A brain specific alternatively spliced isoform of nonmuscle myosin IIA lacks its mechanoenzymatic activities.

Recent genomic studies reported that 90 to 95% of human genes can undergo alternative splicing, by which multiple isoforms of proteins are synthesized. However, the functional consequences of most of the isoforms are largely unknown. Here, we report a novel alternatively spliced isoform of nonmuscle myosin IIA (NM IIA), called NM IIA2, which is generated by the inclusion of 21 amino acids near the actin-binding region (loop 2) of the head domain of heavy chains. Expression of NM IIA2 is found exclusively in the brain tissue, where it reaches a maximum level at 24 h during the circadian rhythm. The actin-dependent Mg2+-ATPase activity and in vitro motility assays reveal that NM IIA2 lacks its motor activities but localizes with actin filaments in cells. Interestingly, NM IIA2 can also make heterofilaments with NM IIA0 (noninserted isoform of NM IIA) and can retard the in vitro motility of NM IIA, when the two are mixed. Altogether, our findings provide the functional importance of a previously unknown alternatively spliced isoform, NM IIA2, and its potential physiological role in regulating NM IIA activity in the brain.

Nitroreductase-Triggered Fluorophore Labeling of Cells and Tissues under Hypoxia.

Several reductases, including nitroreductase, are upregulated under hypoxic conditions characterized by an oxygen-deficient microenvironment. Given that hypoxia is a prominent feature of solid tumors, our investigation focused on developing a bioconjugative probe designed for staining tissue under hypoxic conditions, particularly activated by nitroreductase. This probe, developed using our trigger-release-bioconjugation system rooted in the ortho-quinone methide chemistry, exhibited selective activation by nitroreductase and fluorophore labeling within mitochondria and endoplasmic reticulum. As a result, it displayed sustained fluorescence that persisted even after washing steps in cells and tissues. We applied this innovative probe to stain mouse kidney tissue in an acute kidney injury model induced by inadequate oxygen supply. Among various organ tissues examined, only kidney tissue showed significantly higher fluorescence in the injury model compared with the control tissue, as revealed by two-photon microscopic imaging. This research presents a promising avenue for the development of practical staining agents for image-guided tumor surgery.

Chromone-Based Cd(II) Fluorescent Coordination Polymer Fabricated to Study Optoelectronic and Explosive Sensing Properties.

Here, electrical conductivity and explosive sensing properties of multifunctional chromone-Cd(II)-based coordination polymers (CPs) (1-4) have been explored. The presence of different pseudohalide linkers, thiocyanate ions, and dicyanamide ions resulted in 1D and 3D architecture in the CPs. Thin film devices developed from CPs 1-4 (complex-based Schottky devices, CSD1, CSD2, CSD3, and CSD4, respectively) showed semiconductor behavior. Their conductivity values increased under photo illumination (1.37 × 10-5, 1.85 × 10-5, 1.61 × 10-5, and 2.01 × 10-5 S m-1 under dark conditions and 5.06 × 10-5, 8.78 × 10-5, 7.26 × 10-5, and 10.21 × 10-5 S m-1 under light). The nature of the I-V plots of these thin film devices under light irradiation and dark are nonlinear rectifying, which has been observed in Schottky barrier diodes (SBDs). All four CPs (1-4) exhibited highly selective fluorescence quenching-based sensing properties toward well-known explosives, 2,4-dinitrophenol (DNP) and 2,4,6-trinitrophenol (TNP). The limit of detection (LOD) values are 55, 28, 27, and 31 µM for TNP and 78, 44, 32, and 41 µM for DNP for complexes 1-4, respectively. A structure property correlation has been established to explain optoelectronic and explosive sensing properties.

A novel recurrence-based approach for investigating multiphase flow dynamics in bubble column reactors.

In chemical industries, multiphase flows in a bubble column reactor are frequently observed. The nonlinearity associated with bubble hydrodynamics, such as bubble-bubble and bubble-liquid interactions, gives rise to complex spatiotemporal patterns with increased gas or liquid velocities, which are extremely difficult to model and predict. In the current study, we propose a new, computationally efficient recurrence-based approach involving the angular separation between suitably defined state vectors and implement it on the experimental multiphase flow variables. The experimental dataset that consists of image frames obtained using a high-speed imaging system is generated by varying air and water flow rates in a bubble column reactor setup. The recurrence plots using the new approach are compared with those derived from conventional recurrence, considering standard benchmark problems. Further, using the recurrence plots and recurrence quantification from the new recurrence methodology, we discover a transition from a high recurrence state to a complex regime with very low recurrence for an increase in airflow rate. Determinism exhibits a rise for the decrease in airflow rate. A sharp decline in determinism and laminarity, signifying the quick shift to complex dynamics, is more prominent for spatial recurrence than temporal recurrence, indicating that the rise in airflow rate significantly impacts the spatial location of bubbles. We identify three regimes that appeared as distinct clusters in the determinism-laminarity plane. The bubbly regime, characterized by high values of determinism and laminarity, is separated by an intermediate regime from the slug flow regime, which has low determinism and laminarity.

Strategies to convert organic fluorophores into red/near-infrared emitting analogues and their utilization in bioimaging probes.

Organic fluorophores aided by current microscopy imaging modalities are essential for studying biological systems. Recently, red/near-infrared emitting fluorophores have attracted great research efforts, as they enable bioimaging applications with reduced autofluorescence interference and light scattering, two significant obstacles for deep-tissue imaging, as well as reduced photodamage and photobleaching. Herein, we analyzed the current strategies to convert key organic fluorophores bearing xanthene, coumarin, and naphthalene cores into longer wavelength-emitting derivatives by focussing on their effectiveness and limitations. Together, we introduced typical examples of how such fluorophores can be used to develop molecular probes for biological analytes, along with key sensing features. Finally, we listed several critical issues to be considered in developing new fluorophores.

Dissecting the Crosstalk between Bisulfite and Hypochlorous Acid in the Reaction-Based Fluorescence Detection with Dicyanovinyl-Based Probes.

Fluorescent probes bearing a reactive moiety of 1,1-dicyanovinyl are known to detect several biological species including bisulfite and hypochlorous acid, which, however, possess a selectivity issue among those analytes. Structural modifications of the reactive group for optimal steric and electron effects based on theoretical calculations led us to address the selectivity issue, offering new reactive moieties that provide complete analyte selectivity, including that between bisulfite and hypochlorous acid, in cells as well as in solution.

Comparison of simplicity, convenience, safety, and cost-effectiveness between use of insulin pen devices and disposable plastic syringes by patients with type 2 diabetes mellitus: a cross-sectional study from Bangladesh.

INTRODUCTION: Insulin pen devices and disposable plastic insulin syringes are two common tools for insulin administration. This study aims to compare the simplicity, convenience, safety, and cost-effectiveness of insulin pens versus syringe devices in patients with type 2 diabetes mellitus (T2DM). METHODS: A cross-sectional study was conducted at 14 diabetes clinics throughout Bangladesh from November 2021 to April 2022 among adults with T2DM injecting insulin by pen devices or disposable insulin syringes at least once a day for at least one year by purposive sampling. The simplicity, convenience, and safety of insulin devices were assessed using a structured questionnaire, and the study subjects were scored based on their answers; higher scores indicated a poorer response. Total scores for simplicity, convenience, and safety were obtained by adding the scores for relevant components. Their average monthly medical expense and cost of insulin therapy were recorded. The median values of the total scores and monthly expenses were compared between pen devices and disposable syringe users. RESULTS: 737 subjects were evaluated; 406 were pen users, and 331 were vial syringe users. The pen users had lower median scores for simplicity [6.0 (5.0-8.0) vs. 7.0 (5.0-9.0), p = 0.002], convenience [4.0 (3.0-6.0) vs. 5.0 (4.0-6.0), p < 0.001], and safety [7.0 (6.0-8.0) vs. 7.0 (6.0-9.0), p = 0.008] than vial syringe users. Pen devices were more expensive than vial syringes in terms of average medical expense per month [BDT 5000 (3500-7000) vs. 3000 (2000-5000), p < 0.001], the total cost of insulin therapy per month [BDT 2000 (1500-3000) vs. 1200 (800-1700), p < 0.001] and cost per unit of insulin used [BDT 2.08 (1.39-2.78) vs. 0.96 (0.64-1.39), p < 0.001]. Non-significant differences in favor of pens were observed in HbA1c levels [8.7 (7.8-10) vs. 8.9 (7.9-10)%, p = 0.607] and proportions of subjects having HbA1c < 7% (6.9 vs. 6.3%, p = 0.991). CONCLUSION: Insulin pens are simpler, more convenient, and safe but more expensive than vial syringes. Glycemic control is comparable between pen and syringe users. Long-term follow-up studies are needed to determine the clinical and economic impacts of such benefits of insulin pens.

A General Strategy Toward pH-Resistant Phenolic Fluorophores for High-Fidelity Sensing and Bioimaging Applications.

Aryl alcohol-type or phenolic fluorophores offer diverse opportunities for developing bioimaging agents and fluorescence probes. Due to the inherently acidic hydroxyl functionality, phenolic fluorophores provide pH-dependent emission signals. Therefore, except for developing pH probes, the pH-dependent nature of phenolic fluorophores should be considered in bioimaging applications but has been neglected. Here we show that a simple structural remedy converts conventional phenolic fluorophores into pH-resistant derivatives, which also offer "medium-resistant" emission properties. The structural modification involves a single-step introduction of a hydrogen-bonding acceptor such as morpholine nearby the phenolic hydroxyl group, which also leads to emission bathochromic shift, increased Stokes shift, enhanced photo-stability and stronger emission for several dyes. The strategy greatly expands the current fluorophores' repertoire for reliable bioimaging applications, as demonstrated here with ratiometric imaging of cells and tissues.

A Small-Molecule Fluorescence Probe for Nuclear ATP.

Fluorescence monitoring of ATP in different organelles is now feasible with a few biosensors developed, which, however, show low sensitivity, limited biocompatibility, and accessibility. Small-molecule ATP probes that alleviate those limitations thus have received much attention recently, leading to a few ATP probes that target several organelles except for the nucleus. We disclose the first small-molecule probe that selectively detects nuclear ATP through reversible binding, with 25-fold fluorescence enhancement at pH 7.4 and excellent selectivity against various biologically relevant species. Using the probe, we observed 2.1-3.3-fold and 3.9-7.8-fold higher nuclear ATP levels in cancerous cell lines and tumor tissues compared with normal cell lines and tissues, respectively, which are explained by the higher nuclear ATP level in the mitosis phase. The probe has great potential for studying nuclear ATP-associated biology.

A π-Stacking Based Fluorescent Probe for Labeling of Flavin Analogues in Live Cells through Unusual FRET Process.

The flavin adenine dinucleotide (FAD) is an indispensable coenzyme in live cells. It acts as a catalyst in many redox responsive metabolic reactions, including oxidative phosphorylation in mitochondria. The real-time monitoring of flavin is important to understand the disorder in the metabolic process, redox system, etc. Thus, we have developed a fluorescent probe CPy-1 that noncovalently binds with flavin to exhibit the FRET process. 1H- NMR and docking study indicated that there is a strong hydrophobic interaction between flavins and CPy-1. Also, a π-π stacking between isoalloxazine ring in flavin and quinoline and coumarin moieties of CPy-1 favors self-assembly. The nontoxic probe CPy-1 could distinguish cancer cells from normal cells based on expressions of endogenous FAD.

Rationally Designed Two-Photon Ratiometric Elastase Probe for Investigating Inflammatory Bowel Disease.

Elastase, a serine protease, plays important roles in our body in food digestion and defence against pathogens. Particularly, the elastase present in neutrophils is directly associated with inflammatory bowel disease (IBD). Through a rational approach, we have developed a fluorescent elastase probe that has multiple advantages for biological applications including two-photon ratiometric imaging capability. Using the probe, which is capable of detecting intracellular elastase activity associated with inflammation, we have investigated elastase level changes in various mouse organs under an IBD condition for the first time. The results reveal notably higher elastase levels in the liver and duodenum of the healthy mice than in the other investigated organs. Under the IBD condition, we observed significant elastase level changes in the liver, duodenum, colon, and lung. The downregulation of elastase in the liver under the IBD condition suggests migration of neutrophils into the upregulated organs. The notable upregulation of elastase in the duodenum is explained by self-production of elastase, in addition to the neutrophil migration from the liver. We have observed little elastase level changes in selected organs of immune-deficient mice raised under the normal and IBD conditions, which supports the neutrophil migration as the reason for perturbed elastase activity in the healthy mice. The results also suggest an important role of the liver in maintaining the immune response associated with the inflammation-induced elastase level changes. The probe offers an ideal tool for mapping neutrophil migration in body. Further understanding of the elastase-associated biology and diagnosis of IBD by monitoring affected organs are anticipated using the probe.

Fluorophore Labeling of Proteins: a Versatile Trigger-Release-Conjugation Platform Based on the Quinone Methide Chemistry.

In situ conjugation of fluorescent molecules to biomolecules such as proteins under spatiotemporal control offers a powerful means for studying biological systems. For that purpose, the o-quinone methide chemistry involving a sequence of the trigger-release-conjugation (TRC) process provides a versatile conjugation method. We have developed a new TRC platform bearing a quaternary ammonium salt for the release process, which can be structurally modified and readily synthesized from commonly used aryl alcohol-type organic fluorophores under environmentally benign conditions. We show that different aryl alcohol fluorophores containing the o-(morpholinium)methyl group for the release process allow efficient fluorophore labeling of proteins under both light- and chemical-triggering conditions. The bioconjugation in cells as well as in tissues was further demonstrated with an o-(morpholinium)methyl analogue containing a triggering group sensitive to reactive oxygen species. The new TRC system thus provides a versatile and unique platform for in situ fluorophore labeling of proteins in biological systems under spatiotemporal control.

Biosensors for detection of prostate cancer: a review.

Diagnosis of prostate cancer (PC) has posed a challenge worldwide due to the sophisticated and costly diagnostics tools, which include DRE, TRUS, GSU, PET/CT scan, MRI, and biopsy. These diagnostic techniques are very helpful in the detection of PCs; however, all the techniques have their serious limitations. Biosensors are easier to fabricate and do not require any cutting-edge technology as required for other imaging techniques. In this regard, point-of-care (POC) biosensors are important due to their portability, convenience, low cost, and fast procedure. This review explains the various existing diagnostic tools for the detection of PCs and the limitation of these methods. It also focuses on the recent studies on biosensors technologies as an alternative to the conventional diagnostic techniques for the detection of PCs.

Organoamine Templated Multifunctional Hybrid Metal Phosphonate Frameworks: Promising Candidates for Tailoring Electrochemical Behaviors and Size-Selective Efficient Heterogeneous Lewis Acid Catalysis.

The successful discovery of novel multifunctional metal phosphonate framework materials that incorporate newer organoamines and their utilization as a potential electroactive material for energy storage applications (supercapacitors) and as efficient heterogeneous catalysts are the most enduring challenges at present. From this perspective, herein, four new inorganic-organic hybrid zinc organodiphosphonate materials, namely, [C5H14N2]2[Zn6(hedp)4] (I), [C5H14N2]0.5[Zn3(Hhedp) (hedp)]·2H2O (II), [C6H16N2][Zn3(hedp)2] (III), and [C10H24N4][Zn6(Hhedp)2(hedp)2] (IV) (H4hedp = 1-hydroxyethane 1,1-diphosphonic acid), have been synthesized through the introduction of different organoamines and then structurally analyzed using various techniques. The compounds (I-IV) possess a three-dimensional network through alternate connectivity of zinc ions and diphosphonate ligands, as confirmed using single-crystal X-ray diffraction. The investigations of electrochemical charge storage behaviors of the present compounds indicate that compound III exhibits a high specific capacitance of 190 F g-1 (76 C g-1) at 1 A g-1, while compound II shows an excellent cycling stability of 90.11% even after 5000 cycles at 5 A g-1 in the 6 M KOH solution. Further, the present materials have also been utilized as active heterogeneous Lewis acid catalysts in the ketalization reaction. The screening of various substrate scopes during the catalytic process confirms the size-selective heterogeneous catalytic nature of the framework compounds. To our utmost knowledge, such a size-selective heterogeneous Lewis acid catalytic behavior has been observed for the first time in the amine templated inorganic-organic hybrid framework family. Moreover, the excellent size-selective catalytic efficiencies with the d10 metal system and recyclability performances make the compounds (I-IV) more efficient and promising Lewis acid heterogeneous catalysts.

NAD(P)H Quinone Oxidoreductase-1 in Organ and Tumor Tissues: Distinct Activity Levels Observed with a Benzo-rosol-Based Dual-Excitation and Dual-Emission Probe.

NAD(P)H quinone oxidoreductase-1 (NQO1), a protective enzyme against cellular oxidative stress, is expressed abnormally high in solid tumors and thus recognized as a cancer biomarker. To develop a fluorescent NQO1 probe with practicality, we investigated benzo-rosol fluorophores linked with a known self-immolative quinone substrate. Four probe candidates exhibited ratiometric sensing behavior toward the enzyme, satisfying our orbital mismatch stratagem proposed before, under dual-excitation and dual-emission conditions that alleviate the spectral overlap issue commonly observed with the ratiometric probes based on intramolecular charge-transfer change. Among the candidates, two ester-linked compounds exhibited hydrolytic instability to water or an esterase, discouraging us to develop such ester-linked probes. One ether-linked, hydrolytically stable probe provided brighter cellular fluorescence than the other and thus was applied to ratiometric imaging of NQO1 in cells and tissues. We found that the enzyme activity levels are much different in organ tissues: stomach (56), kidney (22), colon (9.8), testis (7.8), bladder (5.6), lung (1.2), and muscle (1.0). Furthermore, a markedly high enzyme level (14.6-fold) was observed in a xenograft tumor tissue compared with that in a normal tissue, which suggests that such an NQO1 probe is promising for cancer diagnosis and for studying the enzyme-associated biology.

Charge Separated One-Dimensional Hybrid Cobalt/Nickel Phosphonate Frameworks: A Facile Approach to Design Bifunctional Electrocatalyst for Oxygen Evolution and Hydrogen Evolution Reactions.

Two new organoamine templated one-dimensional transition metal phosphonate compounds are synthesized, and their bifunctional electrocatalytic activities are examined in highly alkaline and acidic media. Compared with state-of-the-art materials, the cobalt phosphonate system is a new fabrication of sustainable and highly efficient catalysts toward electrochemical water splitting systems.

An Endeavor in the Reaction-Based Approach to Fluorescent Probes for Biorelevant Analytes: Challenges and Achievements.

The promising features of fluorescence spectroscopy have inspired a quest for fluorescent probes for analysis and monitoring of molecular interactions in biochemical, medical, and environmental sciences. To overcome the competitive supramolecular interactions in aqueous media encountered with conventional molecular-recognition-based probes, the use of reaction-based probes that involve making or breaking of covalent bonds has emerged as a complementary sensing strategy to realize higher selectivity and sensitivity with larger spectroscopic changes. In spite of the enormous efforts, the development of reaction-based fluorescent probes meets with certain challenges in terms of their practical applications, demanding "intelligent design" of probes with an appropriate fluorophore attached to an efficient reactive moiety at the right place. This Account summarizes the results of our efforts made in the development and fine-tuning of reaction-based fluorescent probes toward those goals, classified by the type of analyte (anions, metal cations, and biomolecules) with notes on the challenges and achievements. The reaction-based approach was demonstrated to be powerful for the selective sensing of anions (cyanide and (amino)carboxylates) for the first time, and later it was extended to develop two-photon probes for bisulfite and fluoride ions. The reaction-based approach also enabled selective sensing of noble metal ions such as silver, gold, and palladium along with toxic (methyl)mercury species and paramagnetic copper ions. Furthermore, microscopic imaging and monitoring of biologically relevant species with reaction-based two-photon probes were explored for hydrogen sulfide, hypochlorous acid, formaldehyde, monoamine oxidase enzyme, and ATP.

Ratiometric Imaging of γ-Glutamyl Transpeptidase Unperturbed by pH, Polarity, and Viscosity Changes: A Benzocoumarin-Based Two-Photon Fluorescent Probe.

γ-Glutamyltransferase (GGT) is involved in maintaining the intracellular glutathione levels and, at its elevated levels, is associated with various diseases including cancer and myocardial infarction. To study this enzyme in biological systems, fluorescent probes have received significant attention recently. As fluorescence signal is sensitive to environmental fluctuations; however, it is challenging to address the signal fluctuation issue. Disclosed is the benzocoumarin-based probe that enables ratiometric imaging of GGT activity levels in cells as well as in tissues, essentially unperturbed by medium pH, viscosity, and polarity changes. Validity of the probe is demonstrated by determining the GGT activity level in HeLa cells directly through ratiometric imaging. Furthermore, the probe and its enzymatic product are two-photon absorbing, extending its applicability to tissue: an 8.5-fold higher level of GGT in cancerous tissue over the normal tissue is determined, and the GGT activity levels between different mouse organ tissues are quantitatively compared with the highest level in the kidney. The probe with practicality holds great promise for studying GGT-associated biological processes directly through ratiometric imaging by two-photon microscopy.

The Rim15-endosulfine-PP2ACdc55 signalling module regulates entry into gametogenesis and quiescence via distinct mechanisms in budding yeast.

Quiescence and gametogenesis represent two distinct survival strategies in response to nutrient starvation in budding yeast. Precisely how environmental signals are sensed by yeast cells to trigger quiescence and gametogenesis is not fully understood. A conserved signalling module consisting of Greatwall kinase, Endosulfine and Protein Phosphatase PP2ACdc55 proteins regulates entry into mitosis in Xenopus egg extracts and meiotic maturation in flies. We report here that an analogous signalling module consisting of the serine-threonine kinase Rim15, the Endosulfines Igo1 and Igo2 and the Protein Phosphatase PP2ACdc55, regulates entry into both quiescence and gametogenesis in budding yeast. PP2ACdc55 inhibits entry into gametogenesis and quiescence. Rim15 promotes entry into gametogenesis and quiescence by converting Igo1 into an inhibitor of PP2ACdc55 by phosphorylating at a conserved serine residue. Moreover, we show that the Rim15-Endosulfine-PP2ACdc55 pathway regulates entry into quiescence and gametogenesis by distinct mechanisms. In addition, we show that Igo1 and Igo2 are required for pre-meiotic autophagy but the lack of pre-meiotic autophagy is insufficient to explain the sporulation defect of igo1Δ igo2Δ cells. We propose that the Rim15-Endosulfine-PP2ACdc55 signalling module triggers entry into quiescence and gametogenesis by regulating dephosphorylation of distinct substrates.

Synthesis of a Liposomal MUC1 Glycopeptide-Based Immunotherapeutic and Evaluation of the Effect of l-Rhamnose Targeting on Cellular Immune Responses.

Generation of a CD8(+) response to extracellular antigen requires processing of the antigen by antigen presenting cells (APC) and cross-presentation to CD8(+) T cell receptors via MHC class I molecules. Cross-presentation is facilitated by efficient antigen uptake followed by immune-complex-mediated maturation of the APCs. We hypothesize that improved antigen uptake of a glycopeptide sequence containing a CD8(+) T cell epitope could be achieved by delivering it on a liposome surface decorated with an immune complex-targeting ligand, an l-Rhamnose (Rha) epitope. We synthesized a 20-amino-acid glycopeptide TSAPDT(GalNAc)RPAPGSTAPPAHGV from the variable number tandem repeat region of the tumor marker MUC1 containing an N-terminal azido moiety and a tumor-associated α-N-acetyl galactosamine (GalNAc) at the immunogenic DTR motif. The MUC1 antigen was attached to Pam3Cys, a Toll-like receptor-2 ligand via copper(I)-catalyzed azido-alkyne cycloaddition (CuAAc) chemistry. The Rha-decorated liposomal Pam3Cys-MUC1-Tn 4 vaccine was evaluated in groups of C57BL/6 mice. Some groups were previously immunized to generate anti-Rha antibodies. Anti-Rha antibody expressing mice that received the Rha liposomal vaccine showed higher cellular immunogenicity compared to the control group while maintaining a strong humoral response.