Reversible and irreversible inhibitors of coronavirus Nsp15 endoribonuclease.

The emergence of severe acute respiratory syndrome coronavirus 2, the causative agent of coronavirus disease 2019, has resulted in the largest pandemic in recent history. Current therapeutic strategies to mitigate this disease have focused on the development of vaccines and on drugs that inhibit the viral 3CL protease or RNA-dependent RNA polymerase enzymes. A less-explored and potentially complementary drug target is Nsp15, a uracil-specific RNA endonuclease that shields coronaviruses and other nidoviruses from mammalian innate immune defenses. Here, we perform a high-throughput screen of over 100,000 small molecules to identify Nsp15 inhibitors. We characterize the potency, mechanism, selectivity, and predicted binding mode of five lead compounds. We show that one of these, IPA-3, is an irreversible inhibitor that might act via covalent modification of Cys residues within Nsp15. Moreover, we demonstrate that three of these inhibitors (hexachlorophene, IPA-3, and CID5675221) block severe acute respiratory syndrome coronavirus 2 replication in cells at subtoxic doses. This study provides a pipeline for the identification of Nsp15 inhibitors and pinpoints lead compounds for further development against coronavirus disease 2019 and related coronavirus infections.

Investigating the effect of inquiry-based stress reduction on mortality awareness and interpersonal problems among intensive care unit nurses.

INTRODUCTION: Caring for dying patients is one of the job stressors. Nurses in intensive care units are among the medical staff who have a close interaction with dying patients. Studies have shown that psychological interventions are very helpful in improving thinking about death and its problems. Therefore, this study was conducted to investigate the effect of Inquiry-Based Stress Reduction on mortality awareness and interpersonal problems among intensive care unit nurses in southeastern Iran. MATERIALS AND METHODS: This was a Quasi-experimental study with a pretest-posttest design in southeast of Iran in 2021. Nurses were selected using the convenience sampling method and divided into intervention (n = 32) and control (n = 35) groups using the block randomization method. The intervention group received a two-hour Inquiry-Based Stress Reduction counseling session every week for 6 weeks. Data were gathered using Multidimensional Mortality Awareness Measure and Inventory of Interpersonal Problems before, immediately after, and 6 weeks after the intervention. IBM SPSS Statistics software version 25 was used for data analysis. RESULTS: In the intervention group, the mean scores of Mortality Awareness before, immediately after, and 6 weeks after the intervention were 130.41 ± 5.91, 164.47 ± 8.66, and 163.91 ± 9.29, respectively. Therefore, in the intervention group, the increase of Mortality Awareness mean score was statistically significant (P < 0.001). In the control group, the mean scores of Mortality Awareness before, immediately after, and 6 weeks after intervention were 129.63 ± 5.59, 135.26 ± 11.14, and 132.66 ± 5.62, respectively. Difference between the two groups was significant (P < 0.001). The results also showed that in the intervention group the mean scores of Interpersonal Problems immediately after and 6 weeks after the intervention were lower than before the intervention (P < 0.001). In the control group, Interpersonal Problems increased over time (P < 0.001). Accordingly, the difference between the two groups in terms of Interpersonal Problems during the study was statistically significant (P < 0.001). CONCLUSION: The study results suggest that the Inquiry-Based Stress Reduction is an appropriate intervention method to improve mortality awareness and reduce interpersonal problems in intensive care unit nurses.

Psychological Predictors of Mortality Awareness: Time Perspective, Contentment With Age and Paternal Antipathy and Neglect.

Much research has focused upon the association between mortality awareness and mental ill-health. In this study we attempt to explore positive as well as negative psychological concomitants of mortality awareness. 170 participants were recruited in an online questionnaire study, measuring seven independent variables - marginalisation, childhood adversity, rebelliousness, time perspective, attitudes toward age and stage of life, health attitudes and demographics - and five dependent variables, specifically mortality legacy, mortality fearfulness, mortality acceptance, mortality disempowerment and mortality disengagement. Several significant bivariate associations were found. Follow-up regression analysis observed combined effects of variables accounting for 28% of variance in mortality legacy, 27% for mortality fearfulness, 13% for mortality acceptance, 42% for mortality disempowerment and 25% for mortality disengagement. Time perspective, contentment with age, and paternal antipathy and neglect were the most notable independent predictors. It was concluded that attitudes towards health, stage of life and childhood experiences significantly predict mortality awareness.

Gold nanostars as a colloidal substrate for in-solution SERS measurements using a handheld Raman spectrometer.

The evolution of Raman spectroscopy into a useful analytical technique has been due, in part, to the development of inexpensive, compact instrumentation and advancements in methodologies that enhance Raman intensities. Surface enhanced Raman scattering (SERS) is a primary methodology for quantitative and low detection limit measurements. While a broad array of applications using solid SERS substrates have been demonstrated, in-solution SERS measurements are not as widely pursued. This work seeks to optimize the synthesis of gold nanostars (AuNS) as a colloidal SERS substrate for in-solution measurements using handheld instrumentation. The types and concentrations of two buffers typically used for AuNS synthesis are examined to optimize the SERS intensity of a chemisorbed Raman probe. The observed SERS intensity primarily depends on conditions that allow higher surface coverage of the probe. Conditions that result in AuNS aggregates are found to be most optimal for SERS, similar to other nanoparticle shapes. A method to quantitate methimazole, an anti-hormone pharmaceutical, in urine is developed and reported. The primary impact of this work is the demonstration of the combination of water dispersible substrates and handheld instrumentation for rapid and sensitive analytical measurements.

Evaluation of the electroanalytical performance of carbon-on-gold films prepared by electron-beam evaporation.

Carbon film electrodes can often be used without pretreatment, and their fabrication allows for flexibility in size and shape and for mass production. In this work, we are exploring layered structures comprised of thin films of carbon on gold (eC/Au) prepared by electron-beam evaporation. These extremely flat films are not pyrolyzed and are comprised of mainly amorphous carbon but still exhibit reasonable conductivity due to the underlying gold layer. eC/Au electrodes, without any pretreatment, yield similar heterogeneous electron-transfer rates for benchmark redox systems and significantly lower background current in comparison with polished glassy carbon. Interestingly, they show insignificant adsorption of quinones, which is uncommon for most carbon electrode materials. However, eC/Au is still prone to adsorption of airborne hydrocarbons when exposed to ambient air like most graphitic materials. With reproducibly fast electron transfer kinetics, low background current, negligible adsorption, and ultraflat surface, eC/Au films are a promising candidate for electrochemical and sensor applications.

The interface between phosphatidylinositol transfer protein function and phosphoinositide signaling in higher eukaryotes.

Phosphoinositides are key regulators of a large number of diverse cellular processes that include membrane trafficking, plasma membrane receptor signaling, cell proliferation, and transcription. How a small number of chemically distinct phosphoinositide signals are functionally amplified to exert specific control over such a diverse set of biological outcomes remains incompletely understood. To this end, a novel mechanism is now taking shape, and it involves phosphatidylinositol (PtdIns) transfer proteins (PITPs). The concept that PITPs exert instructive regulation of PtdIns 4-OH kinase activities and thereby channel phosphoinositide production to specific biological outcomes, identifies PITPs as central factors in the diversification of phosphoinositide signaling. There are two evolutionarily distinct families of PITPs: the Sec14-like and the StAR-related lipid transfer domain (START)-like families. Of these two families, the START-like PITPs are the least understood. Herein, we review recent insights into the biochemical, cellular, and physiological function of both PITP families with greater emphasis on the START-like PITPs, and we discuss the underlying mechanisms through which these proteins regulate phosphoinositide signaling and how these actions translate to human health and disease.

Factors Associated with the Development of Posterior Capsule Opacification Requiring Yttrium Aluminum Garnet Capsulotomy.

SIGNIFICANCE: Determining risk factors for posterior capsule opacification will allow for further interventions to reduce the risk of development and thus additional procedures. PURPOSE: The purpose of this study was to investigate risk factors associated with development of clinically significant posterior capsule opacification requiring yttrium aluminum garnet (YAG) capsulotomy. METHODS: Medical records of patients (≥18 years) who underwent cataract surgery between January 1, 2011, and March 31, 2014, at Kresge Eye Institute were reviewed. Three hundred eyes requiring YAG capsulotomy up to 3 years after cataract surgery were included in the YAG capsulotomy group. Three hundred eyes not requiring YAG capsulotomy up to 3 years after cataract surgery were selected via age-matched simple randomization (control group). RESULTS: The YAG capsulotomy group included patients with younger age (65.8 ± 11.3 vs. 70.1 ± 10.6 years, P < .001), more men (42.67 vs. 34.67%, P = .04), fewer patients with hypertension (73.00 vs. 83.00%, P < .001), and more patients with hydrophilic intraocular lenses (74.67 vs. 47.00%, P < .001). Logistic regression analysis demonstrated a negative association between YAG capsulotomy and age (coefficient, -0.04; 95% confidence interval [CI], 0.95 to 0.98; P < .001) and hydrophobic intraocular lenses (coefficient, -1.50; 95% CI, 0.15 to 0.33; P < .001), and a positive association with presence of glaucoma (coefficient, 0.88; 95% CI, 1.39 to 4.17; P = .002). Elapsed time to YAG capsulotomy was sooner in patients with a history of uveitis (95% CI, 5.10 to 9.70 months; P = .02) and insertion of hydrophilic intraocular lenses (95% CI, 18.67 to 21.57 months; P < .001). CONCLUSIONS: Results of this study suggest that development of visually significant posterior capsule opacification is associated with younger age, glaucoma, and hydrophilic intraocular lenses, and it occurs earlier among those with hydrophilic intraocular lenses and a history of uveitis.

Dynamics and energetics of the mammalian phosphatidylinositol transfer protein phospholipid exchange cycle.

Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling. However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITPα, both on membrane bilayers and in solvated systems, informed downstream biochemical analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITPα lipid exchange cycle: (i) interaction of PITPα with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability of PITPα to initiate closure around the PtdCho ligand is accompanied by loss of flexibility of two helix/loop regions, as well as of the C-terminal helix; (iii) the energy barrier of phospholipid extraction from the membrane is lowered by a network of hydrogen bonds between the lipid molecule and PITPα; (iv) the trajectory of PtdIns or PtdCho into and through the lipid-binding pocket is chaperoned by sets of PITPα residues conserved throughout the StART-like PITP family; and (v) conformational transitions in the C-terminal helix have specific functional involvements in PtdIns transfer activity. Taken together, these findings provide the first mechanistic description of key aspects of the PITPα PtdIns/PtdCho exchange cycle and offer a rationale for the high conservation of particular sets of residues across evolutionarily distant members of the metazoan StART-like PITP family.

A surface plasmon resonance based inhibition immunoassay for measurement of steroid hormones.

Quantitative measurement of small-molecule metabolites is now emerging as an effective way to link the metabolite profile to disease state. Surface plasmon resonance (SPR) is a sensing platform that has demonstrated applicability for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. Herein, we utilized an indirect detection format and developed an inhibition immunoassay for the quantitative measurement of 17ß-estradiol (E2) using SPR. One competitor, BSA-E2 conjugate, was immobilized to the SPR chip via the reaction between the primary amino group of the conjugate and the succinimide group (NHS) introduced by the formation of a thiol-NHS monolayer on gold surface. Free E2 molecules compete with BSA-E2 on chip surface for binding sites provided by a monoclonal anti-E2 antibody. It was found the binding affinity of the antibody to BSA-E2 conjugate increases with decreasing surface coverage of BSA-E2 conjugate. Under optimal conditions, a sigmoidal calibration curve with a negative slope and a dynamic range from 10 pM to 2 nM was generated. The detection limit of the immunoassay is estimated to be 0.3 pM. Moreover, the immunoassay exhibits high specificity for E2 detection using estrone (E1) as a potential interference.

Functionalized gold nanoparticle-enhanced competitive assay for sensitive small-molecule metabolite detection using surface plasmon resonance.

The development of sensing systems for the measurement of small molecules is an active area of research. A sensor based approach for the measurement of metabolites can potentially provide the simplicity and portability required for widespread use. Rapid detection and quantitation of small-molecule metabolites can potentially emerge as an effective way to link the metabolite profile to the disease state. Surface plasmon resonance (SPR) combined with molecular recognition elements to deliver high specificity is a sensing platform that has been widely applied for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. The work described here combines a periplasmic binding protein for recognition with target modified gold nanoparticles (AuNPs) in a competitive assay format for folic acid (FA) detection. Specifically, a SPR imaging substrate containing immobilized folate binding protein (FBP) is used to measure the adsorption of FA conjugated AuNPs. The immobilization of the FBP and the binding of the FA conjugated AuNPs are characterized and optimized. It is shown that free FA in solution can be quantitatively measured by competition for the surface binding sites with the functionalized AuNPs. We demonstrate that the dynamic range can be lowered from micromolar to nanomolar by simply decreasing the concentration of FA conjugated AuNPs, thus lowering the limit of detection to 2.9 nM. This type of competitive assay can be applied to a range of small molecules, which paves the way for future multiplexed analysis of metabolites using SPR.

Cellulose nanocrystal-derived hollow mesoporous carbon spheres and their application as a metal-free catalyst.

In this contribution, we demonstrate the fabrication of hollow mesoporous carbon spheres (HCSs) derived from cellulose nanocrystals (CNCs). The HCSs were prepared by templating CNCs onto sacrificial silica spheres followed by heat treatment. Mesoporous carbon spheres result from the removal of the silica spheres by etching. The walls of the HCSs are approximately 4 nm thick and are composed of amorphous and graphitic carbon. The catalytic activity of the HCSs was investigated for the reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) by sodium borohydride (NaBH4). The present investigation reveals the outstanding catalytic activity of these particles. The reaction rate followed pseudo-first order kinetics with k value of 4.72 × 10-3 s-1 and activity parameter of 52.2 s-1 g-1, which showed superior performance compared to that of metal nanoparticle and metal nanoparticle-carbon hybrid based catalysts.

A Fully Integrated CMOS Fluorescence Biochip for DNA and RNA Testing.

Design and successful implementation of a fully-integrated CMOS fluorescence biochip for DNA/RNA testing in molecular diagnostics (MDx) is presented. The biochip includes a 32×32 array of continuous wave fluorescence detection biosensing elements. Each biosensing element is capable of having unique DNA probe sequences, wavelength-selective multi-dielectric emission filter (OD of 3.6), resistive heater for thermal cycling, and a high performance and programmable photodetector. The dimension of each biosensor is 100µm×100µm with a 50µm×50µm Nwell-Psub photodiode acting as the optical transducer, and a ΣΔ modulator based photocurrent sensor. The measured photodetector performance shows ~116 dB detection dynamic range (10fA - 10nA) over the 25°C - 100°C temperature range, while being ~1 dB away from the fundamental shot-noise limit. To empirically demonstrate the compatibility of this biochip with MDx applications, we have successfully utilized the array and its thermal cycling capability to adopt a 7-plex panel for detection of 6 human upper respiratory viruses.

Accuracy of the Air Flow Sphincter Locator system in identifying the lower esophageal sphincter for placement of pH catheters.

The Air Flow Sphincter Locator (AFSL) is marketed as an alternative method to manometry for localizing the lower esophageal sphincter (LES) for pH probe placement. Such a system is desirable due to the additional time, cost, and discomfort associated with dual nasal intubation, but its accuracy has never been assessed. To assess the accuracy of the AFSL in localizing the LES. Fifty consecutive outpatients presenting for pH and manometry studies were included. The upper border of the LES was determined using HRM and the AFSL by two technicians independently. LES locations measured by technicians using AFSL versus manometry, as well as the manometrically determined LES locations by technicians versus MDs were compared. Differences in LES locations determined by HRM as read by MDs versus technicians were small; none were >3 cm, and 92% were within 2 cm. Comparison between LES locations determined by technicians using HRM versus the AFSL revealed that 52% had a difference of 2-3cm and 32% had a difference of >3 cm. Hiatal hernia was associated with a difference in LES location of >3 cm. Excluding patients with hiatal hernia, nonetheless, still produced a >3 cm difference in 24% of studies. Prior reports have suggested that a difference greater than +/-3 cm in pH probe placement is considered unacceptable for clinical studies. Based on our study, the AFSL placed the LES outside of this range in 32% of patients, and may be particularly inaccurate in the setting of a hiatal hernia. This suggests that the device may not be an acceptable alternative to manometry in determining LES location for pH probe placement.

Fungal Isolate Optimized for Biogenesis of Silver Nanoparticles with Enhanced Colloidal Stability.

Understanding the synthesis and properties of nanomaterials is critical for reliable applications. Biological systems, such as fungi, have been described as a "green" alternative to synthesis, yet knowledge gaps exist in terms of production variability, comparison with commercial products, and identifying a clear biological advantage over other synthesis methods. In this study, we evaluated 12 fungal isolates of Fusarium oxysporum for Ag nanoparticle production and characterized the resultant biologically produced (biogenic) nanoparticles. We show evidence that isolate selection, temperature, and pH can influence the quantity, size, and shape of nanoparticles. All F. oxysporum isolates produced Ag nanoparticles, but in varied quantities. Increasing incubation temperature increased the quantity, yet nanoparticle diameter was inversely related to temperature. Variations in pH predominately influenced nanoparticle morphology. A direct comparison with commercial, chemically produced Ag nanoparticles yielded physical similarities; however, important differences in surface chemistry are observed. Biogenic nanoparticles show a greater degree of colloidal stability in high-ionic-strength solutions, pointing to a biological advantage associated with the fungal produced layer (corona) surrounding the nanoparticles. It is clear that understanding the organic layer and interfacial interactions will be beneficial in developing innovative applications, particularly in the field of biosensing.

Diazonium Chemistry for the Bio-Functionalization of Glassy Nanostring Resonator Arrays.

Resonant glassy nanostrings have been employed for the detection of biomolecules. These devices offer high sensitivity and amenability to large array integration and multiplexed assays. Such a concept has however been impaired by the lack of stable and biocompatible linker chemistries. Diazonium salt reduction-induced aryl grafting is an aqueous-based process providing strong chemical adhesion. In this work, diazonium-based linker chemistry was performed for the first time on glassy nanostrings, which enabled the bio-functionalization of such devices. Large arrays of nanostrings with ultra-narrow widths down to 10 nm were fabricated employing electron beam lithography. Diazonium modification was first developed on SiCN surfaces and validated by X-ray photoelectron spectroscopy. Similarly modified nanostrings were then covalently functionalized with anti-rabbit IgG as a molecular probe. Specific enumeration of rabbit IgG was successfully performed through observation of downshifts of resonant frequencies. The specificity of this enumeration was confirmed through proper negative control experiments. Helium ion microscopy further verified the successful functionalization of nanostrings.

Mammalian START-like phosphatidylinositol transfer proteins - Physiological perspectives and roles in cancer biology.

PtdIns and its phosphorylated derivatives, the phosphoinositides, are the biochemical components of a major pathway of intracellular signaling in all eukaryotic cells. These lipids are few in terms of cohort of unique positional isomers, and are quantitatively minor species of the bulk cellular lipidome. Nevertheless, phosphoinositides regulate an impressively diverse set of biological processes. It is from that perspective that perturbations in phosphoinositide-dependent signaling pathways are increasingly being recognized as causal foundations of many human diseases - including cancer. Although phosphatidylinositol transfer proteins (PITPs) are not enzymes, these proteins are physiologically significant regulators of phosphoinositide signaling. As such, PITPs are conserved throughout the eukaryotic kingdom. Their biological importance notwithstanding, PITPs remain understudied. Herein, we review current information regarding PITP biology primarily focusing on how derangements in PITP function disrupt key signaling/developmental pathways and are associated with a growing list of pathologies in mammals.

A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated protein kinase C regulation.

Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and ßII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of full-length Pin1 complexed to the C-terminal tail of PKCßII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a conformation in which it uses the WW and PPIase domains to engage two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, respectively. Hydrophobic motif is a non-canonical Pin1-interacting element. The structural information combined with the results of extensive binding studies and experiments in cultured cells suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.

Dileucine and PDZ-binding motifs mediate synaptic adhesion-like molecule 1 (SALM1) trafficking in hippocampal neurons.

Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in neurite outgrowth and synapse formation. Of the five family members, only SALM1, -2, and -3 contain a cytoplasmic C-terminal PDZ-binding motif. We have found that SALM1 is unique among the SALMs because deletion of its PDZ-binding motif (SALM1ΔPDZ) blocks its surface expression in heterologous cells. When expressed in hippocampal neurons, SALM1ΔPDZ had decreased surface expression in dendrites and the cell soma but not in axons, suggesting that the PDZ-binding domain may influence cellular trafficking of SALMs to specific neuronal locations. Endoglycosidase H digestion assays indicated that SALM1ΔPDZ is retained in the endoplasmic reticulum (ER) in heterologous cells. However, when the entire C-terminal tail of SALM1 was deleted, SALM1 was detected on the cell surface. Using serial deletions, we identified a region of SALM1 that contains a putative dileucine ER retention motif, which is not present in the other SALMs. Mutation of this DXXXLL motif allowed SALM1 to leave the ER and enhanced its surface expression in heterologous cells and neurons. An increase in the number of protrusions at the dendrites and cell body was observed when this SALM1 mutant was expressed in hippocampal neurons. With electron microscopy, these protrusions appeared to be irregular, enlarged spines and filopodia. Thus, enrichment of SALM1 on the cell surface affects dendritic arborization, and intracellular motifs regulate its dendritic versus axonal localization.