A hierarchical fusion strategy of deep learning networks for detection and segmentation of hepatocellular carcinoma from computed tomography images.

BACKGROUND: Automatic segmentation of hepatocellular carcinoma (HCC) on computed tomography (CT) scans is in urgent need to assist diagnosis and radiomics analysis. The aim of this study is to develop a deep learning based network to detect HCC from dynamic CT images. METHODS: Dynamic CT images of 595 patients with HCC were used. Tumors in dynamic CT images were labeled by radiologists. Patients were randomly divided into training, validation and test sets in a ratio of 5:2:3, respectively. We developed a hierarchical fusion strategy of deep learning networks (HFS-Net). Global dice, sensitivity, precision and F1-score were used to measure performance of the HFS-Net model. RESULTS: The 2D DenseU-Net using dynamic CT images was more effective for segmenting small tumors, whereas the 2D U-Net using portal venous phase images was more effective for segmenting large tumors. The HFS-Net model performed better, compared with the single-strategy deep learning models in segmenting small and large tumors. In the test set, the HFS-Net model achieved good performance in identifying HCC on dynamic CT images with global dice of 82.8%. The overall sensitivity, precision and F1-score were 84.3%, 75.5% and 79.6% per slice, respectively, and 92.2%, 93.2% and 92.7% per patient, respectively. The sensitivity in tumors < 2 cm, 2-3, 3-5 cm and > 5 cm were 72.7%, 92.9%, 94.2% and 100% per patient, respectively. CONCLUSIONS: The HFS-Net model achieved good performance in the detection and segmentation of HCC from dynamic CT images, which may support radiologic diagnosis and facilitate automatic radiomics analysis.

Enhancing Efficacy of Albumin-Bound Paclitaxel for Human Lung and Colorectal Cancers through Autophagy Receptor Sequestosome 1 (SQSTM1)/p62-Mediated Nanodrug Delivery and Cancer therapy.

Selective autophagy is a defense mechanism by which foreign pathogens and abnormal substances are processed to maintain cellular homeostasis. Sequestosome 1 (SQSTM1)/p62, a vital selective autophagy receptor, recruits ubiquitinated cargo to form autophagosomes for lysosomal degradation. Nab-PTX is an albumin-bound paclitaxel nanoparticle used in clinical cancer therapy. However, the role of SQSTM1 in regulating the delivery and efficacy of nanodrugs remains unclear. Here we showed that SQSTM1 plays a crucial role in Nab-PTX drug delivery and efficacy in human lung and colorectal cancers. Nab-PTX induces SQSTM1 phosphorylation at Ser403, which facilitates its incorporation into the selective autophagy of nanoparticles, known as nanoparticulophagy. Nab-PTX increased LC3-II protein expression, which triggered autophagosome formation. SQSTM1 enhanced Nab-PTX recognition to form autophagosomes, which were delivered to lysosomes for albumin degradation, thereby releasing PTX to induce mitotic catastrophe and apoptosis. Knockout of SQSTM1 downregulated Nab-PTX-induced mitotic catastrophe, apoptosis, and tumor inhibition in vitro and in vivo and inhibited Nab-PTX-induced caspase 3 activation via a p53-independent pathway. Ectopic expression of SQSTM1 by transfection of an SQSTM1-GFP vector restored the drug efficacy of Nab-PTX. Importantly, SQSTM1 is highly expressed in advanced lung and colorectal tumors and is associated with poor overall survival in clinical patients. Targeting SQSTM1 may provide an important strategy to improve nanodrug efficacy in clinical cancer therapy. This study demonstrates the enhanced efficacy of Nab-PTX for human lung and colorectal cancers via SQSTM1-mediated nanodrug delivery.

Different synaptic mechanisms of intermittent and continuous theta-burst stimulations in a severe foot-shock induced and treatment-resistant depression in a rat model.

Treatment-resistant depression (TRD) is a condition wherein patients with depression fail to respond to antidepressant trials. A new form of repetitive transcranial magnetic stimulation (rTMS), called theta-burst stimulation (TBS), which includes intermittent theta-burst stimulation (iTBS) and continuous theta-burst stimulation (cTBS), is non-inferior to rTMS in TRD treatment. However, the mechanism of iTBS and cTBS underlying the treatment of TRD in the prefrontal cortex (PFC) remains unclear. Hence, we applied foot-shock stress as a traumatic event to develop a TRD rat model and investigated the different mechanisms of iTBS and cTBS. The iTBS and cTBS treatment were effective in depressive-like behavior and active coping behavior. The iTBS treatments improved impaired long-term potentiation and long-term depression (LTD), whereas the cTBS treatment only improved aberrant LTD. Moreover, the decrease in mature brain-derived neurotrophic factor (BDNF)-related protein levels were reversed by iTBS treatment. The decrease in proBDNF-related protein expression was improved by iTBS and cTBS treatment. Both iTBS and cTBS improved the decreased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and downregulation of mammalian target of the rapamycin (mTOR) signaling pathway. The iTBS produces both excitatory and inhibitory synaptic effects, and the cTBS only produces inhibitory synaptic effects in the PFC.

The Relationship Between Colles' Fractures and Leukocytosis in the Emergency Department.

In Taiwan, emergency physicians often perform wrist joint reduction and cast immobilization before orthopedic surgeons arrange for surgical management. Prophylactic antibiotics can decrease the risk of wound infection and have been routinely employed in orthopedic surgery. In Taiwan, emergency physicians also regularly perform blood investigations and administer prophylactic antibiotics to prevent infection if the patient exhibits leukocytosis. However, pain and pressure also cause leukocytosis, making it difficult to discern if the cause is infection or injury. Therefore, we explored the relationship between Colles' fractures and leukocytosis to determine if antibiotic treatment is necessary for this type of injury.

One-kilojoule pulsed-power generator for laboratory space sciences.

This paper reports on the assembly of a compact, low-cost, pulsed-power facility used for plasma studies. The construction uses two modules placed on opposite sides of the test chamber to minimize the system impedance and improve access to test samples. The stored energy is 1  kJ with a peak current of 135  kA and a 1592  ns quarter-period time. Up until now, an imploding conical-wire array has been studied by using time-integrated (visible) imaging, and time-resolved laser imaging, providing a measure of the plasma jet speed in the range of 170  km/s. Our future plans will continue to investigate high-energy-density plasmas that are relevant to the space environment, fusion, and spectroscopy.

Effects of Artemisia annua on experimentally induced leucocytozoonosis in chickens.

The biting midge Culicoides arakawae is the vector for the parasite Leucocytozoon caulleryi. Birds infected with L. caulleryi develop leucocytozoonosis. Given the food safety concern regarding drug residue in eggs, discovering a natural alternative to antibiotics is a worthy of exploration. Thus, we investigated the effects of the antimalarial herb Artemisia annua on experimentally induced leucocytozoonosis in chickens. We reared C. arakawae in the laboratory. Eggs were cultured, developing into larvae, pupae, and imagoes. Female midges sucked the blood of sick chickens and then were ground into a solution injected into healthy chickens. The control group was given empty capsules daily, whereas the 2 experimental groups were given 40 mg/kg sulfadimethoxine or 0.5 g of A. annua powder. Leucocytozoon gametocytes were detected in chicken blood through Giemsa staining. PCR detected the cytochrome b gene of L. caulleryi in the infected chickens. No significant among-group differences in body weight gain were observed before d 14 postinoculation (P > 0.05). Body weight gain in the control group was significantly lower from day 14 to 28 postinoculation (P < 0.05). After day 14, rectal temperature in the experimental groups decreased significantly compared with that in the control group. Lower rates of pale comb and green feces were observed in the animals receiving treatment from day 0. The experimental groups had a higher recovery rate and recovered earlier than did the control group. By day 31, all the animals had recovered. PCR detected L. caulleryi in the infected chickens with high sensitivity and accuracy. The animals receiving A. annua exhibited increased weight gain and reduced parasite concentrations in the blood. This in turn reduced mortality and the occurrence of pale comb and green feces. The findings are informative for research on leucocytozoonosis.

Targeting EGFR and Monitoring Tumorigenesis of Human Lung Cancer Cells In Vitro and In Vivo Using Nanodiamond-Conjugated Specific EGFR Antibody.

Nanoprobes provide advantages for real-time monitoring of tumor markers and tumorigenesis during cancer progression and development. Epidermal growth factor receptor (EGFR) is a key protein that plays crucial roles for tumorigenesis and cancer therapy of lung cancers. Here, we show a carbon-based nanoprobe, nanodiamond (ND), which can be applied for targeting EGFR and monitoring tumorigenesis of human lung cancer cells in vitro and in vivo. The optimal fluorescent intensities of ND particles were observed in the human lung cancer cells and nude mice under in vivo imaging system. The fluorescence signal of ND particles can be real-time detected in the xenografted human lung tumor formation of nude mice. Moreover, the ND-conjugated specific EGFR antibody cetuximab (Cet) can track the location and distribution of EGFR proteins of lung cancer cells in vitro and in vivo. ND-Cet treatment increased cellular uptake ability of nanocomposites in the EGFR-expressed cells but not in the EGFR-negative lung cancer cells. Interestingly, single ND-Cet complex can be directly observed on the protein G bead by immunoprecipitation and confocal microscopy. Besides, the EGFR proteins were transported to lysosomes for degradation. Together, this study demonstrates that ND-conjugated Cet can apply for targeting EGFR and monitoring tumorigenesis during lung cancer progression and therapy.

CirPred, the first structure modeling and linker design system for circularly permuted proteins.

BACKGROUND: This work aims to help develop new protein engineering techniques based on a structural rearrangement phenomenon called circular permutation (CP), equivalent to connecting the native termini of a protein followed by creating new termini at another site. Although CP has been applied in many fields, its implementation is still costly because of inevitable trials and errors. RESULTS: Here we present CirPred, a structure modeling and termini linker design method for circularly permuted proteins. Compared with state-of-the-art protein structure modeling methods, CirPred is the only one fully capable of both circularly-permuted modeling and traditional co-linear modeling. CirPred performs well when the permutant shares low sequence identity with the native protein and even when the permutant adopts a different conformation from the native protein because of three-dimensional (3D) domain swapping. Linker redesign experiments demonstrated that the linker design algorithm of CirPred achieved subangstrom accuracy. CONCLUSIONS: The CirPred system is capable of (1) predicting the structure of circular permutants, (2) designing termini linkers, (3) performing traditional co-linear protein structure modeling, and (4) identifying the CP-induced occurrence of 3D domain swapping. This method is supposed helpful for broadening the application of CP, and its web server is available at http://10.life.nctu.edu.tw/CirPred/ and http://lo.life.nctu.edu.tw/CirPred/ .

A secondary structure-based position-specific scoring matrix applied to the improvement in protein secondary structure prediction.

Protein secondary structure prediction (SSP) has a variety of applications; however, there has been relatively limited improvement in accuracy for years. With a vision of moving forward all related fields, we aimed to make a fundamental advance in SSP. There have been many admirable efforts made to improve the machine learning algorithm for SSP. This work thus took a step back by manipulating the input features. A secondary structure element-based position-specific scoring matrix (SSE-PSSM) is proposed, based on which a new set of machine learning features can be established. The feasibility of this new PSSM was evaluated by rigid independent tests with training and testing datasets sharing <25% sequence identities. In all experiments, the proposed PSSM outperformed the traditional amino acid PSSM. This new PSSM can be easily combined with the amino acid PSSM, and the improvement in accuracy was remarkable. Preliminary tests made by combining the SSE-PSSM and well-known SSP methods showed 2.0% and 5.2% average improvements in three- and eight-state SSP accuracies, respectively. If this PSSM can be integrated into state-of-the-art SSP methods, the overall accuracy of SSP may break the current restriction and eventually bring benefit to all research and applications where secondary structure prediction plays a vital role during development. To facilitate the application and integration of the SSE-PSSM with modern SSP methods, we have established a web server and standalone programs for generating SSE-PSSM available at http://10.life.nctu.edu.tw/SSE-PSSM.

Coupled nuclear-electronic decay dynamics of O2 inner valence excited states revealed by attosecond XUV wave-mixing spectroscopy.

Multiple Rydberg series converging to the O2+c4Σ-u state, accessed by 20-25 eV extreme ultraviolet (XUV) light, serve as important model systems for the competition between nuclear dissociation and electronic autoionization. The dynamics of the lowest member of these series, the 3sσg state around 21 eV, has been challenging to study owing to its ultra-short lifetime (<10 fs). Here, we apply transient wave-mixing spectroscopy with an attosecond XUV pulse to investigate the decay dynamics of this electronic state. Lifetimes of 5.8 ± 0.5 fs and 4.5 ± 0.7 fs at 95% confidence intervals are obtained for v = 0 and v = 1 vibrational levels of the 3s Rydberg state, respectively. A theoretical treatment of predissociation and electronic autoionization finds that these lifetimes are dominated by electronic autoionization. The strong dependence of the electronic autoionization rate on the internuclear distance because of two ionic decay channels that cross the 3s Rydberg state results in the different lifetimes of the two vibrational levels. The calculated lifetimes are highly sensitive to the location of the 3s potential with respect to the decay channels; by slight adjustment of the location, values of 6.2 and 5.0 fs are obtained computationally for the v = 0 and v = 1 levels, respectively, in good agreement with experiment. Overall, an intriguing picture of the coupled nuclear-electronic dynamics is revealed by attosecond XUV wave-mixing spectroscopy, indicating that the decay dynamics are not a simple competition between isolated autoionization and predissociation processes.

Rail-gap switch with a multistep high-voltage triggering system.

A rail-gap switch with a multistep triggering system was developed. The rail-gap switch consisted of two rail-like electrodes and a knife-edge electrode parallel to each other. It was assembled from many pieces and filled with unpressurized-flowing dry air. Good alignments between all electrodes were achieved by using a special jig and the knife-edge electrode as the spatial reference. Furthermore, to trigger the rail-gap switch, a multistep triggering system was used. The triggering system consisted of three components: an optical trigger-pulse generator, a slow high-voltage trigger-pulse generator using an ignition coil for a car, and finally, a fast high-voltage trigger-pulse generator using a three-stage Marx generator. The triggering system generated a negative high-voltage trigger pulse of less than -40 kV with a falling speed of -6.6 ± 0.4 kV/ns. The falling speed was fast enough to initiate multichannel discharges in the rail-gap switch. Finally, the rail-gap switch was tested using a test bench consisting of a 0.5-µF capacitor bank charged to 20 kV. The rail-gap switch was triggered by the multistep triggering system robustly with a delay of 180 ns. The delay between the time, when the peak current of the test bench occurred, and the trigger pulse was 890 ns with a jitter of 20 ns, i.e., ∼2% uncertainty in time. The inductance of the rail-gap switch was ∼80 nH obtained from the discharge tests. The rail-gap switch with the multistep high-voltage triggering system is suitable for any pulsed-power systems with current rise times in the order of 1 µs.

A multi-targeting strategy to ameliorate high-fat-diet- and fructose-induced (western diet-induced) non-alcoholic fatty liver disease (NAFLD) with supplementation of a mixture of legume ethanol extracts.

NAFLD (non-alcoholic fatty liver disease) is a multifactorial liver disease related to multiple causes or unhealthy conditions, including obesity and chronic inflammation. The accumulation of excess triglycerides, called steatosis, is known as a hallmark of an imbalance between the rates of hepatic fatty acid uptake/synthesis and oxidation/export. Furthermore, occurrence of NAFLD may lead to a cocktail of disease consequences caused by the altered metabolism of glucose, lipids, and lipoproteins, for instance, insulin resistance, type II diabetes, nonalcoholic steatohepatitis (NASH), liver fibrosis, and even hepatocarcinogenesis. Due to the complexity of the occurrence of NAFLD, a multi-targeting strategy is highly recommended to effectively address the issue and combat the causal loop. Ethanol extracts of legumes are popular supplements due to their richness and diversity in phytochemicals, especially isoflavones and anthocyanins. Although many of them have been reported to have efficacy in the treatment of different metabolic syndromes and obesity, there have not been many studies on them as a supplemental mixture. In this study, the alleviative effects of selected legume ethanol extracts (CrE) on high-fat-diet- and fructose-induced obesity, liver steatosis, and hyperglycemia are discussed. As revealed by the findings, CrE not only ameliorated obesity in terms of weight gained and enlargement of adipose tissue, but also significantly reduced the incidence of steatosis via phosphorylation of AMPK, resulting in inhibition of the downstream SREBP-1c/FAS pathway and an increase in an indicator of ß-oxidation (carnitine palmitoyl transferase 1a, CPT1A). Furthermore, CrE dramatically alleviated inflammatory responses, including both plasma and hepatic TNF-α, IL-6, and MCP-1 levels. CrE also had attenuating effects on hyperglycemia and insulin resistance and significantly reduced the fasting glucose level, fasting insulin level, and plasma leptin, and it exhibited positive effects in the Oral glucose tolerance test (OGTT) and Homeostatic Model Assessment for Insulin Resistance (HOMA-IR). At the molecular level, CrE could activate the PI3K/Akt/Glut2 pathway, which indicated an increase in insulin sensitivity and glucose uptake. Taken together, these results suggest that ethanol extracts of legumes could be potential supplements for metabolic syndromes, and their efficacy and effectiveness might facilitate the multi-targeting strategy required to mitigate NAFLD.

Targeted photoresponsive carbazole-coumarin and drug conjugates for efficient combination therapy in leukemia cancer cells.

Phototriggered drug delivery systems (PTDDSs) facilitate controlled delivery of drugs loaded on photoactive platform to the target region under light stimulation. The present study investigated the synthesis and efficacy of carbazole-coumarin (CC)-fused heterocycles as a PTDDS platform for the photocontrolled release of a chemotherapeutic agent, chlorambucil, in an in vitro model of human breast and leukemia cancer cells. CC-fused heterocycles were constructed using 4-hydroxycarbazole as the starting material, and further modification of these heterocycles yielded two CC derivatives. CC-7 with an additional - COOH group and CC-8 with the triphenylphosphonium (TPP) group, a mitochondria-targeting ligand introduced in the carbazole ring, dissolved in polar solvents and exhibited emission bands at 360 and 450 nm, respectively. The results indicate that visible light of 405 nm triggers the photolysis of the CC-drug conjugate and efficiently delivers the drug in both in vitro cancer cell models. Cytotoxicity evaluation indicates the suppression of proliferation of both types of cells treated with CC-8 under synergy effect combining drug potency and photosensitization. Further, the lower IC50 of CC-8 toward leukemia cells suggests the efficacy of the TPP ligand in increasing the bioavailability of CC-drug conjugates in leukemia treatment. Studies on mitochondria-targeting drug delivery systems are required for improving the performance of anticancer drugs.

The Making of a Flight Feather: Bio-architectural Principles and Adaptation.

The evolution of flight in feathered dinosaurs and early birds over millions of years required flight feathers whose architecture features hierarchical branches. While barb-based feather forms were investigated, feather shafts and vanes are understudied. Here, we take a multi-disciplinary approach to study their molecular control and bio-architectural organizations. In rachidial ridges, epidermal progenitors generate cortex and medullary keratinocytes, guided by Bmp and transforming growth factor ß (TGF-ß) signaling that convert rachides into adaptable bilayer composite beams. In barb ridges, epidermal progenitors generate cylindrical, plate-, or hooklet-shaped barbule cells that form fluffy branches or pennaceous vanes, mediated by asymmetric cell junction and keratin expression. Transcriptome analyses and functional studies show anterior-posterior Wnt2b signaling within the dermal papilla controls barbule cell fates with spatiotemporal collinearity. Quantitative bio-physical analyses of feathers from birds with different flight characteristics and feathers in Burmese amber reveal how multi-dimensional functionality can be achieved and may inspire future composite material designs. VIDEO ABSTRACT.

Triplet vs πσ* state mediated N-H dissociation of aniline.

UV-excited aromatic molecules with N-H/O-H moieties often possess an important nonradiative relaxation pathway, from an optically bright ππ* state to a dark dissociative πσ* state. We apply a new time-selected photofragment translational spectroscopy method to disclose a previously unknown triplet-mediated N-H dissociation of aniline prevented by the multiphoton dissociative ionization in conventional methods. We further determined the branching fractions of aniline dissociated in the πσ*, triplet, and ground states at 248 nm. Additionally, we selectively captured the population changes in the singlet and triplet states with ionization from different laser wavelengths, 355 or 266 nm, in time-resolved photoion yields. The combination of experimental data enables us to uniquely determine the relative ionization cross sections of the singlet and triplet states at an ionization laser wavelength of 266 nm and allows us to extensively measure the rate constants of intersystem crossing and the branching fractions at various excitation wavelengths.

Characteristic Distance of Resonance Energy Transfer Coupled with Surface Plasmon Polaritons.

We investigate resonance energy transfer (RET) between a donor-acceptor pair above a gold surface (including bulk and thin-film systems) and explore the distance/frequency dependence of RET enhancements using the theory we developed previously. The mechanism of RET above a gold surface can be attributed to the effects of mirror dipoles, surface plasmon polaritons (SPPs), and retardation. To clarify these effects on RET, we analyze the enhancements of RET by the mirror method, the decomposition of s- and p-polarization, and the SPP dispersion of charge-symmetric and charge-antisymmetric modes. We find a characteristic distance (approximately 1/10 of the wavelength) that can be used to classify the dominant effect on RET. Moreover, the characteristic distance can be shortened by narrowing the thickness of the thin-film systems, indicating that SPPs can enhance the rate of RET at a short range. The charge-symmetric and charge-antisymmetric modes of the thin films also allow us to engineer the maximum RET enhancement. We hope that our analysis inspires further investigation into the mechanism of RET coupled with SPPs and its applications.

Excited-state dissociation dynamics of phenol studied by a new time-resolved technique.

Phenol is an important model molecule for the theoretical and experimental investigation of dissociation in the multistate potential energy surfaces. Recent theoretical calculations [X. Xu et al., J. Am. Chem. Soc. 136, 16378 (2014)] suggest that the phenoxyl radical produced in both the X and A states from the O-H bond fission in phenol can contribute substantially to the slow component of photofragment translational energy distribution. However, current experimental techniques struggle to separate the contributions from different dissociation pathways. A new type of time-resolved pump-probe experiment is described that enables the selection of the products generated from a specific time window after molecules are excited by a pump laser pulse and can quantitatively characterize the translational energy distribution and branching ratio of each dissociation pathway. This method modifies conventional photofragment translational spectroscopy by reducing the acceptance angles of the detection region and changing the interaction region of the pump laser beam and the molecular beam along the molecular beam axis. The translational energy distributions and branching ratios of the phenoxyl radicals produced in the X, A, and B states from the photodissociation of phenol at 213 and 193 nm are reported. Unlike other techniques, this method has no interference from the undissociated hot molecules. It can ultimately become a standard pump-probe technique for the study of large molecule photodissociation in multistates.

MicroRNA-29a mitigation of toll-like receptor 2 and 4 signaling and alleviation of obstructive jaundice-induced fibrosis in mice.

Cholestasis and hepatitis can cause continuous liver damage that may ultimately result in liver fibrosis. In a previous study, we demonstrated that microRNA-29a (miR-29a) protects against liver fibrosis. Toll-like receptor 2 (TLR2) and TLR4 are pattern recognition receptors of bacterial lipoprotein and lipopolysaccharide, both of which participate in activating hepatic stellate cells and liver fibrosis. The purpose of this study is to characterize the biological influence of miR-29a on TLR2 and TLR4 signaling in livers injured with bile duct ligation (BDL). We performed BDL on both miR-29a transgenic mice (miR-29aTg) and wild-type mice to induce cholestatic liver injury. Primary HSCs were transfected with a miR-29a mimic and inhibitor. In the wild-type mice, the BDL demonstrated significant α-smooth muscle actin fibrotic matrix formation and hepatic high mobility group box-1 expression. However, in the miR-29aTg mice, these factors were significantly reduced. Furthermore, miR-29a overexpression reduced the BDL exaggeration of TLR2, TLR4, MyD88, bromodomain-containing protein 4 (BRD4), phospho-p65 as well as proinflammatory cytokines, IL-1ß, MCP-1, TGF-ß, and TNF-α. In vitro, miR-29a mimic transfection reduced α-SMA, BRD4,TLR2, and TLR4 expressions in HSCs. This study provides new molecular insight into the ability of miR-29a to inhibit TLR2 and TLR4 signaling, which thus slows the progression of cholestatic liver deterioration.

MicroRNA-29a mitigation of endoplasmic reticulum and autophagy aberrance counteracts in obstructive jaundice-induced fibrosis in mice.

Hepatic fibrosis was caused by a number of signaling pathways that damage liver integrity. We have previously shown that microRNA-29a (miR-29a) protects against liver fibrosis. Aberrant endoplasmic reticulum (ER) and autophagy function reportedly exaggerate hepatic disorders. The aim of this study was to characterize the biological influence of miR-29a on ER function in injured livers with bile duct ligation (BDL). We performed BDL on miR-29a transgenic mice (miR-29aTg) and wild-type mice to induce cholestatic liver injury. Rat T6 cells were transfected with miR-29a mimic and tunicamycin. Compared to the wild-type mice, the BDL deterioration of liver function in terms of total bilirubin, alanine transaminase, and aspartate transaminase activity in the miR-29aTg mice was significantly reduced. Affected livers in the miR-29aTg mice demonstrated a slight fibrotic matrix formation. miR-29a over-expression reduced the BDL disturbance of the expressions of inositol-requiring kinase 1alpha, double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase, spliced-X-box binding protein 1 (sXBP1), CCAAT/enhancer-binding protein homologous protein (CHOP), ULK, LC3BII, p62, and cleaved caspase-8, 9 and 3. In vitro, T6 cells exposed to tunicamycin by increasing abundances of CHOP, sXBP1, cleaved caspase-3, and LC3BII were diminished in the cell cultures transfected with the miR-29a mimic. On the other hand, we observed that miR-29a signaling protected liver tissues from BDL-mediated metabolic dysfunction and excessive fibrosis histopathology. This study provides new molecular insight into the miR-29a stabilization of ER integrity that slows the progression of cholestatic liver deterioration. Impact statement Long-term hepatic damage caused by hepatitis and cholestasis can accelerate fibrosis matrix over-production, which is a harmful process attributed to the dysregulation of a number of cellular and molecular events. The purpose of this study is to characterize the biological influence of miR-29a on endoplasmic reticulum (ER) function in bile duct ligation (BDL)-injured livers. To the best of our knowledge, this report is the first demonstration that miR-29a over-expression diminishes BDL provocation of ER stress (unfolded protein response, UPR) effector protein expression. This work also demonstrates that miR-29a decreased caspases protein expression in cholestatic livers, while an increase in miR-29a function reduced sXBP1 and CHOP expressions in T6 cells in mice. Analyses of this study highlight that controlling miR-29a signaling can serve as an innovative strategy in the future for microRNA regulation of ER homeostasis to combat cholestasis induction hepatic disorders.

Controlling Ligand Spacing on Surface: Polyproline-Based Fluorous Microarray as a Tool in Spatial Specificity Analysis and Inhibitor Development for Carbohydrate-Protein Interactions.

Multivalent carbohydrate-protein interactions are essential for many biological processes. Convenient characterization for multivalent binding property of proteins will aid the development of molecules to manipulate these processes. We exploited the polyproline helix II (PPII) structure as molecular scaffolds to adjust the distances between glycan ligand attachment sites at 9, 18, and 27 Å on a peptide scaffold. Optimized fluorous groups were also introduced to the peptide scaffold for immobilization to the microarray surface through fluorous interaction to control the orientation of the helical scaffolds. Using lectin LecA and antibody 2G12 as model proteins, the binding preference to the 27 Å glycopeptide scaffold, matched the distance of 26 Å between its two galactose binding sites on LecA and 31 Å spacing between oligomannose binding sites on 2G12, respectively. We further demonstrate this microarray system can aid the development of inhibitors by transforming the selected surface-bound scaffold into multivalent ligands in solution. This strategy can be extended to analyze proteins that lacking structural information to speed up the design of potent and selective multivalent ligands.