Parallel, Continuous Monitoring and Quantification of Programmed Cell Death in Plant Tissue.

Accurate quantification of hypersensitive response (HR) programmed cell death is imperative for understanding plant defense mechanisms and developing disease-resistant crop varieties. Here, a phenotyping platform for rapid, continuous-time, and quantitative assessment of HR is demonstrated: Parallel Automated Spectroscopy Tool for Electrolyte Leakage (PASTEL). Compared to traditional HR assays, PASTEL significantly improves temporal resolution and has high sensitivity, facilitating detection of microscopic levels of cell death. Validation is performed by transiently expressing the effector protein AVRblb2 in transgenic Nicotiana benthamiana (expressing the corresponding resistance protein Rpi-blb2) to reliably induce HR. Detection of cell death is achieved at microscopic intensities, where leaf tissue appears healthy to the naked eye one week after infiltration. PASTEL produces large amounts of frequency domain impedance data captured continuously. This data is used to develop supervised machine-learning (ML) models for classification of HR. Input data (inclusive of the entire tested concentration range) is classified as HR-positive or negative with 84.1% mean accuracy (F1 score = 0.75) at 1 h and with 87.8% mean accuracy (F1 score = 0.81) at 22 h. With PASTEL and the ML models produced in this work, it is possible to phenotype disease resistance in plants in hours instead of days to weeks.

Plasmonic Titanium Nitride Nanohole Arrays for Refractometric Sensing.

Group IVB metal nitrides have attracted great interest as alternative plasmonic materials. Among them, titanium nitride (TiN) stands out due to the ease of deposition and relative abundance of Ti compared to those of Zr and Hf metals. Even though they do not have Au or Ag-like plasmonic characteristics, they offer many advantages, from high mechanical stability to refractory behavior and complementary metal oxide semiconductor-compatible fabrication to tunable electrical/optical properties. In this study, we utilized reactive RF magnetron sputtering to deposit plasmonic TiN thin films. The flow rate and ratio of Ar/N2 and oxygen scavenging methods were optimized to improve the plasmonic performance of TiN thin films. The stoichiometry and structure of the TiN thin films were thoroughly investigated to assess the viability of the optimized operation procedures. To assess the plasmonic performance of TiN thin films, periodic nanohole arrays were perforated on TiN thin films by using electron beam lithography and reactive ion etching methods. The resulting TiN periodic nanohole array with varying periods was investigated by using a custom microspectroscopy setup for both reflection and transmission characteristics in various media to underline the efficacy of TiN for refractometric sensing.

Characterization of FcγRIa (CD64) as a Ligand Molecule for Site-Specific IgG1 Capture: A Side-By-Side Comparison with Protein A.

Fc γ receptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin-biotin interaction, and His-tagged FcγRIa capture. The His-tagged FcγRIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate FcγRIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the FcγRIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI revealed that the association rate (ka 50-80 × 105 M-1 s-1) increased in comparison to His capture method (1.9-2.4 × 105 M-1 s-1). In addition, the dissociation rate (kd 10-5 s-1) seemed slower over the His capture method (10-4 s-1) and provided stability on the chip surface during the dissociation phase. The KD values for FcγRIa were found in the picomolar range (2.1-10.33 pM from steady-state affinity analysis and 37.5-46.2 pM from kinetic analysis) for IgG1-type antibodies. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification.

SELEX against whole-cell bacteria resulted in lipopolysaccharide binding aptamers.

Nucleic acid aptamers are target-specific oligonucleotides selected from combinatorial libraries through an iterative in vitro screening process known as Systemic Evolution of Ligands by Exponential Enrichment (SELEX). In this report, the selection of bacteria differentiating ssDNA aptamer candidates from a combinatorial library through the whole-cell SELEX method was performed. The enriched SELEX pool was sequenced using Illumina Next-Generation Sequencing (NGS) technology and analyzed for the most abundant sequences using CLC Genomics Workbench. The sequencing data resulted in several oligonucleotide families from which three individual sequences were chosen per SELEX based on the copy numbers. The binding performance of the selected aptamers was assessed by flow cytometry and fluorescence spectroscopy, and the binding constants were estimated using binding saturation curves. Varying results were obtained from two independent SELEX procedures where the SELEX against the model gram-negative bacterium Escherichia coli provided more selective sequences while the SELEX library used against gram-positive bacterium Listeria monocytogenes did not evolve as expected. The sequences that emerged from E. coli SELEX were shown to bind Lipopolysaccharide residues (LPS) and inhibit LPS-induced macrophage polarization. Thus, it can be said that, performed whole-cell SELEX could be resulted as the selection of aptamers which can bind LPS and inhibit LPS induced inflammation response and thus can be candidates for the inhibition of bacterial infections. In future studies, the selected aptamer sequences could be structurally and chemically modified and exploited as potential diagnostic tools and therapeutic agents as LPS antagonists.

Endothelial Dysfunction and Ischemia-Modified Albumin Levels in Males with Diabetic and Nondiabetic Erectile Dysfunction.

In this study, we aimed to determine endothelial dysfunction and ischemia-modified albumin (IMA) levels in patients diagnosed with erectile dysfunction (ED) and to examine the relationship between these and diabetes disease. 86 male patients (46 patients with diabetes, age: 51.5 ± 9.2 and 40 patients with nondiabetes (control group), age: 54.78 ± 12.2) were included in the study. IMA, a new indicator of tissue ischemia and oxidative stress, was checked. Superoxide dismutase (SOD) activity, another oxidative stress indicator, was examined. Endothelin-1 (ET-1), one of the parameters of endothelial dysfunction, was measured. Additionally, endothelial function was evaluated with flow-mediated vasodilatation (FMD). Student's t-test was used for statistical evaluation. p values less than 0.05 were considered statistically significant. SOD activity was significantly lower in the diabetic group than in the control group, and ET-1 was significantly higher (p < 0.001). IMA was found to be significantly higher in the diabetic group than the control group (p < 0.001). FMD was significantly lower in diabetic group compared to the control group (p < 0.002). According to our findings, the co-occurrence of erectile dysfunction and diabetes demonstrates a complex condition that includes endothelial dysfunction, oxidative stress, and tissue ischemia. When the correlation of indicators, which are markers, was examined, the severity of the co-occurrence of diabetes and erectile dysfunction was again demonstrated.

Nanoplasmonic biosensors: Theory, structure, design, and review of recent applications.

Nanoplasmonic biosensing shows an immense potential to satisfy the needs of the global health industry - low-cost, fast, and portable automated systems; highly sensitive and real-time detection; multiplexing and miniaturization. In this review, we presented the theory of nanoplasmonic biosensing for popular detection schemes - SPR, LSPR, and EOT - and underline the consideration for nanostructure design, material selection, and their effects on refractometric sensing performance. Later, we covered the bottom-up and top-down nanofabrication methods for nanoplasmonic biosensors. Subsequently, we reviewed the recent examples of nanoplasmonic biosensors over a wide range of clinically relevant analytes in the diagnosis and prognosis of a wide range of diseases and conditions such as biomarker proteins, infectious bacteria, viral agents. Finally, we discussed the challenges of nanoplasmonic biosensing toward clinical translation and proposed strategic avenues to be competitive against current clinical detection methods. Hopefully, nanoplasmonic biosensing can realize its potential through successful demonstrations of clinical translation in the upcoming years.

The Association Between Delayed Graft Function and Renal Resistive Index in Kidney Transplant Recipients.

Background and objectives Delayed graft function (DGF) may increase the risk for kidney graft dysfunction. Renal resistive index (RRI) in Doppler ultrasonography is useful in measuring blood flow changes in kidneys which is indicative of tubulointerstitial damage. Most of the diseases in DGF etiology are related to tubulointerstitium and arteries of the kidneys. In this study, we investigated whether there is a relationship between delayed graft function and renal resistive index in kidney transplant recipients (KTR). Materials and methods Patients who underwent kidney transplantation were included in this retrospective study. KTR were divided into two groups as DGF (+) and DGF (-). Comparison of RRI values of DGF (+) and DGF (-) groups according to the measurements at different times. Results The findings showed that both RRI measurements (post-transplant in the first week and the end of the first year) of the DGF (+) group were higher than DGF (-) group (p=0.001 and p=0.003, respectively). The interaction of measurements and DGF did not have an effect on RRI (p>0.05). Conclusion The value of RRI in the DGF (-) group was lower than DGF (+) group in the first week after kidney transplantation.

The relationship between premature ejaculation and the timing of pre-adult circumcision.

We aimed to investigate the relationship between premature ejaculation and the age when men had been circumcised before adulthood. A total of 2,768 sexually active male patients aged between 18 and 65 years were included in this study. A multicentre study was conducted prospectively with the participation of 20 centres. A survey consisting of 12 questions prepared by the researchers, as well as the validated Turkish versions of the five-item Premature Ejaculation Diagnostic Tool, was administered to all participants. The study included 1,603 participants who met the inclusion criteria. There was no significant difference in the Premature Ejaculation Diagnostic Tool and self-reported ejaculation time between the participants who had been circumcised at different ages during childhood. Remembering circumcision experience with fear or anxiety did not increase the risk of sexual dysfunction compared to the participants who described their experience with happiness or with no particular emotion. There was no significant difference in Premature Ejaculation Diagnostic Tool scores or the self-reported ejaculation time of the participants circumcised at different ages. The age of childhood circumcision, having a fearful or anxious circumcision experience, does not affect the risk of premature ejaculation in adult life.

Evaluating the Relationship Between Burnout Levels and Compassion Fatigue, Emotional Intelligence, and Communication Skills of Organ Transplant Coordinators.

AIM: The aim was to determine the effects of compassion fatigue, emotional intelligence levels, and communication skills of organ transplant coordinators on burnout. METHODS: This descriptive correlational study included 104 organ transplant coordinators. Data were collected via a survey that consisted of demographic information, the Copenhagen Burnout Inventory, the Emotional Intelligence Scale, and the Compassion Fatigue Scale. FINDINGS: A positive relationship was reported between burnout (mean, 39.42 [SD, 19.54]) and compassion fatigue (mean, 2.26 [SD, 0.86]) measurements of the organ transplant coordinators (r = 0.432) and a negative and significant relationship existed between burnout and emotional intelligence (mean, 3.53 [SD, 0.58]) measurements (r = -0.372). However, no correlation was found between communication skills (mean, 4.27 [SD, 0.52]) and burnout (r = -0.184; P > .05) except a negative correlation with client-related burnout measurements (r = -0.222; P < .05). As a result of the 2-step regression analysis, the regression-squared value predicted the joint effect of compassion fatigue and emotional intelligence on burnout (R2 = .285). CONCLUSIONS: Organ transplant coordinators reported moderate levels of burnout and compassion fatigue and high levels of emotional intelligence and communication skills. Compassion fatigue caused burnout, while emotional intelligence decreased. Additionally, transplant coordinators' communication skills were related to client-related burnout.

A Rare Complication of Amplatz Sheath: Amplatz Sheath Rupture During Percutaneous Nephrolithotomy.

Introduction: Amplatz sheaths are hollow tubes that serve as the portal for the insertion of the nephroscope during percutaneous nephrolithotomy (PCNL). Breakage of this tube during the procedure is rare, but when it does occur it should be recognized and addressed promptly. Case Presentation: A 46-year-old Caucasian male patient was scheduled for PCNL. The Amplatz sheath was inserted in the usual manner over a balloon dilator and nephroscopy was performed. Profuse bleeding was encountered early. Upon meticulous endoscopic navigation, the broken Amplatz tube was recognized and replaced. This allowed us to identify and remove the fragment of the Amplatz tube, followed by stone fragmentation and removal. Conclusion: Our experience highlights the importance of recognizing this rare complication of a broken Amplatz sheath that should be managed promptly and effectively through endoscopic means without the need to abort the planned PCNL.

Aptamer and nanomaterial based FRET biosensors: a review on recent advances (2014-2019).

Fluorescence resonance energy transfer, one of the most powerful phenomena for elucidating molecular interactions, has been extensively utilized as a biosensing tool to provide accurate information at the nanoscale. Numerous aptamer- and nanomaterial-based FRET bioassays has been developed for detection of a large variety of molecules. Affinity probes are widely used in biosensors, in which aptamers have emerged as advantageous biorecognition elements, due to their chemical and structural stability. Similarly, optically active nanomaterials offer significant advantages over conventional organic dyes, such as superior photophysical properties, large surface-to-volume ratios, photostability, and longer shelf life. In this report (with 175 references), the use of aptamer-modified nanomaterials as FRET couples is reviewed: quantum dots, upconverting nanoparticles, graphene, reduced graphene oxide, gold nanoparticles, molybdenum disulfide, graphene quantum dots, carbon dots, and metal-organic frameworks. Tabulated summaries provide the reader with useful information on the current state of research in the field. Graphical abstract Schematic representation of a fluorescence resonance energy transfer-based aptamer nanoprobe in the absence and presence of a given target molecule (analyte). Structures are not drawn to their original scales.

Plasmonic Selection of ssDNA Aptamers against Fibroblast Growth Factor Receptor.

In this work, we describe the selection of ssDNA aptamers targeting fibroblast growth factor receptor binding protein 3 K650E, which has roles in cell division, growth, and differentiation through the kinase cascade. The selection process was based on the label-free, real-time monitoring of binding interactions by surface plasmon resonance, allowing for convenient manipulation of the selection rounds. Next generation sequencing data provided four major motif families from which nine individual sequences were selected based on their abundance levels. Electrophoretic mobility shift assays revealed binding of the selected aptamers to the target protein without significant interference from fibroblast growth factor receptor binding protein 2, indicating the selectivity of the aptamers. The dissociation constant at equilibrium for the best aptamer candidate, SU-3, was found to be (28.2 ± 19.6) × 10-9 M (n = 5) using a single-cycle kinetic analysis method. Advantages of the experimental setup and potential applications of the selected aptamers are discussed.

Conformation-mediated Förster resonance energy transfer (FRET) in blue-emitting polyvinylpyrrolidone (PVP)-passivated zinc oxide (ZnO) nanoparticles.

Homopolymers, such as polyvinylpyrrolidone (PVP), are commonly used to passivate the surface of blue-light emitting ZnO nanoparticles during colloid nucleation and growth. However, although PVP is known to auto-fluoresce at 400nm, which is near the absorption edge of ZnO, the impact of PVP adsorption characteristics on the surface of ZnO and the surface-related photophysics of PVP-capped ZnO nanoparticles is not well understood. To investigate, we have synthesized ZnO nanoparticles in solvents containing PVP of 3 concentrations-0.5, 0.7, and 0.11gmL-1. Using time-domain NMR, we show that the adsorbed polymer conformation differs with polymer concentration-head-to-tail under low concentration (e.g., 0.05gmL-1) and looping, then train-like, with increasing concentration (e.g., 0.07gmL-1 and 0.11gmL-1, respectively). When the surface-adsorbed PVP is entrained, the surface states of ZnO are passivated and radiative emission from surface trap states is suppressed, allowing emission to be dominated by exciton transitions in the UV (ca. 310nm). Moreover, the reduced proximity between the PVP molecule and the ZnO gives rise to increased efficiency of energy transfer between the exciton emission of ZnO and the HOMO-LUMO absorption of PVP (ca. 400nm). As a result, light emission in the blue is enhanced in the PVP-capped ZnO nanoparticles. We thus show that the emission properties of ZnO can be tuned by controlling the adsorbed PVP conformation on the ZnO surface via the PVP concentration in the ZnO precipitation medium.

Nitric oxide and asymmetric dimethyl arginine (ADMA) levels in an experimental hydronephrotic kidney caused by unilateral partial ureteral obstruction.

AIM: Our aim is to measure asymmetric dimethyl arginine and nitric oxide levels in rats with induced unilateral acute ureteral obstruction to research the effects on the kidney. MATERIAL AND METHODS: The study included 21 adolescent (average age 6 weeks) Sprague-Dawley male rats weighing between 240-290g divided at random into 3 groups. Group-1: Control group (n=6): underwent no procedures. Group-2: Sham group (n=6): underwent the same procedures as the experimental group without ureter and psoas muscle dissection. Group-3: Group with induced partial unilateral ureteral obstruction (n=9). All rats were sacrificed after 12 weeks. Superoxide dismutase enzyme activity and nitrite and nitrate salt levels were measured in renal tissue. Plasma nitrite-nitrate and ADMA levels were examined. RESULTS: In the experimental group histopathological changes observed included renal pelvis dilatation, flattened papillae, sclerotic glomerulus and fibrosis. In the experimental group tissue SOD and blood ADMA levels were higher than the control and sham groups (p<0.05) while tissue NO and plasma NO values were lower than in the sham and control groups (p<0.05). CONCLUSION: Oxidative stress and disruption of NO synthesis play an important role in renal function and histopathological changes after obstructive renal disease. To prevent renal complications developing after obstructive nephropathy we believe that a new strategy may be research on reducing ADMA.

Nitric oxide and asymmetric dimethyl arginine (ADMA) levels in an experimental hydronephrotic kidney caused by unilateral partial ureteral obstruction

ABSTRACT Aim Our aim is to measure asymmetric dimethyl arginine and nitric oxide levels in rats with induced unilateral acute ureteral obstruction to research the effects on the kidney. Material and Methods The study included 21 adolescent (average age 6 weeks) Sprague-Dawley male rats weighing between 240-290g divided at random into 3 groups. Group-1: Control group (n=6): underwent no procedures. Group-2: Sham group (n=6): underwent the same procedures as the experimental group without ureter and psoas muscle dissection. Group-3: Group with induced partial unilateral ureteral obstruction (n=9). All rats were sacrificed after 12 weeks. Superoxide dismutase enzyme activity and nitrite and nitrate salt levels were measured in renal tissue. Plasma nitrite-nitrate and ADMA levels were examined. Results In the experimental group histopathological changes observed included renal pelvis dilatation, flattened papillae, sclerotic glomerulus and fibrosis. In the experimental group tissue SOD and blood ADMA levels were higher than the control and sham groups (p<0.05) while tissue NO and plasma NO values were lower than in the sham and control groups (p<0.05). Conclusion Oxidative stress and disruption of NO synthesis play an important role in renal function and histopathological changes after obstructive renal disease. To prevent renal complications developing after obstructive nephropathy we believe that a new strategy may be research on reducing ADMA.

Dual-excitation upconverting nanoparticle and quantum dot aptasensor for multiplexed food pathogen detection.

In this report, a dual-excitation sensing method was developed using aptamer-functionalized quantum dots and upconverting nanoparticles, exhibiting Stokes and anti-Stokes type excitation profiles, respectively. Conjugation of the aptamer-functionalized luminescent nanoparticles with the magnetic beads, comprising short DNA sequences that were partially complementary to the aptamer sequences, enabled facile separation of the analyte-free conjugates for fluorescent measurement. UV-Visible spectroscopy, Circular Dichroism spectroscopy, Dynamic Light Scattering and Polyacrylamide Gel Electrophoresis techniques were used to characterize the aptamer probes developed. The target-specific luminescent conjugates were applied for multiplex detection of model food pathogens, Salmonella typhimurium, and Staphylococcus aureus, in which the fluorescent emission spectra were obtained under UV excitation at 325nm for quantum dots and NIR excitation at 980nm for upconverting nanoparticles, respectively. The dual-excitation strategy was aimed to minimize cross-talk between the luminescent signals for multiplexed detection, and yielded limit of detection values of 16 and 28cfumL(-1) for Staphylococcus aureus, and Salmonella typhimurium, respectively. By employing a greater number of quantum dots and upconverting nanoparticles with non-overlapping fluorescent emissions, the proposed methodology might be exploited further to detect several analytes, simultaneously.

Alterations in iris structure and pupil size related to alpha-1 adrenergic receptor antagonists use: implications for floppy iris syndrome.

PURPOSE: To evaluate structural alterations of iris and pupil diameters (PDs) in patients using systemic α-1-adrenergic receptor antagonists (α-1ARAs), which are associated with intraoperative floppy iris syndrome (IFIS). METHODS: Eighty-eight eyes of 49 male were evaluated prospectively. Patients were assigned to 2 different groups. Study group included 23 patients taking any systemic α-1ARAs treatment, and control group included 26 patients not taking any systemic α-1ARAs treatment. All patients underwent anterior segment optical coherence tomography to evaluate iris thickness at the dilator muscle region (DMR) and at the sphincter muscle region (SMR). The PD was measured using a computerized infrared pupillometer under scotopic and photopic illumination. RESULTS: The study group included 46 eyes of 23 patients and the control group included 42 eyes of 26 patients. Most treated patients were on tamsulosin (16/23). Mean age was similar in the study and control groups (61.9±7.1 vs. 60.3±8, 2 years, nonsignificant). DMR (506.5±89.4 vs. 503.6±83.5 µm), SMR (507.8±78.1 vs. 522.1±96.4 µm) and the DMR/SMR ratio (1.0±0.15 vs. 0.99±0.23 µm) was similar in the study and control groups and these differences were nonsignificant. Scotopic PDs were also similar in both groups (3.99±1.11 vs. 3.74±1.35, nonsignificant). A significantly reduced photopic PD (2.89±0.55 vs. 3.62±0.64, P<0.001) and an increased scotopic/photopic PD (1.42±0.44 vs. 1.02±0.30, P<0.001) were found in the study group. CONCLUSIONS: Evaluating PD alterations might be more useful than evaluating iris structural alterations in predicting IFIS. There is still a need for a reliable method that will determine the possibility of IFIS.

Unexpected severe hepatotoxicity of ciprofloxacine: two case reports.

Ciprofloxacin is a fluoroquinolone antibiotic that has a relatively low rate of occurence of adverse side effects. However, increasing evidences suggest that ciprofloxacin may cause unexpected severe liver damage. Especially, the risk of hepatotoxicity is significantly higher in elderly men receiving drug for a long time. In this article, 2 cases of unexpected severe hepatoxicity of ciprofloxacin are presented.