in cancer diagnosis and therapy.

Advances in Methane Emission Estimation in Livestock: A Review of Data Collection Methods, Model Development and the Role of AI Technologies.

This review examines the significant role of methane emissions in the livestock industry, with a focus on cattle and their substantial impact on climate change. It highlights the importance of accurate measurement and management techniques for methane, a potent greenhouse gas accounting for 14-16% of global emissions. The study evaluates both conventional and AI-driven methods for detecting methane emissions from livestock, particularly emphasizing cattle contributions, and the need for region-specific formulas. Sections cover livestock methane emissions, the potential of AI technology, data collection issues, methane's significance in carbon credit schemes, and current research and innovation. The review emphasizes the critical role of accurate measurement and estimation methods for effective climate change mitigation and reducing methane emissions from livestock operations. Overall, it provides a comprehensive overview of methane emissions in the livestock industry by synthesizing existing research and literature, aiming to improve knowledge and methods for mitigating climate change. Livestock-generated methane, especially from cattle, is highlighted as a crucial factor in climate change, and the review underscores the importance of integrating precise measurement and estimation techniques for effective mitigation.

Antifibrotic effect of MMP13-encoding plasmid DNA delivered using polyethylenimine shielded with hyaluronic acid.

The imbalanced expression of matrix metalloproteinases (MMPs) is associated with liver fibrosis, one of the most common chronic liver diseases. Enhanced expression of MMPs by gene therapy is emerging as a promising antifibrotic strategy, but the effectiveness of this approach depends on reliable systems for delivering MMP genes. Here, we evaluated a newly designed hyaluronic acid (HA)-shielded delivery system for systemic administration of plasmid DNA encoding MMP13 (pMMP13), and tested whether the enhanced expression of MMP13 ameliorates liver fibrosis in mice. In the CCl(4)-induced liver fibrosis model, systemic administration of pMMP13 using HA and polyethylenimine (PEI) significantly increased the expression of MMP13 and reduced collagen deposition. Moreover, following delivery of pMMP13 in a HA-shielded PEI complex, the serum levels of aspartate transaminase were reduced to levels approaching those in untreated normal mice. These results indicate that the delivery of pMMP13 using HA-shielded PEI enhances the efficiency of MMP13 expression in the liver, and highlight the potential of pMMP13 gene therapy as an antifibrotic strategy.

Magnetic resonance imaging of superparamagnetic iron oxide-labeled macrophage infiltrates in acute-phase renal ischemia-reperfusion mouse model.

Macrophages play a key role in the initial pathogenesis of kidney ischemia-reperfusion (I-R) injury, but the mechanism of their spatial and temporal recruitment from circulation remains uncertain. This study aimed to evaluate the feasibility of magnetic resonance imaging (MRI) for detecting intravenously administered superparamagnetic iron oxide (SPIO)-labeled macrophages in an experimental renal I-R mouse model. Unilateral kidney I-R mice were imaged with a 4.7-T MRI scanner before and after administration of SPIO-labeled macrophages (RAW 264.7). On MR images, adoptive transfer of SPIO-labeled macrophages in the acute phase (1-2 days after I-R) caused a band-shaped signal-loss zone resulting from macrophage infiltrations, in the outer medullary region of injured kidneys. MRI detection of macrophages homing to an injured kidney may facilitate early detection and investigation of the pathogenesis of acute kidney injury and be a strategy for determining the treatment of acute renal failure. From the Clinical Editor: This study evaluated the feasibility of magnetic resonance imaging for detecting superparamagnetic iron oxide (SPIO)-labeled macrophages in a renal ischemia-reperfusion mouse model. Similar strategies in humans may facilitate early detection and stratification of acute kidney injury.

The complete mitochondrial genome of Korean indigenous stag beetle, Dorcus koreanus.

In this study, the mitogenome sequences of Korean indigenous stag beetle, Dorcus koreanus, was completely determined by next-generation sequencing analysis. The complete mitogenome of D. koreanus has 15,421 bp in length and consists of 13 protein-coding genes (PCGs), 22 tRNAs, two rRNAs, and one control region. The gene orders and content were identical with previously recorded mitogenomes of Lucanidae species. Phylogenetic analysis based on mitogenome dataset, consisting PCGs was revealed the taxonomical position in the family Lucanidae.

rGO nanomaterial-mediated cancer targeting and photothermal therapy in a microfluidic co-culture platform.

We developed the microfluidic co-culture platform to study photothermal therapy applications. We conjugated folic acid (FA) to target breast cancer cells using reduced graphene oxide (rGO)-based functional nanomaterials. To characterize the structure of rGO-based nanomaterials, we analyzed the molecular spectrum using UV-visible and Fourier-transform infrared spectroscopy (FT-IR). We demonstrated the effect of rGO-FA-based nanomaterials on photothermal therapy of breast cancer cells in the microfluidic co-culture platform. From the microfluidic co-culture platform with breast cancer cells and human umbilical vein endothelial cells (HUVECs), we observed that the viability of breast cancer cells treated with rGO-FA-based functional nanomaterials was significantly decreased after near-infrared (NIR) laser irradiation. Therefore, this microfluidic co-culture platform could be a potentially powerful tool for studying cancer cell targeting and photothermal therapy.

Combinatorial biophysical cue sensor array for controlling neural stem cell fate.

Biophysical cues, such as electrical stimulus, mechanical feature, and surface topography, enable the control of neural stem cell (NSC) differentiation and neurite outgrowth. However, the effect of these biophysical cues on NSC behavior has not been fully elucidated. In the present study, we developed an innovative combinatorial biophysical cue sensor array combining a surface modified nanopillar array with conductive hydrogel micropatterns. The micro/nanopattern comprised silicon oxide-coated polyurethane nanopillar arrays on a flexible film and conductive hydrogel micropatterns including polyethylene glycol (PEG) hydrogel, silver nanowires (AgNW), and reduced graphene oxide (rGO). A computational fluid dynamic (CFD) model was used to optimize the design parameters of the nanopillar arrays. In the study, we successfully demonstrated that SiO2-coated nanopillar array enhanced the differentiation of NSCs and efficiently regulated neuronal behavior, such as neurite outgrowths, by conductive hydrogel micropatterns combined with electrical stimuli. Therefore, our innovative combinatorial biophysical cue sensor array to control NSC behavior via electrical stimuli can be potentially useful to study neurodegenerative and neurological disorder therapy applications.

Hyaluronic acid complexed to biodegradable poly L-arginine for targeted delivery of siRNAs.

BACKGROUND: Small interfering RNA (siRNA) has been recognized as a new therapeutic drug to treat various diseases by inhibition of oncogene or viral gene expression. Because hyaluronic acid (HA) has been described as a biocompatible biomaterial, we tested the nanoparticles formed by electrostatic complexation of negatively-charged HA and cationic poly L-arginine (PLR) for siRNA delivery systems. METHODS: Different electrostatic complexes of HA and PLR (HPs) were formulated: HP101 with 50% (w/w) HA and HP110 with 9% (w/w) HA. RESULTS: Gel retardation assays showed that HP101 and HP110 could form complexes with siRNAs. The diameters of these complexes were less than 200 nm. Cellular delivery efficiency of siRNAs by HPs depended on cell surface CD44 density. The HP-mediated delivery of siRNAs was highest in WM266.4 cells followed by B16F10 cells and COS-7 cells, in parallel with CD44 surface densities of these cell lines. TC(50) values (i.e. the HP concentrations at which 50% of cells were viable after treatment) were used as indicators of cytotoxicity. HP101 showed TC(50) values that were 2-fold and 23-fold higher than those of HP110 and PLR, respectively. After delivery into cells, siRNA exerted target-specific RNA interference effects on mRNA and protein levels. Three days after treatment of red fluorescent protein (RFP)-expressing B16F10 cells with RFP-specific siRNA complexed to HP101, cellular fluorescence signals were reduced. Intratumoral administration of RFP-specific siRNA via HP101 delivery significantly reduced the expression of RFP in tumor tissues. CONCLUSIONS: HP101 may function as a biocompatible polymeric carrier of siRNAs and have possible application to localized siRNA delivery in vivo.

Dual Stimuli-Triggered Nanogels in Response to Temperature and pH Changes for Controlled Drug Release.

Poly-N-isopropyl acrylamide (PNIPAM) nanogels have been modified with different acrylic acid (AAc) contents for the efficient control of lower critical solution temperature (LCST). In this study, PNIPAM-co-AAc nanogels nanogels showed two volume phase transitions in comparison with PNIPAM. The transition temperature of PNIPAM nanogels was increased with AAc contents. The controlled drug release performance of PNIPAM-co-AAc nanogels loaded with ß-lapachone was attributed to the AAc content ratio and was efficiently triggered in response to temperature and pH. Moreover, a colorimetric cell proliferation assay and direct fluorescence-based live/dead staining were used to confirm the concurrence on drug release profiles. Finally, PNIPAM-co-AAc20 showed a relatively low level of drug release in the range of acidic to neutral pH at body temperature, while maximizing drug release at basic pH. Therefore, we demonstrated that the PNIPAM-based nanogel with the temperature- and pH-responsive features could be a promising nanocarrier for potential intestine-specific drug delivery.

Development of a theranostic prodrug for colon cancer therapy by combining ligand-targeted delivery and enzyme-stimulated activation.

The high incidence of colorectal cancer worldwide is currently a major health concern. Although conventional chemotherapy and surgery are effective to some extent, there is always a risk of relapse due to associated side effects, including post-surgical complications and non-discrimination between cancer and normal cells. In this study, we developed a small molecule-based theranostic system, Gal-Dox, which is preferentially taken up by colon cancer cells through receptor-mediated endocytosis. After cancer-specific activation, the active drug Dox (doxorubicin) is released with a fluorescence turn-on response, allowing both drug localization and site of action to be monitored. The therapeutic potency of Gal-Dox was also evaluated, both in vivo and ex vivo, thus illustrating the potential of Gal-Dox as a colorectal cancer theranostic with great specificity.

Generation of uniform-sized multicellular tumor spheroids using hydrogel microwells for advanced drug screening.

Even though in vitro co-culture tumor spheroid model plays an important role in screening drug candidates, its wide applications are currently limited due to the lack of reliable and high throughput methods for generating well-defined and 3D complex co-culture structures. Herein, we report the development of a hydrogel microwell array to generate uniform-sized multicellular tumor spheroids. Our developed multicellular tumor spheroids are structurally well-defined, robust and can be easily transferred into the widely used 2D culture substrates while maintaining our designed multicellular 3D-sphere structures. Moreover, to develop effective anti-cancer therapeutics we integrated our recently developed gold-graphene hybrid nanomaterial (Au@GO)-based photothermal cancer therapy into a series of multicellular tumor spheroid co-culture system. The multicellular tumor spheroids were harvested onto a two-dimensional (2D) substrate, under preservation of their three-dimensional (3D) structure, to evaluate the photothermal therapy effectiveness of graphene oxide (GO)-wrapped gold nanoparticles (Au@GO). From the model of co-culture spheroids of HeLa/Ovarian cancer and HeLa/human umbilical vein endothelial cell (HUVEC), we observed that Au@GO nanoparticles displayed selectivity towards the fast-dividing HeLa cells, which could not be observed to this extent in 2D cultures. Overall, our developed uniform-sized 3D multicellular tumor spheroid could be a powerful tool for anticancer drug screening applications.

In vivo imaging of ß-galactosidase stimulated activity in hepatocellular carcinoma using ligand-targeted fluorescent probe.

Development of targeted, selective, and noninvasive fluorescent probes for in vivo visualization of tumor-associated overexpressed enzymes are highly anticipated for cancer diagnosis and therapy. Herein, we developed a noninvasive fluorescent probe (DCDHF-ßgal) for the sensitive detection, and in vivo visualization of ß-galactosidase in hepatocyte HepG2 cells and its xenograft model. As a model system for in vivo targeted imaging, DCDHF-ßgal possessing galactose unit selectively target hepatocyte and monitor the ß-galactosidase activity with deep tissue penetration, and low background interference. DCDHF-ßgal was activated by intracellular ß-galactosidases as the driving force for the release of NIR fluorophore, thereby exhibiting ratiometric optical response. Initial fluorescence emission measured at 615 nm was changed to fluorescence at 665 nm upon activation of DCDHF-ßgal with ß-galactosidase. Ratiometric fluorescence detection of ß-galactosidase was also observed in hepatocellular carcinoma cells and tumor xenograft. The noninvasive in vivo optical imaging facilitated by targeted and enzyme-activated imaging agent would be useful in various biomedical and diagnostic applications.

Acid-triggered release of doxorubicin from a hydrazone-linked Gd(3+)-texaphyrin conjugate.

The hydrazone-based Gd(3+)-texaphyrin doxorubicin conjugate 1, releases active doxorubicin at acidic pH values, allowing its components to be followed by two complementary imaging methods, namely Off-On fluorescence enhancement and MR imaging. It thus acts as a promising theranostic agent.

Hypoxia-directed and activated theranostic agent: Imaging and treatment of solid tumor.

Hypoxia, a distinguished feature of various solid tumors, has been considered as a key marker for tumor progression. Inadequate vasculature and high interstitial pressures result in relatively poor drug delivery to these tumors. Herein, we developed an antitumor theranostic agent, 4, which is activated in hypoxic conditions and can be used for the diagnosis and treatment of solid tumors. Compound 4, bearing biotin, a tumor-targeting unit, and SN38, an anticancer drug, proved to be an effective theranostic agent for solid tumors. SN38 plays a dual role: as an anticancer drug for therapy and as a fluorophore for diagnosis, thus avoids an extra fluorophore and limits cytotoxicity. Compound 4, activated in the hypoxic environment, showed high therapeutic activity in A549 and HeLa cells and spheroids. In vivo imaging of solid tumors confirmed the tumor-specific localization, deep tissue penetration and activation of compound 4, as well as the production of a strong anticancer effect through the inhibition of tumor growth in a xenograft mouse model validating it as a promising strategy for the treatment of solid tumors.

In Vivo Tracking of Phagocytic Immune Cells Using a Dual Imaging Probe with Gadolinium-Enhanced MRI and Near-Infrared Fluorescence.

A novel dual imaging probe for in vivo magnetic resonance imaging (MRI) and optical imaging was developed by combining gadolinium (Gd)-chelating MR probe and a near-infrared (NIR) fluorophore, aza-BODIPY (AB; BODIPY = boron-dipyrromethene). This aza-BODIPY-based bimodal contrast agent (AB-BCA) showed a significant fluorescence emission around the NIR range and an enhanced longitudinal relaxivity in MR modality. The probe was easily delivered to phagocytic cells of the innate immune system, together with macrophages and dendritic cells (DCs), and presented high-performance fluorescence and MR imaging without obvious cytotoxicity. For in vivo visualization of AB-BCA using MRI and optical imaging, bone marrow-derived DCs were labeled and injected into the footpad of mice, and labeled DCs were tracked in vivo. We observed the migration of AB-BCA-labeled DCs into the lymph nodes via lymphatic vessels using NIR fluorescence and T1-weighted MR images. This dual-modality imaging probe was used for noninvasive monitoring of DC migration into lymph nodes and could be useful for investigating advanced cellular immunotherapy.

Comparison of diagnostic accuracy between CellprepPlus® and ThinPrep® liquid-based preparations in effusion cytology.

Liquid-based cytology (LBC) is being increasingly used for body fluid specimens and has improved diagnostic accuracy when compared to conventional smears. We compared the diagnostic accuracy and cellular morphologic features between CellprepPlus® LBC and ThinPrep® LBC in effusion cytology. One hundred and eighty body fluid specimens, consisting of 119 pleural fluid specimens, 59 peritoneal fluid specimens, and 2 pericardial fluid specimens, were obtained from 166 patients. Equal volumes of body fluid from each specimen were used in the CellprepPlus® and ThinPrep® preparations. Sensitivity, specificity, and positive and negative predictive values were evaluated. In addition, we selected 16 specimens from patients with metastatic adenocarcinoma, confirmed them by both LBC preparations, and measured the size of the nucleus in the tumor cells in these specimens. The sensitivity of the CellprepPlus® and ThinPrep® methods was 73.1% and 50.0%, respectively. The specificity and positive predictive values were 100% for both LBC methods, and the negative predictive values of the CellprepPlus® and ThinPrep® methods were 90.9% and 83.3%, respectively. The average nuclear size of the tumor cells was calculated as 20.87 µm using the CellprepPlus® method and 15.08 µm using the ThinPrep® method (P < 0.05). The CellprepPlus® method provided better diagnostic accuracy of effusion cytology compared to the ThinPrep® method and revealed the characteristic morphological features of tumor cells, including large and hypochromatic nuclei, prominent nucleoli, distinct nuclear membranes, and high cellularity.