Prognostic Factors for Recovery from Left Recurrent Laryngeal Nerve Palsy After Minimally Invasive McKeown Esophagectomy: A Retrospective Study.

BACKGROUND: Recurrent laryngeal nerve (RLN) palsy is a serious complication of esophagectomy that affects the patient's phonation and the ability to prevent life-threatening aspiration events. The aim of this single-center, retrospective study was to investigate the clinical course of left RLN palsy and to identify the main prognostic factors for recovery. METHODS: The study cohort consisted of 85 patients who had developed left RLN palsy after minimally invasive McKeown esophagectomy. Vocal cord function was assessed in all participants through laryngoscopic examinations, both in the immediate postoperative period and during follow-up. Permanent palsy was defined as no evidence of recovery after 6 months. Univariate and multivariable logistic regression analyses were applied to evaluate the associations between different variables and the outcome of palsy. RESULTS: Twenty-two (25.8%) patients successfully recovered from left RLN palsy. On multivariable logistic regression analysis, active smoking (odds ratio [OR] 0.335, p = 0.038) and the use of thoracoscopic surgery (vs. robotic surgery; OR 0.264, p = 0.028) were identified as independent unfavorable predictors for recovery from palsy. The estimated rates of recovery derived from a logistic regression model for patients harboring two, one, or no risk factors were 13.16%, 31.15-34.75%, and 61.39%, respectively. CONCLUSION: Only one-quarter of patients who had developed left RLN palsy after minimally invasive McKeown esophagectomy were able to fully recover. Smoking habits and the surgical approach were identified as key determinants of recovery. Patients harboring adverse prognostic factors are potential candidates for early intervention strategies.

Improving the Photostability of Semiconducting Polymer Dots Using Buffers.

The photostability of fluorescent probes is critical in biological imaging, especially for long-term observational analyses. Here, we describe a simple and universal method to improve the photostability of semiconducting polymer dots (Pdots) and other fluorescent probes by using buffers. Using Pdots as a model system, we found that HEPES or MES buffer can improve the photostability of Pdots by a factor of 20. Through a systematic study, we show that Pdot photobleaching is dominated by photoinduced radicals which can be quenched by the piperazine or morpholine structures of these buffers, which act as radical scavengers. For conditions where choice of buffer is limited, we designed fluorescent polymers conjugated with radical scavengers to improve Pdot photostability. We then demonstrate a practical application in which HEPES buffer is used to improve the photostability of Pdots during cell imaging.

Single-Molecule Flow Platform for the Quantification of Biomolecules Attached to Single Nanoparticles.

We describe here a flow platform for quantifying the number of biomolecules on individual fluorescent nanoparticles. The platform combines line-confocal fluorescence detection with near nanoscale channels (1-2 µm in width and height) to achieve high single-molecule detection sensitivity and throughput. The number of biomolecules present on each nanoparticle was determined by deconvolving the fluorescence intensity distribution of single-nanoparticle-biomolecule complexes with the intensity distribution of single biomolecules. We demonstrate this approach by quantifying the number of streptavidins on individual semiconducting polymer dots (Pdots); streptavidin was rendered fluorescent using biotin-Alexa647. This flow platform has high-throughput (hundreds to thousands of nanoparticles detected per second) and requires minute amounts of sample (∼5 µL at a dilute concentration of 10 pM). This measurement method is an additional tool for characterizing synthetic or biological nanoparticles.

Photostable Ratiometric Pdot Probe for in Vitro and in Vivo Imaging of Hypochlorous Acid.

Developing probes for the detection of reactive oxygen species (ROS), a hallmark of many pathophysiological process, is imperative to both understanding the precise roles of ROS in many life-threatening diseases and optimizing therapeutic interventions. We herein report an all-in-one fluorescent semiconducting polymer based far-red to near-infrared (NIR) Pdot nanoprobe for the ratiometric detection of hypochlorous acid (HOCl). The fabrication takes the advantage of flexible polymer design by incorporating target-sensitive and target-inert fluorophores into a single conjugated polymer to avoid leakage or differential photobleaching problems existed in other nanoprobes. The obtained nanoprobe has improved performance in HOCl sensing, such as high brightness, ideal far-red to NIR optical window, excellent photostability, self-referenced ratiometric response, fast response, and high selectivity. The dual-emission property allows the sensitive imaging of HOCl fluctuations produced in living macrophage cells and peritonitis of living mice with high contrast. This study not only provides a powerful and promising nanoprobe to be potentially used in the investigations of in situ HOCl status of diseases in living systems but also puts forward the design strategy of a new category of ratiometric fluorescent probes facilitating precise and reliable measurement in biological systems.

Optically Encoded Semiconducting Polymer Dots with Single-Wavelength Excitation for Barcoding and Tracking of Single Cells.

Multiplexed optical encoding is emerging as a powerful technique for high-throughput cellular analysis and molecular assays. Most of the developed optical barcodes, however, either suffer from large particle size or are incompatible with most commercial optical instruments. Here, a new type of nanoscale fluorescent barcode (Pdot barcodes) was prepared from semiconducting polymers. The Pdot barcodes possess the merits of small size (∼20 nm in diameter), narrow emission bands (full-width-at-half-maximum (fwhm) of 30-40 nm), three-color emissions (blue, green, and red) under single-wavelength excitation, a high brightness, good pH and thermal stability, and efficient cellular uptake. The Pdot barcodes were prepared using a three-color and six-intensity encoding strategy; for ratiometric readout of the barcodes, one of the colors might be used as an internal reference. We used the Pdot barcodes to label 20 sets of cancer cells and then distinguished and identified each set based on the Pdot barcodes using flow cytometry. We also monitored and tracked single cells labeled with different Pdot barcodes, even through rounds of cell division. These results suggest Pdot barcodes are strong candidates for discriminating different labeled cell and for long-term cell tracking.

Lanthanide-Coordinated Semiconducting Polymer Dots Used for Flow Cytometry and Mass Cytometry.

Simultaneous monitoring of biomarkers as well as single-cell analyses based on flow cytometry and mass cytometry are important for investigations of disease mechanisms, drug discovery, and signaling-network studies. Flow cytometry and mass cytometry are complementary to each other; however, probes that can satisfy all the requirements for these two advanced technologies are limited. In this study, we report a probe of lanthanide-coordinated semiconducting polymer dots (Pdots), which possess fluorescence and mass signals. We demonstrated the usage of this dual-functionality probe for both flow cytometry and mass cytometry in a mimetic cell mixture and human peripheral blood mononuclear cells as model systems. The probes not only offer high fluorescence signal for use in flow cytometry, but also show better performance in mass cytometry than the commercially available counterparts.

Metabolomic characterization of rhubarb species by capillary electrophoresis and ultra-high-pressure liquid chromatography.

This study developed CE and ultra-high-pressure LC (UHPLC) methods coupled with UV detectors to characterize the metabolomic profiles of different rhubarb species. The optimal CE conditions used a BGE with 15 mM sodium tetraborate, 15 mM sodium dihydrogen phosphate monohydrate, 30 mM sodium deoxycholate, and 30% ACN v/v at pH 8.3. The optimal UHPLC conditions used a mobile phase composed of 0.05% phosphate buffer and ACN with gradient elution. The gradient profile increased linearly from 10 to 21% ACN within the first 25 min, then increased to 33% ACN for the next 10 min. It took another 5 min to reach the 65% ACN, then for the next 5 min, it stayed unchanged. Sixteen samples of Rheum officinale and Rheum tanguticum collected from various locations were analyzed by CE and UHPLC methods. The metabolite profiles of CE were aligned and baseline corrected before chemometric analysis. Metabolomic signatures of rhubarb species from CE and UHPLC were clustered using principle component analysis and distance-based redundancy analysis; the clusters were not only able to discriminate different species but also different cultivation regions. Similarity measurements were performed by calculating the correlation coefficient of each sample with the authentic samples. Hybrid rhizome was clearly identified through similarity measurement of UHPLC metabolite profile and later confirmed by gene sequencing. The present study demonstrated that CE and UHPLC are efficient and effective tools to identify and authenticate herbs even coupled with simple detectors.

Human Posture Transition-Time Detection Based upon Inertial Measurement Unit and Long Short-Term Memory Neural Networks.

As human-robot interaction becomes more prevalent in industrial and clinical settings, detecting changes in human posture has become increasingly crucial. While recognizing human actions has been extensively studied, the transition between different postures or movements has been largely overlooked. This study explores using two deep-learning methods, the linear Feedforward Neural Network (FNN) and Long Short-Term Memory (LSTM), to detect changes in human posture among three different movements: standing, walking, and sitting. To explore the possibility of rapid posture-change detection upon human intention, the authors introduced transition stages as distinct features for the identification. During the experiment, the subject wore an inertial measurement unit (IMU) on their right leg to measure joint parameters. The measurement data were used to train the two machine learning networks, and their performances were tested. This study also examined the effect of the sampling rates on the LSTM network. The results indicate that both methods achieved high detection accuracies. Still, the LSTM model outperformed the FNN in terms of speed and accuracy, achieving 91% and 95% accuracy for data sampled at 25 Hz and 100 Hz, respectively. Additionally, the network trained for one test subject was able to detect posture changes in other subjects, demonstrating the feasibility of personalized or generalized deep learning models for detecting human intentions. The accuracies for posture transition time and identification at a sampling rate of 100 Hz were 0.17 s and 94.44%, respectively. In summary, this study achieved some good outcomes and laid a crucial foundation for the engineering application of digital twins, exoskeletons, and human intention control.

Cholesterol twists the transmembrane Di-Gly region of amyloid-precursor protein.

Nearly 95% of Alzheimer's disease (AD) occurs sporadically without genetic linkage. Aging, hypertension, high cholesterol content, and diabetes are known nongenomic risk factors of AD. Aggregation of Aß peptides is an initial event of AD pathogenesis. Aß peptides are catabolic products of a type I membrane protein called amyloid precursor protein (APP). Aß40 is the major product, whereas the 2-residue-longer version, Aß42, induces amyloid plaque formation in the AD brain. Since cholesterol content is one risk factor for sporadic AD, we aimed to explore whether cholesterol in the membrane affects the structure of the APP transmembrane region, thereby modulating the γ-secretase cutting behavior. Here, we synthesized several peptides containing the APP transmembrane region (sequence 693-726, corresponding to the Aß22-55 sequence) with one or two Cys mutations for spin labeling. We performed three electron spin resonance experiments to examine the structural changes of the peptides in liposomes composed of dioleoyl phosphatidylcholine and different cholesterol content. Our results show that cholesterol increases membrane thickness by 10% and peptide length accordingly. We identified that the di-glycine region of Aß36-40 (sequence VGGVV) exhibits the most profound change in response to cholesterol compared with other segments, explaining how the presence of cholesterol affects the γ-secretase cutting site. This study provides spectroscopic evidence showing how cholesterol modulates the structure of the APP transmembrane region in a lipid bilayer.

Controllable-Swelling Microneedle-Assisted Ultrasensitive Paper Sensing Platforms for Personal Health Monitoring.

Microneedle (MN) patches, which allow the extraction of skin interstitial fluid (ISF) without a pain sensation, are powerful tools for minimally invasive biofluid sampling. Herein, an MN-assisted paper-based sensing platform that enables rapid and painless biofluid analysis with ultrasensitive molecular recognition capacity is developed. First, a controllable-swelling MN patch is constructed through the engineering of a poly(ethylene glycol) diacrylate/methacrylated hyaluronic acid hydrogel; it combines rapid, sufficient extraction of ISF with excellent structural integrity. Notably, the analyte molecules in the needles can be recovered into a moist cellulose paper through spontaneous diffusion. More importantly, the paper can be functionalized with enzymatic colorimetric reagents or a plasmonic array, enabling a desired detection capacity-for example, the use of paper-based surface-enhanced Raman spectroscopy sensors leads to label-free, trace detection (sub-ppb level) of a diverse set of molecules (cefazolin, nicotine, paraquat, methylene blue). Finally, nicotine is selected as a model drug to evaluate the painless monitoring of three human volunteers. The changes in the nicotine levels can be tracked, with the levels varying significantly in response to the metabolism of drug in different volunteers. This as-designed minimally invasive sensing system should open up new opportunities for precision medicine, especially for personal healthcare monitoring.

Folic acid inhibits endothelial cell proliferation through activating the cSrc/ERK 2/NF-κB/p53 pathway mediated by folic acid receptor.

Folate is important for normal cell division. Folate deficiency has been implicated in various diseases, including atherosclerosis, neural tube defects, and cancer. However, the effect of folate on angiogenesis was unclear. The aim of this study was to investigate the anti-angiogenic action of folic acid (FA). FA (0-10 µmol/L) concentration-dependently decreased DNA synthesis and proliferation in cultured human umbilical venous endothelial cells (HUVEC). Western blot analyses demonstrated that the levels of p21, p27 and p53 protein in HUVEC were increased by FA. The FA-inhibited [3H]thymidine incorporation was completely blocked when the expressions of p21 and p27 were knocked-down together. Knock-down of p53 prevented the FA-induced increases in p21 and p27 protein level. The levels of phosphorylated Src (p-Src) and p-Src-FA receptor (FR) complex in HUVEC were increased by FA. Knock-down of FR reduced the FA-induced increases of p-Src and p53. The FA-induced increases of p21, p27 and p53 protein levels were abolished when cSrc was knocked-down. FA also increased NF-κB nuclear translocation and binding onto the p53 promoter. The FA-induced up-regulation of the p53 promoter activity was prevented by knocked-down of ERK. Matrigel angiogenesis assay in mice demonstrate the anti-angiogenic effect of FA in vivo. In conclusion, our data indicate that FA bound to FR in HUVEC, subsequently activated the cSrc/ERK 2/NF-κB/p53 signaling pathway, which in turn up-regulated the expression of p21 and p27, and finally resulted in cell cycle arrest at the G0/G1 phase. In the present study, we uncover a completely novel role of FA for anti-angiogenesis.

Visual semiquantification via the formation of phase segregation.

Colorimetry, one of the central themes in contemporary chemistry, generally relies on spectrometers to quantify specimens of interest. Developed herein is a rapid screening scheme to determine whether the amount of an analyte falls into a diagnostic concentration range by the naked eye. This is particularly important for applications under circumstances where instruments are not readily available. The aforementioned goal is demonstrated by utilizing copper-thiol chemistry as a model system in which polymerization occurs within a certain range of copper-to-cysteine mole ratios; hence, the targeted range of copper concentration is tunable by adjusting the amount of cysteine. Within and outside the range, the solutions appear cloudy and homogeneous, respectively. The reaction mechanism is proposed and scrutinized. The detection scheme is applied successfully on samples of human serum (15.7-23.6 µM) and pond water (<3.0 ppm or 47 µM) with a handy laser pointer.

On the size evolution of gold-monolayer-protected clusters by ligand place-exchange reactions: the effect of headgroup-gold interactions.

To functionalize Au MPCs (monolayer-protected gold clusters), ligand place-exchange reactions provide a convenient route in which the initial capping ligands are displaced by mixing the MPCs with an excess amount of incoming ones. However, literature reports show that the diameters of the modified products do not always stay unchanged. Because of the diversity of experimental conditions carried out in the documented studies, there is still a lack of comprehensive understanding concerning the size evolution. Herein, carefully controlled and examined parameters include the initial size of MPCs [Au(101)(PPh(3))(21)Cl(5)], the reaction time, the concentration of incoming ligands, a homologous series of incoming ligands, and the anchoring headgroups. The results show that the final particle size is determined by the strength of headgroup-gold adsorption which is correlated with the curvature of the final product in a thermodynamic model derived from Gibbs-Thomson equation.

Author Correction: Immunofluorescence can assess the efficacy of mTOR pathway therapeutic agent Everolimus in breast cancer models.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunofluorescence can assess the efficacy of mTOR pathway therapeutic agent Everolimus in breast cancer models.

When breast cancer patients start to exhibit resistance to hormonal therapy or chemotherapy, the mTOR inhibitor everolimus can be considered as an alternative therapeutic agent. Everolimus can deregulate the PI3K/AKT/mTOR pathway and affect a range of cellular functions. In some patients, the agent does not exhibit the desired efficacy and, even worse, not without the associated side effects. This study assessed the use of immunofluorescence (IF) as a modality to fill this unmet need of predicting the efficacy of everolimus prior to administration. Cell viability and MTT assays based on IF intensities of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 on breast cancer cells (Hs578T, MCF7, BT474, MDA-MB-231) and patient-derived cell culture from metastatic sites (ABC-82T and ABC-16TX1) were interrogated. Results show that independent pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 IF expressions can classify data into different groups: everolimus sensitive and resistant. The combined IF baseline intensity of these proteins is predictive of the efficacy of everolimus, and their intensities change dynamically when cells are resistant to everolimus. Furthermore, mTOR resistance is not only consequence of the AKT/mTOR pathway but also through the LKB1 or MAPK/ERK pathway. The LKB1 and pho-GSK3ß may also be potential predictive markers for everolimus.

Progesterone receptor activation is required for folic acid-induced anti-proliferation in colorectal cancer cell lines.

Previously, we demonstrated that folic acid (FA) could inhibit proliferation of colorectal cancer cell lines through activating the folate receptor (FR)α/cSrc/ERK1/2/NFκB/p53 pathway and anti-COLO-205 tumor growth in vivo. Since we recently also demonstrated that female sex hormones could affect the FA's action in regulating endothelial cell proliferation and migration, the aim of this study was to investigate the effect of progesterone (P4) on the FA-induced anti-proliferation in colorectal cancer cells. Treatment with FA significantly reduced the proliferation of the P4 receptor (PR)-positive colon cancer cell lines, COLO-205, HT-29 and LoVo, but did not significantly affect the proliferation of the PR-negative colon cancer cell lines, HCT116 and DLD-1. Pre-treatment with Org 31710, a PR specific antagonist, abolished the FA-induced proliferation inhibition and activation in the signaling pathway involved in regulating proliferation inhibition in these PR positive colorectal cancer cell lines. The involvement of PR in the FA-induced activation of cSrc and up-regulations in cell cycle inhibitory proteins (p21, p27 and p53) was confirmed by knock-down of PR expression using the siRNA technique. Importantly, we show direct protein interaction between FR and PR in COLO-205. Moreover, treatment with FA induced PR activation in COLO-205. Taken together, these data suggest that FA induced proliferation inhibition in colon cancer cells through activation of PR. This finding might explain some of the controversies of FA's effects on cancer growth and provide valuable reference for clinical applications of FA in treating colorectal cancer.

Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots.

The efficient selection and isolation of individual cells of interest from a mixed population is desired in many biomedical and clinical applications. Here we show the concept of using photoswitchable semiconducting polymer dots (Pdots) as an optical 'painting' tool, which enables the selection of certain adherent cells based on their fluorescence, and their spatial and morphological features, under a microscope. We first develop a Pdot that can switch between the bright (ON) and dark (OFF) states reversibly with a 150-fold contrast ratio on irradiation with ultraviolet or red light. With a focused 633-nm laser beam that acts as a 'paintbrush' and the photoswitchable Pdots as the 'paint', we select and 'paint' individual Pdot-labelled adherent cells by turning on their fluorescence, then proceed to sort and recover the optically marked cells (with 90% recovery and near 100% purity), followed by genetic analysis.

Fluorometric determination of ammonium ion by ion chromatography using postcolumn derivatization with o-phthaldialdehyde.

A postcolumn fluorometric derivatization method for the determination of trace amounts of ammonium ion (microg/L level) under matrices with high concentrations of sodium and amino acids has been developed. In this method, ammonium ion was determined by ion chromatography combined with fluorometric detection (IC-FL) in less than 16 min. IC was performed in a high-capacity cation-exchange Dionex IonPac CS16 analytical column (250 mm x 5 mm) under isocratic conditions with 30 mM methanesulfonic acid (MSA) as mobile phase at flow-rate 1.0 mL/min. To remove amino acid interference, the postcolumn derivatization based on the reaction of ammonia with o-phthaldialdehyde (OPA) and sulfite was applied. The excitation and emission wavelengths were 364 and 425 nm, respectively. The effects of pH, reaction temperature and time, OPA-reagent composition and concentration, and sample matrix were studied. The linear range and detection limit of this method were similar to the standard method. The IC-FL method with a postcolumn fluorometric derivatization allows the routine determination of ammonium ion in extreme matrices where the ratios of sodium and amino acids to ammonium are up to 2,800,000:1 and 28,000:1, respectively.