A bio-inspired nano-material recapitulating the composition, ultra-structure, and function of the glycosaminoglycan-rich extracellular matrix of nucleus pulposus.

The nucleus pulposus (NP) of intervertebral disc represents a soft gel consisting of glycosaminoglycans (GAGs)-rich extracellular matrix (ECM). Significant loss of GAGs and normal functions are the most prevalent changes in degenerated disc. Attempts targeted to incorporate GAGs into collagen fibrous matrices have been made but the efficiency is very low, and the resulting structures showed no similarity with native NP. Inspired by the characteristic composition and structures of the ECM of native NP, here, we hypothesize that by chemically modifying the collagen (Col) and hyaluronic acid (HA) and co-precipitating with GAGs, a bio-inspired nano-material recapitulating the composition, ultra-structure and function of the GAG-rich ECM will be fabricated. Compositionally, the bio-inspired nano-material namely Aminated Collagen-Aminated Hyaluronic Acid-GAG (aCol-aHA-GAG) shows a record high GAG/hydroxyproline ratio up to 39.1:1 in a controllable manner, out-performing that of the native NP. Ultra-structurally, the nano-material recapitulates the characteristic 'nano-beads' (25 nm) and 'bottle-brushes' (133 nm) features as those found in native NP. Functionally, the nano-material supports the viability and maintains the morphological and phenotypic markers of bovine NP cells, and shows comparable mechanical properties of native NP. This work contributes to the development of a compositionally, structurally, and functionally biomimetic nano-material for NP tissue engineering.

Identification of bHLH family genes in Agaricus bisporus and transcriptional regulation of arginine catabolism-related genes by AbbHLH1 after harvest.

Basic helix-loop-helix (bHLH) transcription factors (TFs) are widely distributed in eukaryotes and play an important role in biological growth and development. The identification and functional analyses of bHLH genes/proteins in edible mushrooms (Agaricus bisporus) have yet to be reported. In the present study, we identified 10 putative bHLH members carrying the conserved bHLH domains. Phylogenetic analyses revealed that the 10 AbbHLHs were the closest to sequences of species belonging to 7 different fungal subgroups, which was supported by loop length, intron patterns, and key amino acid residues. The substantial increase after harvest and continuously elevated expression of AbbHLH1 during the development until the disruption of mushroom velum, and the preferential expression in cap and gill tissues suggest the important function of AbbHLH1 in postharvest development of A. bisporus. The relationship of arginine catabolism-related genes with the early stage of postharvest continuing development also was revealed by expression determination. Subcellular localization showed that AbbHLH1 could be localized in nucleus. Importantly, the electrophoretic mobility shift and dual-luciferase reporter assays showed that AbbHLH1 activated the promoters of AbOAT, AbSPDS, and AbSAMDC and suppressed the expression of AbARG, AbUREA, and AbODC, probably for the modulation of arginine catabolism and thus control of postharvest mushroom development. Taken together, the available data provide valuable functional insight into the role of AbbHLH proteins in postharvest mushrooms.

Neural substrates of behavioral inhibitory control during the two-choice oddball task: functional neuroimaging evidence.

Background: Behavioral inhibitory control (BIC) depicts a cognitive function of inhibiting inappropriate dominant responses to meet the context requirement. Despite abundant research into neural substrates of BIC during the go/no-go and stop signal tasks, these tasks were consistently shown hard to isolate neural processes of response inhibition, which is of primary interest, from those of response generation. Therefore, it is necessary to explore neural substrates of BIC using the two-choice oddball (TCO) task, whose design of dual responses is thought to produce an inhibition effect free of the confounds of response generation. Objective: The current study aims at depicting neural substrates of performing behavioral inhibitory control in the two-choice oddball task, which designs dual responses to balance response generation. Also, neural substrates of performing BIC during this task are compared with those in the go/no-go task, which designs a motor response in a single condition. Methods: The present study integrated go/no-go (GNG) and TCO tasks into a new Three-Choice BIC paradigm, which consists of standard (75%), deviant (12.5%), and no-go (12.5%) conditions simultaneously. Forty-eight college students participated in this experiment, which required them to respond to standard (frequent) and deviant stimuli by pressing different keys, while inhibiting motor response to no-go stimuli. Conjunction analysis and ROI (region of interest) analysis were adopted to identify the unique neural mechanisms that subserve the processes of BIC. Results: Both tasks are effective in assessing BIC function, reflected by the significantly lower accuracy of no-go compared to standard condition in GNG, and the significantly lower accuracy and longer reaction time of deviant compared to standard condition in TCO. However, there were no significant differences between deviant and no-go conditions in accuracy. Moreover, functional neuroimaging has demonstrated that the anterior cingulate cortex (ACC) activation was observed for no-go vs. standard contrast in the GNG task, but not in deviant vs. standard contrast in the TCO task, suggesting that ACC involvement is not a necessary component of BIC. Second, ROI analysis of areas that were co-activated in TCO and GNG showed co-activations in the right inferior frontal cortex (triangle and orbital), with the signals in the TCO task significantly higher than those in the GNG task. Conclusions: These findings show that the designed responses to both standard and deviant stimuli in the TCO task, compared to the GNG task, produced a more prominent prefrontal inhibitory processing and extinguished an unnecessary component of ACC activation during BIC. This implies that prefrontal involvement, but not that of ACC, is mandatory for the successful performance of inhibiting prepotent behaviors.

Thermo-Induced Coalescence of Dual Cores in Double Emulsions for Single-Cell RT-PCR.

Single-cell reverse-transcription polymerase chain reaction (RT-PCR) has shown significant promise for transcriptional profiling of heterogeneous cells. However, currently developed microfluidic droplet-based methodologies for single-cell RT-PCR often require complex chip design to accommodate the associated multistep processes as well as customized detection platforms for high-throughput analysis. Herein, we proposed a dual-core double emulsion (DE)-based method to streamline the single-cell RT-PCR through thermo-induced coalescence of the dual cores. The dual-core DEs were produced by pairing two water-in-oil single emulsions containing a single-cell/lysis buffer and RT-PCR mix, respectively. After complete lysis of single cells in one of the cores, the dual-core DEs were merged by gentle heating, made possible by the optimized glycerol concentration present in the cores. Upon the coalescence of dual cores, the alkaline lysis buffer present in the core of the cell lysate was neutralized by the reaction buffer presented in the RT-PCR core, allowing TaqMan assay-based RT-PCR to occur effectively within the DEs. To demonstrate the potential of this streamlined dual-core platform, AKR1B10-positive A549 cells and AKR1B10-negative HEK293 cells were investigated via the TaqMan assay. Subsequently, specific transcript of AKR1B10 was readily available for quantitative profiling at the single-cell level using a commercially available flow cytometer in a high-throughput manner.

A high spatial resolution osteogenic differentiation in human mesenchymal stem cells induced by femtosecond laser.

A variety of physical and chemical methods have been developed in research laboratories for the induction of stem cell differentiation. However, the use of exogenous chemicals and materials may limit their widespread utility in clinics. To develop a clean and precise induction approach with minimal invasion, we reported here that 1-second stimulation by a tightly focused femtosecond laser (fsL) (140 mW/µm2 , 200 fs) can modulate the signaling systems in human mesenchymal cells, such as intracellular calcium and reactive oxygen species. Upon stimulation on an automatic platform, hMSCs were found to express osteoblastic markers and form calcium-rich deposits. Moreover, tissue mineralization was observed when the fsL-illuminated hMSCs were ectopically transplanted into nude mice. Collectively, we described a novel and non-contact optical stimulation method for cell differentiation with high spatiotemporal resolution.

Double emulsion-pretreated microwell culture for the in vitro production of multicellular spheroids and their in situ analysis.

Multicellular spheroids have served as a promising preclinical model for drug efficacy testing and disease modeling. Many microfluidic technologies, including those based on water-oil-water double emulsions, have been introduced for the production of spheroids. However, sustained culture and the in situ characterization of the generated spheroids are currently unavailable for the double emulsion-based spheroid model. This study presents a streamlined workflow, termed the double emulsion-pretreated microwell culture (DEPMiC), incorporating the features of (1) effective initiation of uniform-sized multicellular spheroids by the pretreatment of double emulsions produced by microfluidics without the requirement of biomaterial scaffolds; (2) sustained maintenance and culture of the produced spheroids with facile removal of the oil confinement; and (3) in situ characterization of individual spheroids localized in microwells by a built-in analytical station. Characterized by microscopic observations and Raman spectroscopy, the DEPMiC cultivated spheroids accumulated elevated lipid ordering on the apical membrane, similar to that observed in their Matrigel counterparts. Made possible by the proposed technological advancement, this study subsequently examined the drug responses of these in vitro-generated multicellular spheroids. The developed DEPMiC platform is expected to generate health benefits in personalized cancer treatment by offering a pre-animal tool to dissect heterogeneity from individual tumor spheroids.

Extraction of Functional Mitochondria Based on Membrane Stiffness.

The abnormal functionality of mitochondria has been linked to many life-threatening diseases such as cancers, failure of cardiovascular functions, and neurodegenerative disorders. Therefore, in vitro analysis of mitochondria has garnered great interest for understanding the mechanism of mitochondrial dysfunction-related disease development and therapeutics. However, due to the intrinsic heterogeneity of cell membrane stiffness, it remains challenging to standardize the protocols for the extraction of mitochondria and adequate disruption of the cellular membrane while retaining the functionality of mitochondria. We have previously developed a microfluidics-based cell shredder capable of serving the purpose. In this protocol, we describe the step-by-step procedures to empirically identify the threshold shear stress using this microfluidics-based cell shredder for mitochondrial extraction. The optimal shear stress to disrupt human embryonic kidney cell (HEK 293) and mice muscle cell (C2C12) has been characterized at around 16.4 Pa, whereas cell lines with stiffer membrane stiffness, for example, neuroblastoma cells (SH-SY5Y), require 27.4 Pa to effectively lyse the cells. This protocol also provides detailed procedures to determine the quality of extracted mitochondria based on the membrane potential and the integrity of extracted mitochondria. A comparison with the widely employed Dounce homogenizer has shown that the proposed microscale cell shredder can yield at least 40% more functional mitochondria and retain higher integrity regarding extracted mitochondria than the counterparts extracted from Dounce homogenizer, especially for low cell concentrations (5-20 × 104 cells/mL) and small sample volume (<200 µL).

Effect of cognitive behavior therapy combined with exercise intervention on the cognitive bias and coping styles of diarrhea-predominant irritable bowel syndrome patients.

BACKGROUND: Irritable bowel syndrome (IBS) is a common digestive system disease with a high incidence rate and is common in women. The cause of IBS remains unclear. Some studies have shown that mental and psychological diseases are independent risk factors for IBS. At present, the treatment of IBS is mainly symptomatic treatment. Clinically, doctors also use cognitive behavioral therapy to improve patients' cognitive ability to diseases and clinical symptoms. In recent years, exercise therapy has attracted more and more attention from scholars. Improving the symptoms of IBS patients through psychosomatic treatment strategy may be a good treatment method. AIM: To explore the effects of an intervention of cognitive behavioral therapy combined with exercise (CBT+E) on the cognitive bias and coping styles of patients with diarrhea-predominant irritable bowel syndrome (IBS-D); and to provide a theoretical reference for the management of IBS. METHODS: Sixty IBS-D patients and thirty healthy subjects were selected. The 60 IBS-D patients were randomly divided into experimental and control groups. The experimental group was treated with the CBT+E intervention, while the control group was treated with conventional drugs without any additional intervention. The cognitive bias and coping styles of the participants were evaluated at baseline and after 6 wk, 12 wk and 24 wk using the Automatic Thoughts Questionnaire (ATQ), Dysfunctional Attitudes Scale (DAS) and Pain Coping Style Questionnaire (CSQ) instruments, and the intervention effect was analyzed using SPSS 17.0 statistical software. RESULTS: At baseline, the scores on the various scales showed that all subjects had cognitive bias and adverse coping styles. The IBS Symptom Severity Scale (IBS-SSS) scores, ATQ total scores, DAS scores and CSQ scores of the two groups were not significantly different (P > 0.05). Compared with baseline, after 6 wk of the CBT+E intervention, there were significant differences in the ATQ scores, the dependence and total scores on the DAS, and the catastrophization, distraction and prayer scores on the CSQ (P < 0.05). After 12 wk, there were significant differences in the scores for perfectionism on the DAS and in the scores for reinterpretation, neglect and pain behavior on the CSQ in the experimental group (P < 0.05). After 24 wk, there were significant differences in the vulnerability, dependence, perfectionism, and total scores on the DAS and in the catastrophization, distraction and prayer scores on the CSQ in the experimental group (P < 0.01). The IBS-SSS scores were negatively correlated with the ATQ and DAS total scores (P < 0.05) but were positively correlated with the CSQ total score (P < 0.05). CONCLUSION: Intervention consisting of CBT+E can correct the cognitive bias of IBS-D patients and eliminate their adverse coping conditions. CBT+E should be promoted for IBS and psychosomatic diseases.

Biocompatibility Profile and In Vitro Cellular Uptake of Self-assembled Alginate Nanoparticles.

Polymeric nanoparticles could offer promising controlled drug delivery. The biocompatibility is of extreme importance for future applications in humans. Self-assembled polymeric nanoparticles based on phenylalanine ethyl ester (PAE)-modified alginate (Alg) had been successfully prepared and characterized in our lab. However, little is known about their interaction with cells and other biological systems. In this study, nanoparticles (NPs) based on PAE-Alg conjugates (PEA-NPs) with different degree of substitution (DS) were prepared and investigated. Our results showed that PEA-NPs had no effects on the proliferation of the human intestinal epithelial Caco-2 cells at concentrations up to 1000 µg/mL. Furthermore, the in vitro cellular uptake profile of PEA-NPs, concerning several parameters involved in the application of therapeutic or diagnostic NPs, such as NPs concentration, time and temperature, was described. Different NPs have been adopted for cellular uptake studies and the NPs internalized into Caco-2 cells were quantified. Cellular uptake efficiency could reach 60% within 4 h. PEA-NPs also showed greater cell permeability than oleoyl alginate ester nanoparticles (OAE-NPs) previously prepared in our lab. Our studies reveal that NPs based on PEA conjugate are promising nanosystems for cellular delivery.

Endoplasmic Reticulum-Localized Two-Photon-Absorbing Boron Dipyrromethenes as Advanced Photosensitizers for Photodynamic Therapy.

Two advanced boron dipyrromethene (BODIPY) based photosensitizers have been synthesized and characterized. With a glibenclamide analogous moiety, these compounds can localize in the endoplasmic reticulum (ER) of HeLa human cervical carcinoma cells and HepG2 human hepatocarcinoma cells. The BODIPY π skeleton is conjugated with two styryl or carbazolylethenyl groups, which can substantially red-shift the Q-band absorption and fluorescence emission and impart two-photon absorption (TPA) property to the chromophores. The TPA cross section of the carbazole-containing analogue reaches a value of 453 GM at 1010 nm. These compounds also behave as singlet oxygen generators with high photostability. Upon irradiation at λ > 610 nm, these photosensitizers cause photocytotoxicity to these two cell lines with IC50 values down to 0.09 µM, for which the cell death is triggered mainly by ER stress. The two-photon photodynamic activity of the distyryl derivative upon excitation at λ = 800 nm has also been demonstrated.

Disulfide-Linked Dendritic Oligomeric Phthalocyanines as Glutathione-Responsive Photosensitizers for Photodynamic Therapy.

A series of disulfide-linked dendritic phthalocyanines were synthesized by using the CuI -catalyzed alkyne-azide cycloaddition reaction as the key step. Whereas these compounds were essentially nonaggregated in N,N-dimethylformamide, they were stacked in citrate solution (pH 7.4, with 1 % Cremophor EL), as shown by the broad appearance of their Q-band absorption. Having two-to-six zinc(II) phthalocyanine units in a molecule, these compounds were significantly self-quenched, particularly in citrate solution. Both the fluorescence intensity and singlet-oxygen generation efficiency were significantly lower than those of the monomeric counterparts, and the self-quenching efficiency increased as the number of phthalocyanine units increased. Upon interaction with 5 mm glutathione (GSH) in citrate solution, the fluorescence intensity of these compounds increased as a result of cleavage of the disulfide linkages and separation of the phthalocyanine units, which thereby reduced the self-quenching effect. The "on/off" ratios were found to be 7, 18, 23, and 21 for the dimeric (PC2), trimeric (PC3), tetrameric (PC4), and hexameric (PC6) systems, respectively. GSH also enhanced the fluorescence emission inside human colon adenocarcinoma HT29 cells and promoted the formation of singlet oxygen of these compounds. Upon irradiation, their half maximal inhibitory concentration (IC50 ) values were found to be in the range of 0.18 to 0.38 µm. Finally, the biodistribution and activation of PC2 and PC6 were also examined in HT29 tumor-bearing nude mice. For both compounds, the fluorescence intensity per unit area at the tumor was found to grow gradually during the first 24 h. Whereas the intensity then dropped for PC2, the intensity for PC6 remained steady over the following 6 d, which might have been a result of the enhanced permeability and retention effect arising from the larger molecular mass of the hexameric system.

Demarcating the membrane damage for the extraction of functional mitochondria.

Defective mitochondria have been linked to several critical human diseases such as neurodegenerative disorders, cancers and cardiovascular disease. However, the detailed characterization of mitochondria has remained relatively unexplored, largely due to the lack of effective extraction methods that may sufficiently retain the functionality of mitochondria, particularly when limited amount of sample is considered. In this study, we explore the possibility of modulating hydrodynamic stress through a cross-junction geometry at microscale to selectively disrupt the cellular membrane while mitochondrial membrane is secured. The operational conditions are empirically optimized to effectively shred the cell membranes while keeping mitochondria intact for the model mammalian cell lines, namely human embryonic kidney cells, mouse muscle cells and neuroblastoma cells. Unsurprisingly, the disruption of cell membranes with higher elastic moduli (neuroblastoma) requires elevated stress. This study also presents a comparative analysis of total protein yield and concentrations of extracted functional mitochondria with two commercially available mitochondria extraction approaches, the Dounce Homogenizer and the Qproteome® Mitochondria Isolation Kit, in a range of cell concentrations. Our findings show that the proposed "microscale cell shredder" yields at least 40% more functional mitochondria than the two other approaches and is able to preserve the morphological integrity of extracted mitochondria, particularly at low cell concentrations (5-20 × 104 cells/mL). Characterized by its capability of rapidly processing a limited quantity of samples (200 µL), demarcating the membrane damage through the proposed microscale cell shredder represents a novel strategy to extract subcellular organelles from clinical samples.

A cell-selective glutathione-responsive tris(phthalocyanine) as a smart photosensitiser for targeted photodynamic therapy.

A biotin-conjugated disulfide-linked tris(phthalocyanine) has been synthesised and characterised. As shown by electronic absorption and steady-state fluorescence spectroscopy methods, the compound remains non-aggregated in N,N-dimethylformamide, but is significantly aggregated in phosphate buffered saline with 1% Cremophor EL. The reduction in fluorescence intensity and singlet oxygen generation efficiency as compared with the monomeric counterpart suggests that the tris(phthalocyanine) exhibits an excellent self-quenching effect, particularly in an aqueous medium. Upon interaction with glutathione, both the fluorescence emission and singlet oxygen production can be restored as a result of the cleavage of the disulfide linkages, thereby releasing the phthalocyanine units and reducing their aggregation and self-quenching effects. With a biotin moiety, this tris(phthalocyanine) is preferentially taken up by the biotin-receptor-positive HeLa cells and activated by the intracellular glutathione, resulting in fluorescence recovery and photocytotoxicity with an IC50 value of 0.68 µM. This compound can therefore act as a dual functional photosensitiser.

pH-Responsive Dimeric Zinc(II) Phthalocyanine in Mesoporous Silica Nanoparticles as an Activatable Nanophotosensitizing System for Photodynamic Therapy.

An acid-cleavable acetal-linked zinc(II) phthalocyanine dimer with an azido terminal group (cPc) was prepared and conjugated to alkyne-modified mesoporous silica nanoparticles via copper(I)-catalyzed alkyne-azide cycloaddition reaction. For comparison, an amine-linked analogue (nPc) was also prepared as a non-acid-cleavable counterpart. These dimeric phthalocyanines were significantly self-quenched due to the close proximity of the phthalocyanine units inside the mesopores, resulting in much weaker fluorescence emission and singlet oxygen generation, both in N,N-dimethylformamide and in phosphate-buffered saline (PBS), compared with the free molecular counterparts. Under acidic conditions in PBS, the cPc-encapsulated nanosystem was activated in terms of fluorescence emission and singlet oxygen production. After internalization into human colon adenocarcinoma HT29 cells, it exhibited much higher intracellular fluorescence and photocytotoxicity compared to the nanosystem entrapped with nPc. The activation of this nanosystem was also demonstrated in tumor-bearing nude mice. The intratumoral fluorescence intensity increased gradually over 24 h, while for the nPc counterpart the fluorescence remained very weak. The results suggest that this nanosystem serves as a promising activatable nanophotosensitizing agent for photodynamic therapy.

A biotin-conjugated glutathione-responsive FRET-based fluorescent probe with a ferrocenyl BODIPY as the dark quencher.

An efficient ferrocenyl BODIPY based dark quencher has been developed and employed to construct a FRET-based fluorescent probe that contains a biotin moiety as a potential directing ligand for cancer cells and a glutathione-cleavable disulfide linker connecting the quencher and a distyryl BODIPY fluorophore. This molecular probe is deactivated in the native form through FRET followed by intramolecular charge transfer due to the ferrocenyl unit. However, upon interaction with glutathione in phosphate buffered saline and inside cancer cells, the fluorescence emission is significantly increased due to detachment of the fluorophore from the quencher. As shown by flow cytometry, this probe also exhibits preferential uptake by the biotin-receptor-expressing A549 human lung adenocarcinoma epithelial cells over the Chinese hamster ovary CHO-K1 cells used as the negative control. On the basis that both biotin receptor and GSH level are often overexpressed or elevated in cancer cells, this dual functional fluorescent probe serves as a promising agent for cancer imaging.

Nanoparticles based on phenylalanine ethyl ester-alginate conjugate as vitamin B2 delivery system.

Phenylalanine ethyl ester (PAE)-alginate (Alg) conjugate (PAE-Alg, PEA) was synthesized and formation of an amide bond between PAE and Alg was confirmed by Fourier transformed-infrared and (1)H nuclear magnetic resonance spectroscopy. The degree of PAE substitution was 3.5-4.7 (PAE group per hundred sugar residues of Alg) which was determined by elemental analysis. The critical aggregation concentration values determined for PEA conjugates PEA1, PEA2, and PEA3 were 0.20, 0.12, and 0.10 mg/ml, respectively. The particle size of PEA nanoparticles (PEA-NPs) decreased from 425 nm to 226 nm with the increasing degree of PAE substitution. Vitamin B2 (VB2), as a model nutrient, was encapsulated into the nanoparticles. The drug-loading content increased with increasing degree of PAE substitution. The maximum VB2 loading capacity and loading efficiency of PEA3 nanoparticles were 3.53 ± 0.03% and 91.48 ± 0.80%, respectively. The in vitro release behavior of VB2 from the PEA-NPs showed a biphasic release profile with an initial burst release of about 40-50% of VB2 in the first 10 h followed by a steady and continuous release phase for the following 50 h in PBS, pH 7.4. The human colorectal carcinoma cell line was used to investigate the cytotoxicity of PEA-NPs. Our results showed that various concentrations of nanoparticles did not cause significant cytotoxicity against cell lines at normal concentrations.