Upconversion nanoparticle-based fluorescence resonance energy transfer sensing of programmed death ligand 1 using sandwich epitope-imprinted polymers.

Programmed death ligand 1 (PD-L1) has been shown to suppress the anti-tumor immune response of some lung cancer patients, and thus PD-L1 expression may be a valuable predictor of the efficacy of anti-PD-1/PD-L1 monoclonal therapy in such patients. In this work, a sandwich approach to fluorescence resonance energy transfer (FRET) was used with green-emitting Yb3+/Ho3+-doped upconversion nanoparticles (UCNPs) and a rhodamine-conjugated conductive polymer as donor and acceptor, respectively. Yb3+/Ho3+-doped UCNPs were synthesized and then coated with poly(ethylene-co-vinyl alcohol), pEVAL, imprinted with PD-L1 peptide. Epitope-imprinted composite nanoparticles were characterized by dynamic light scattering, superconducting quantum interference magnetometry, and atomic force microscopy. Poly(triphenylamine rhodamine-3-acetic acid-co-3,4-ethoxylenedioxythiophene)s copolymers (p(TPAR-co-EDOT)) were imprinted with various epitopes of PD-L1 by in situ electrochemical polymerization. The epitope-imprinted polymer-coated electrodes were then characterized by scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. Finally, the sandwich sensing of various PD-L1 concentrations with peptide-imprinted p(TPAR-co-EDOT)-coated substrate and UCNP-containing magnetic peptide-imprinted pEVAL nanoparticles by FRET was conducted to measure the concentration of PD-L1 in A549 lung cancer cell lysate.

Peptide Selection of MMP-1 for Electrochemical Sensing with Epitope-Imprinted Poly(TPARA-co-EDOT)s.

Instead of molecularly imprinting a whole protein molecule, imprinting protein epitopes is gaining popularity due to cost and solubility issues. Belonging to the matrix metalloproteinase protein family, MMP-1 is an interstitial collagenase that degrades collagen and may be involved in cell migration, cell proliferation, the pro-inflammatory effect, and cancer progression. Hence, it can serve as a disease protein biomarker and thus be useful in early diagnosis. Herein, epitopes of MMP-1 were identified by screening its crystal structure. To identify possible epitopes for imprinting, MMP-1 was cleaved in silico with trypsin, pepsin at pH = 1.3, and pepsin at pH > 2.0 using Peptide Cutter, generating peptide fragments containing 8 to 12 amino acids. Five criteria were applied to select the peptides most suitable as potential epitopes for MMP-1. The triphenylamine rhodanine-3-acetic acid (TPARA) functional monomer was synthesized to form a stable pre-polymerization complex with a selected template epitope. The complexed functional monomer was then copolymerized with 3,4-ethoxylenedioxythiophene (EDOT) using potentiodynamic electropolymerization onto indium−tin−oxide (ITO) electrodes. The composition of the molecularly imprinted poly(TPARA-co-EDOT) (MIP) was optimized by maximizing the film's electrical conductivity. Cyclic voltammetry was used to determine MMP-1 concentration in the presence of the Fe(CN)63−/Fe(CN)64− redox probe actuating the "gate effect." A calibration curve was constructed and used to determine the usable concentration range and the limit of detection as ca. 0.001 to 10.0 pg/mL and 0.2 fg/mL MMP-1, respectively. Finally, the MMP-1 concentration in the A549 human lung (carcinoma) culture medium was measured, and this determination accuracy was confirmed using an ELISA assay.

Embedded Upconversion Nanoparticles in Magnetic Molecularly Imprinted Polymers for Photodynamic Therapy of Hepatocellular Carcinoma.

In this work, high-temperature pyrolysis was used to prepare both the core and shell of lantha-nide-doped UCNPs with lithium yttrium tetrafluoride (LiYF4) to enhance the green luminescence. Merocyanine 540 (MC540)-grafted magnetic nanoparticles were incorporated in the PD-L1 pep-tide-imprinted poly(ethylene-co-vinyl alcohol) particles, which were formed by precipitation in a non-solvent. UCNPs in the non-solvent bath were thus entrapped in the imprinted particles to generate composite nanoparticles for the targeting and photodynamic therapy of PD-L1 in tumor cells. Finally, the in vitro cytotoxicity of the nanoparticles in HepG2 human liver cancer cells was evaluated with the continuous administration of MC540/MNPs@MIPs/UCNPs under irradiation by an NIR laser. To understand the delivery of the UCNP-embedded molecularly imprinted pol-ymers, the intrinsic and extrinsic pathways were also investigated.

Cellular reprogramming with multigene activation by the delivery of CRISPR/dCas9 ribonucleoproteins via magnetic peptide-imprinted chitosan nanoparticles.

Induced pluripotent stem cells are usually derived by reprogramming transcription factors (OSKM), such as octamer-binding transcription factor 4 (OCT4), (sex determining region Y)-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and cellular proto-oncogene (c-Myc). However, the genomic integration of transcription factors risks the insertion of mutations into the genome of the target cells. Recently, the clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) system has been used to edit genomes. In this work, dCas9-VPR (dCas9 with a gene activator, VP64-p65-Rta (VPR), fused to its c-terminus) and guide RNA (gRNA) combined to form ribonucleoproteins, which were immobilized on magnetic peptide-imprinted chitosan nanoparticles. These were then used to activate OSKM genes in human embryonic kidney (HEK) 293T cells. Four pairs of gRNAs were used for the binding site recognition to activate the OSKM genes. Transfected HEK293T cells were then prescreened for the high expression of OSKM proteins by immunohistochemistry images. The optimal gRNAs for OSKM expression were identified using quantitative real-time polymerase chain reaction and the staining of OSKM proteins. Finally, we found that the activated expression of one of the OSKM genes is up to three-fold higher than that of the other genes, enabling precise control of the cell differentiation.

Epitope recognition of magnetic peptide-imprinted chitosan composite nanoparticles for the extraction of CRISPR/dCas9a proteins from transfected cells.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) technology is a powerful method for genetic modification (and regulation) that is of great current interest. The development of new, economical methods of detecting and extracting Cas9 (and/or dCas9) from transfected cells is thus an important advance. In this work, we employed molecular imprinting, using two peptides from the Cas9 protein, to make magnetic peptide-imprinted chitosan nanoparticles. dCas9 was encoded in a plasmid which was then transfected into human embryonic kidney (HEK293T) cells. The expression of dCas9 protein was measured by using total protein kits. Finally, the imprinted nanoparticles were used to extract dCas9 from transfected cell homogenates.

Paradoxical Relationship Between Glycated Hemoglobin and Longitudinal Change in Physical Functioning in Older Adults: A Prospective Cohort Study.

BACKGROUND: Hyperglycemia with high hemoglobin A1c (HbA1c) levels is associated with significant health risks. However, the relationship between HbA1c levels and the physical functioning status in later life remains uncertain and so is the possible underlying mechanism. METHODS: We conducted a prospective study of 2,565 initially well-functioning community-dwelling older adult aged 55 years and older from the Healthy Aging Longitudinal Study in Taiwan. Each participant received baseline measurements of blood HbA1c and inflammatory markers levels and repeated assessments of physical functioning over a mean follow-up period of 5.3 years. We used generalized linear mixed-effects regression to estimate the adjusted changes in the odds ratio for self-reported physical functioning impairment and Short Physical Performance Battery (SPPB) score according to baseline HbA1c levels (categorized into 0.5% increments from <5.5% to ≥7.0%). RESULTS: HbA1c levels showed a U-shaped relationship with changes in the odds ratio for physical functioning impairment and SPPB score (p for quadratic term < .001). Compared with participants with an HbA1c of 5.5% to <6.0%, those with an HbA1c of <5.5% or ≥7.0% had a higher annual increase in the odds ratio for physical functioning impairment (odds ratio [95% confidence interval] per year, 1.25 [1.04-1.50] and 1.21 [1.04-1.41]) and a higher annualized decrease in SPPB score (coefficient [95% confidence interval], -0.05 [-0.10 to 0.00] and -0.04 [-0.08 to 0.00]). These relationships were nonlinear only in participants with high soluble interleukin-6 receptor levels (>48,124 pg/mL; p for interaction < .05). CONCLUSIONS: High and low HbA1c levels at baseline are associated with faster physical functioning decline, particularly among individuals with elevated circulating soluble interleukin-6 receptor, a sign of enhanced interleukin-6 trans-signaling.

Gold-decorated magnetic nanoparticles modified with hairpin-shaped DNA for fluorometric discrimination of single-base mismatch DNA.

The authors describe the use of gold-decorated magnetic nanoparticles (Au/MNPs) in discriminating DNA sequences with a single-base (guanine) mismatch. The Au/MNPs were characterized through dynamic light scattering, X-ray diffraction, superconducting quantum interference device, and UV/visible spectroscopy. They were then conjugated to a probe oligomer consisting of a hairpin-shaped DNA sequence carrying two signalling fluorophores: fluorescein at its 3' end and pyrene in the loop region. When interacting with the target DNA sequences, the hybridized probe-target duplex renders the pyrene signal (at excitation/emission wavelengths of 345/375 nm) either quenched or unquenched. Quenching (or nonquenching) of the pyrene fluorescence depends on the presence of a guanine (or a nonguanine) nucleotide at the designated polymorphic site. The linear range of hybridization in these Au/MNPs is from 0.1 nM to 1.0 µM of ssDNA. Conceivably, this system may serve as a single-nucleotide polymorphism probe. Graphical Abstract Schematic presentation of probe-conjugated Au/MNP preparation (upper panel) and working principle to discriminate DNA with or without single-base (guanine) mismatch sequences at the polymorphic sites (lower panel). Py denotes pyrene-hooked pyrrolocytidine; F denotes fluorescein.

Clinical characteristics and treatment outcomes of patients with tubo-ovarian abscess at a tertiary care hospital in Northern Taiwan.

BACKGROUND/PURPOSE: Controversy exists regarding the need for surgical intervention in patients with tubo-ovarian abscess (TOA). This study was aimed at investigating the clinical characteristics and treatment outcomes in patients with TOA at a tertiary care hospital in Taiwan. METHODS: The medical records of 83 patients who presented at the hospital with TOA between January 1, 2006, and December 31, 2007, were retrospectively reviewed. Outcomes of patients who received medical treatment alone or underwent surgical intervention were analyzed using univariate and logistic regression analyses. RESULTS: Among the 83 patients with TOA, 13 patients (15.7%) underwent surgical intervention, and 70 patients (84.3%) received medical treatment alone. Significant variables related to surgical treatment in the univariate analysis were length of stay (short vs. long; t = -2.267, p = 0.026), department of admission (emergency room vs. outpatient department; χ(2) = 7.459, p = 0.006), number of live births (nulliparous vs. multiparous; χ(2) = 18.202, p = 0.001), and C-reactive protein (CRP) level (high vs. low; t = -2.250, p = 0.028). Logistic regression analysis performed to determine influential factors for surgical treatment showed that the operation odds ratio of three to four live births versus no live births was 33.995 (p = 0.043) and that of two live births versus no live births was 13.598 (p = 0.026). CONCLUSION: Patients with TOA who underwent surgery had a longer duration of hospitalization. Among the patients who underwent surgical intervention, those admitted to the emergency room had higher CRP levels and were more likely to be multiparous.

Mortality predictors of Pneumocystis jirovecii pneumonia in human immunodeficiency virus-infected patients at presentation: Experience in a tertiary care hospital of northern Taiwan.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) remains the leading cause of opportunistic infections and deaths among human immunodeficiency virus (HIV)-infected patients. We would like to identify the predictors of mortality of these patients at initial presentation, and assist clinicians to aware the patients in risk of mortality earlier. METHODS: From 1997 to 2009, adults with HIV infection and a discharge diagnosis of PJP at Mackay Memorial Hospital were included in this retrospective study. Patients' demographic data and laboratory data were analyzed by reviewing the medical records. RESULTS: Eighty-five patients were included in this study. The overall mortality rate was 37.7%. Univariate analysis revealed several host factors significantly related to mortality, including age, systolic blood pressure, diastolic blood pressure, partial pressure of oxygen in arterial blood (PaO(2)), percentage of lymphocyte, percentage of CD4 lymphocyte, CD4 counts, serum total protein, serum albumin, and blood urea nitrogen. Multivariate analysis identified three independent predictors associated with mortality, i.e. systolic blood pressure ≤110 mmHg [adjusted odds ratio (AOR) 3.88; 95% confidence interval (CI) 1.17-12.83; p = 0.03], PaO(2) at room air ≤60 mmHg (AOR 4.97; 95% CI 1.34-18.23; p = 0.01), and lymphocytes ≤10% (AOR 8.19; 95% CI 1.48-45.36; p = 0.02). With these predictors, we can stratify patients into three groups with increasing risks for mortality, ≤one predictor (mortality rate 14%), any two predictors (47%), and three predictors (75%). CONCLUSIONS: HIV-infected patients with PJP can be clinically stratified by three prognostic variables identified by multivariate analysis. Early recognition of patients in higher risk can assist clinicians to prevent rapid deterioration and seek for better outcomes.

Aerosolized colistin for the treatment of multidrug-resistant Acinetobacter baumannii pneumonia: experience in a tertiary care hospital in northern Taiwan.

BACKGROUND/PURPOSE: Ventilator-associated pneumonia (VAP) due to multidrug-resistant (MDR) Acinetobacter baumannii in critically ill patients presents an emerging challenge to clinicians. Administration of aerosolized colistin as an adjunctive therapy is one therapeutic option mentioned in limited evidence-based studies. This study aimed to evaluate the effectiveness of adjunctive aerosolized colistin treatment for VAP due to MDR pathogens. METHODS: We retrospectively reviewed the medical records of patients who had received aerosolized colistin for treatment of VAP due to MDR A. baumannii in our hospital from August to December 2008. RESULTS: Forty-five patients were enrolled in our study. The mean age was 71 +/- 15 years. The mean Acute Physiological and Chronic Health Evaluation II (APACHE II) scores on the day of intensive care unit admission and on the first day of aerosolized colistin administration were 22.5 +/- 6.7 and 18.9 +/- 5.7, respectively. The mean duration of intensive care unit stay was 34 +/- 16 days. The mean daily dosage of aerosolized colistin was 4.29 +/- 0.82 million IU, and the mean duration of administration was 10.29 days. Seventeen patients (37.8%) had a favorable microbiological outcome and 26 (57.8%) showed a clinical response. Mortality due to all causes was 42.2%. No adverse effects related to inhaled colistin were recorded. CONCLUSION: Aerosolized colistin may be considered as an adjunct to intravenous treatments in patients with VAP due to colistin-susceptible MDR A. baumannii in critically ill patients.

Synchronous double primary lung cancers via p53 pathway induced by heavy smoking.

Differences in the histological manifestation of synchronous lung cancers are rare. Synchronous multiple primary lung cancers (SMPLC) are associated with long-term tobacco use, which could independently lead to mutations in the p53 and K-ras genes. We present the case of an 82-year-old man who smoked 30 cigarettes daily for the past 60 years. CT of the chest showed a right upper lobe mass. Bronchoscopy revealed an intra-lumen nodular lesion in the right lower lobe bronchus. The diagnoses of small cell lung carcinoma (SCLC) of the right upper lobe and non-small cell lung carcinoma (NSCLC) of the right lower lobe were confirmed by the morphologic features and the detected immunoreactivity. Immunohistochemical analyses showed a strong positive reaction for p53 in samples of the SCLC and NSCLC. The cancers had a different phenotype, but similar genetic abnormalities may have developed due to the carcinogens in the cigarettes.