Decrease In Fluorescence Intensity Research Articles

Designing Tough Hydrogel Shells for Glucose Sensing.

Conventional hydrogel microcapsules often suffer from inadequate mechanical stability, hindering their use. Here, water-cored double-network (DN) hydrogel shells are designed, formed by polyacrylamide and calcium alginate networks using triple-emulsion templates. These DN hydrogel shells offer robust mechanical stability, optical transparency, and a precisely-defined cut-off threshold. The feasibility of this platform is demonstrated through the development of a fluorometric glucose sensor. Glucose oxidase is enclosed within the water core, while a pH-responsive fluorescent dye is incorporated into the DN shells. Glucose diffuses into the core through the DN shells, where the glucose oxidase converts glucose into gluconic acid, leading to pH reduction and a subsequent decrease in fluorescence intensity of DN shells. Additionally, the pH-sensitive colorant dissolved in the medium enables visual pH assessment. Thus, glucose levels can be determined using both fluorometric and colorimetric methods. Notably, the DN shells exhibit exceptional stability, enduring intense mechanical stress and cycles of drying and rehydration without leakage. Moreover, the DN shells act as effective barriers, safeguarding glucose oxidase against proteolysis by large disruptive proteins, like pancreatin. This versatile DN shell platform extends beyond glucose oxidase encapsulation, serving as a foundation for various capsule sensors utilizing enzymes and heterogeneous catalysts.

Cucurbit[8]uril-Based Supramolecular Probe for the Detection of 3-Nitrotyrosine in Human Serum and Plasma.

The biomarker 3-nitrotyrosine (3-NT) is widely recognized as an indicator of renal oxidative stress injury, making its detection crucial for the early identification of renal insufficiency. This study presents the design and synthesis of a tetraphenylstyrene imidazole derivative (TIPE-MI), which is utilized to create a supramolecular probe in conjunction with cucurbit[8]uril (Q[8]) through host-guest interactions. The resulting supramolecular self-assembly exhibits excellent optical properties and has been employed for the specific detection of 3-NT through fluorescence quenching. The introduction of 3-NT resulted in a decreased fluorescence intensity of the yellow fluorescent probe, which gradually transitioned from bright yellow to light yellow and then became colorless as the 3-NT concentration was increased. A portable detection platform was devised to augment the efficiency of detection. In order to facilitate biological applications, we have substantiated the probe's exceptional precision in detecting 3-NT in biological samples, encompassing human serum and plasma. The probe also exhibited negligible cytotoxicity. The accumulation of the probe in renal cells elicited a fluorescence signal, thereby indicating the prospective viability of this system for visual detection with renal cytocompatibility.

Rational design of anthocyanidins-directed near-infrared two-photon fluorescent probes.

Recently, two-photon fluorescent probes based on anthocyanidin molecules have attracted extensive attention due to their outstanding photophysical properties. However, there are only a few two-photon excited fluorescent probes that really meet the requirements of relatively long emission wavelengths (>600 nm), large two-photon absorption (TPA) cross-sections (300 GM), significant Stokes shift (>80 nm), and high fluorescence intensity. Herein, the photophysical properties of a series of anthocyanidins with the same substituents but different fluorophore skeletons are investigated in detail. Compared with b-series molecules, a-series molecules with a six-membered ring in the backbone have a slightly higher reorganization energy. This results in more energy loss upon light excitation, enabling the reaction products to detect NTR through a larger Stokes shift. More importantly, there is very little decrease in fluorescence intensity as the Stokes shift increases. These features are extremely valuable for high-resolution NTR detection. In light of this, novel 2a-n (n = 1-5) compounds are designed, which are accomplished by inhibiting the twisted intramolecular charge transfer (TICT) effect through alkyl cyclization, azetidine ring and extending π conjugation. Among them, 2a-3 gains a long emission spectrum (λem = 691.4 nm), noticeable TPA cross-section (957 GM), and large Stokes shift (110 nm), indicating that it serves as a promising candidate for two-photon fluorescent dyes. It is hoped that this work will offer some insightful theoretical direction for the development of novel high performance anthocyanin fluorescent materials.

Conformational change-based fluorometric aptasensor for sensitive cadmium(II) detection in fruits and vegetables.

Cadmium (Cd2+) is a highly toxic heavy metal that can accumulate in the human body through contaminated food and water, posing great health risks. In this study, a label-free fluorescent aptasensor based on SYBR Green I (SGI) for the rapid and sensitive detection of Cd2+ in food samples was designed. The aptasensor utilizes a Cd2+-specific aptamer (Cd-(21)) and its complementary strand (CSCd-(21)) to form a double-stranded DNA (dsDNA) structure in the absence of Cd2+. SGI intercalates into the dsDNA, resulting in a strong fluorescence signal. In the presence of Cd2+, the aptamer undergoes a conformational change, preventing the formation of dsDNA and leading to a decrease in fluorescence intensity. Under optimized conditions, the aptasensor exhibited a linear response to Cd2+ concentrations ranging from 0.11 to 157.37 ng mL-1, with a limit of detection (LOD) of 0.07 ng mL-1. The aptasensor demonstrated high specificity and was successfully applied to detect Cd2+ in fruits and vegetables, with satisfactory recovery rates (95-111%). The proposed aptasensor provides a promising tool for the rapid and sensitive detection of Cd2+ in food.

Probing cell proliferation: Considerations for dye selection.

Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.

High-throughput assay exploiting disorder-to-order conformational switches: application to the proteasomal Rpn10:E6AP complex.

Conformational switching is pervasively driven by protein interactions, particularly for intrinsically disordered binding partners. We developed a dually orthogonal fluorescence-based assay to monitor such events, exploiting environmentally sensitive fluorophores. This assay is applied to E3 ligase E6AP, as its AZUL domain induces a disorder-to-order switch in an intrinsically disordered region of the proteasome, the so-named Rpn10 AZUL-binding domain (RAZUL). By testing various fluorophores, we developed an assay appropriate for high-throughput screening of Rpn10:E6AP-disrupting ligands. We found distinct positions in RAZUL for fluorophore labeling with either acrylodan or Atto610, which had disparate spectral responses to E6AP binding. E6AP caused a hypsochromic shift with increased fluorescence of acrylodan-RAZUL while decreasing fluorescence intensity of Atto610-RAZUL. Combining RAZUL labeled with either acrylodan or Atto610 into a common sample achieved robust and orthogonal measurement of the E6AP-induced conformational switch. This approach is generally applicable to disorder-to-order (or vice versa) transitions mediated by molecular interactions.

A TURN-OFF FLUORESCENT CHEMOSENSOR FOR DETECTING FORMALDEHYDE BASED ON PYRIDINE DERIVATIVE

Formaldehyde in solution, commonly known as formalin, is often utilized. In Indonesia, there is widespread misuse of formalin as a food preservative. Formaldehyde has been identified as a carcinogenic substance by the International Agency for Research on Cancer (IARC) and the Environmental Protection Agency (EPA). Based on this background, the present study developed a sensor compound capable of detecting formaldehyde obviously. The compound 3'-(4-(3,4-dimethoxyphenyl)pyridine-2,6-diyl)dianiline (ChP-2A) has been successfully synthesized through the reduction reaction of the compound 4-(3,4-dimethoxyphenyl)-2,6-bis(3-nitrophenyl)pyridine (ChP-0A) using 80% hydrazine hydrate and 10% Pd/C as a catalyst. The ChP-2A compound in acetonitrile exhibits a significant decrease in fluorescence intensity (turn-off) after the addition of formaldehyde, and it has been successfully applied in the form of a test paper for the detection of formaldehyde qualitatively

Modern methods of the diagnostic of pathology of pregnancy and the postpartum period

Postpartum purulent-inflammatory diseases are currently a serious problem. Purpose - to develop and implement of a new modern method of the diagnosis, monitoring and assessment of health of women during pregnancy, before childbirth and in the postpartum period - the method of fluorescence spectroscopy - to ensure the provision of highly qualified medical care, including the effective treatment. Materials and methods. The research material consisted of blood serum samples of 265 patients. There were 40 pregnant women (the Group 1), 40 women with an uncomplicated postpartum period (the Group 2), 170 - with postpartum purulent-inflammatory diseases (the Group 3), 15 patients with sepsis (the Group 4). Diagnostic methods: clinical, laboratory, instrumental, method of fluorescence spectroscopy, statistical (logistic regression and ROC analysis methods). Results. In the Group 1 there is an increase in the intensity of fluorescence to 1.3 r.u. For the Group 2 the intensity of fluorescence was within 0.86-1.0 r.u. For the Group 3, the decrease in fluorescence intensity from 1 to 0.35 r.u. was recorded. For the Group 4 in addition to decreasing the fluorescence intensity, a noticeable shift of the fluorescence spectra of blood serum into the long-wave range was recorded. Conclusions. A reliable prognostic model of the development of postpartum purulent-inflammatory diseases was created. The modern approach for the diagnosis and effective control of treatment within the framework of the method of fluorescent spectroscopy and biomarkers using infusions of donor albumin solutions is proposed. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interest was declared by the authors.

Effect of dissolved ozone flotation thickening process on coliform bacteria and antibiotics simultaneous abatement: A pilot-scale study

This study focused on the removal of the total coliforms, fecal coliforms and four target antibiotics in the dissolved ozone flotation (DOF) thickening sludge process. Additionally, the thickened effluent chromaticity and its effect on thickened sludge hydrolysis process were investigated. Ozonation in the DOF process could inactivate coliforms by oxidizing cellular components and destroying genetic material, as well as altering the chemical structure of antibiotics, leading to the degradation of antibiotics. At an O3 dosage of 16 mg/g TS, the concentration of total coliforms and fecal coliforms decreased by 2.2 log and 2.4 log, corresponding to an overall removal rate of 99.4 % and 99.7 %, respectively. The total degradation rate of four target antibiotics (tetracycline (TC), oxytetracycline (OTC), norfloxacin (NOR), ofloxacin (OFL)) were 66.5 %, 68.8 %, 53.3 % and 57.5 %, respectively. The chromaticity removal rate of the thickened effluent reached 95 %. Analysis of fluorescence spectra indicated alterations in the fluorescence properties of dissolved organic matter, resulting in a decrease in fluorescence intensity by ozonation. The thickened sludge had higher hydrolysis rates, resulting in a greater production of volatile fatty acids (VFAs). This was mainly attributed to the increased amount of soluble protein and carbohydrate in the substrate after DOF treatment, which was more conducive for the rapid conversion of hydrolysis into VFAs during the initial stage. These results provided new ideas for upgrading and transforming the thickening process of wastewater treatment plants (WWTPs).

Effect of ultrasonic treatment on enzymes: Decoupling the relation between the ultrasonic driven conformational change and enzyme activity

Considering the growing interest in enzyme-based extraction technology as a safe and eco-friendly extraction technique, along with the relatively high cost associated with enzymatic applications, it became necessary to explore novel strategies aimed to improve enzyme activity. In this study, the impact of ultrasonic treatment on commercial cellulase and pectinase was investigated. As this effect may be influenced by various ultrasonic and enzyme-related parameters, changes in enzyme conformation were explored under optimal and non-optimal enzyme conditions. The intrinsic fluorescence spectrum was utilized as a tool for monitoring these changes. Additionally, the enzyme’s catalytic potential was also assessed under the same conditions. Results indicated that the impact of ultrasonic treatment on enzyme conformation primarily depends on the total ultrasonic energy delivered to the system, rather than other ultrasonic parameters such as power, sample volume, treatment time, or duty cycle. The maximum relative decrease in intrinsic fluorescence intensity of Pectinex® Ultra Clear (PUC) and Pectinex® Ultra SPL (PUS) after ultrasonic treatment was approximately 51% and 55%, respectively, while the decrease induced by thermal denaturation was 25% and 30% respectively. Furthermore, a blue shift in the fluorescence spectrum of both pectinases was observed upon sonication for all process conditions indicating a change in enzyme conformation. However, ultrasonic treatment did not result in a significant change in enzyme activity, suggesting that these conformational adjustments may occur in regions other than the active sites. Moreover, ultrasonicated pectinases and cellulases did not exhibit any improvement in their catalytic potential under either optimal or non-optimal conditions.

A Ratiometric Fluorescent Sensor Based on Chelation-Enhanced Fluorescence of Carbon Dots for Zinc Ion Detection

Zinc ion, one of the most important transition metal ions in living organisms, plays a crucial role in the homeostasis of the organism. The disorder of zinc is associated with many major diseases. It is highly desirable to develop selective and sensitive methods for the real-time detection of zinc ions. In this work, double-emitting fluorescent carbon dots (CDs) are prepared by a solvothermal method using glutathione, L-aspartic acid, and formamide as the raw materials. The carbon dots specifically recognize zine ions and produce a decrease in fluorescence intensity at 684 nm and an increase at 649 nm, leading to a ratiometric fluorescent sensor for zinc detection. Through surface modification and spectral analysis, the surface groups including carboxyl, carbonyl, hydroxyl, and amino groups, and C=N in heterocycles of CDs are revealed to synergistically coordinate Zn2+, inducing the structural changes in the emission site. The CDs can afford a low limit of detection of ~5 nM for Zn2+ detection with good linearity in the range of 0.02–5 μM, showing good selectivity as well. The results from real samples including fetal bovine serum, milk powder, and zinc gluconate oral solution indicated the good applicability of the CDs in the determination of Zn2+.

Fluorescence detection of amantadine based on competitive β-Cyclodextrin host-guest inclusion process

Cyclodextrin-based fluorescent probes are promising candidates for chemical or biological sensing due to their wonderful sensitivity and selectivity. Typically, their recognition towards special analysts ascribed from hydrophobic cavity of cyclodextrins. In this work, a cyclodextrin-based fluorescent probe (DASP-β-CD) was synthesized by covalently linking trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium (DASP) and β-cyclodextrin (β-CD). In aqueous dispersion, the DASP-β-CD molecules could form intermolecular inclusion complex which would then generate supramolecular aggregates when increasing DASP-β-CD concentration. However, after adding amantadine (AMD) to DASP-β-CD supramolecular system, AMD molecules would compete with DASP for the cavity site, leading to the destroy of DASP-β-CD intermolecular inclusion complex and disassembly of supramolecular aggregates. Moreover, this AMD-induced collapse of DASP-β-CD intermolecular inclusion complex would also result in the decrease of fluorescence intensity of DASP-β-CD. Based on this, a simple fluorescent assay for the detection of AMD was proposed. This approach provides a rapid, sensitive and selective method for AMD detection and may help designing other cyclodextrin-based sensing strategies for medical analysis and environmental monitoring.

PDT-Induced Variations of Radachlorin Fluorescence Lifetime in Living Cells In Vitro

Variations in the fluorescence lifetimes of Radachlorin photosensitizers in HeLa and A549 cells, caused by photodynamic treatment, were studied using fluorescence lifetime imaging microscopy (FLIM). An analysis of FLIM images of the cells demonstrated a substantial decrease in the mean Radachlorin fluorescence lifetime and intensity as a result of UV irradiation of the photosensitized cells at different doses, with higher doses causing a more pronounced decrease in the mean fluorescence lifetime in cells. The post-treatment decrease in Radachlorin fluorescence intensity was accompanied by the appearance of an additional rapidly decaying fluorescence component and a nonlinear decrease in the weighted fluorescence lifetime obtained from double-exponential fits of time-resolved fluorescence signals. Experiments performed in the aqueous solutions of the photosensitizer revealed similar irreversible changes in the Radachlorin fluorescence lifetime and intensity. Therefore, the observed phenomena occurred most likely due to the photodegradation of the photosensitizer molecules and can be applied for dosimetry and monitoring of irradiation doses in different areas of malignant tissues in the course of photodynamic treatment.

Humidity evolution in cement-based materials is monitored using fluorescent molecular humidity-sensitive responses

In situ detection technology for cement-based materials typically produces unclear physical meanings, which limits research on durability issues such as heat-moisture transfer, dry-wet cycles and microcrack propagation. Taking the external thermal insulation system of building exterior walls as an example, this paper explores in-situ fluorescence sensing techniques for moisture evolution of cementitious materials using the quenching effect caused by the aggregation of fluorescent molecules. This study includes the monitoring of fluorescence intensity under humidity evolution and a stability study of fluorescence intensity under different temperatures and humidity cycles. Results showed that the gradual transition of the polymeric state of fluorescent molecules led to the first increase and then decrease of fluorescence intensity with increasing humidity (0 % RH-95 % RH). The fluorescence intensity peaked at 65 % RH, decreases at high humidity due to aggregated caused quenching effects, and maintained good stability after 8 humidity cycles. With regard to ambient temperature, the fluorescence intensity decreased with increasing temperature (5 °C – 40 °C), and the fluorescence intensity peaked at 5 °C. The temperature sensitivity of the fluorescence is weakened by the methyl effect of the rhodamine 6G fluorescent molecules and the thermostatic properties of the insulation boards. Fluorescence sensing technology for humidity evolution of cement-based materials has moisture sensitivity and fluorescence stability, and thus has a wide range of applications in the sensing of humidity evolution of civil engineering materials.

Studying the optoelectronic properties and emission quenching dynamics of poly‐TPD by incorporating ZnO nanoparticles and multiwalled carbon nanotubes

AbstractIn this study, the optoelectronic properties and emission dynamic quenching of poly‐TPD were investigated upon incorporating ZnO nanoparticles and multiwalled carbon nanotubes (MWCNTs). A solution blending method was utilized to successfully prepare poly‐TPD incorporated with various contents of ZnO/MWCNT nanocomposites. The optoelectronic properties of the nanocomposites were analyzed using UV–Vis and photoluminescence spectrophotometry. The incorporation of the nanocomposites resulted in a decrease in transmittance, reflectance, and absorption edge, indicating a reduction in the poly‐TPD's bandgap. Parameters such as extinction coefficient, Urbach energy, and charge carrier density also decreased with addition of the nanocomposites, suggesting increased scattering, disorder, and the presence of defect states. The decrease in bandgap from 2.970 to 2.852 eV with increasing the nanocomposite content confirmed the emergence of new electronic states. Furthermore, the nature of the transitions changed from direct allowed for pure poly‐TPD to direct forbidden upon incorporation of the nanocomposites. The inclusion of the nanocomposites also led to a decrease in refractive index and fluorescence intensity. The observed fluorescence quenching primarily exhibited dynamic characteristics, with a Stern–Volmer constant of 5.14 L/g and quenching rate constants surpassing the minimum threshold for efficient quenching. The increase in charge transfer rate constants with the nanocomposite content indicated enhanced quenching efficiency and charge transfer, likely due to the increased surface area and presence of defects. These findings suggest that poly‐TPD incorporated with ZnO/MWCNT nanocomposites displays promising properties for applications in optoelectronic devices.

Effect and mechanism of Danggui Buxue Decoction-containing serum in mitigating H9c2 cell injury caused by exposure to intermittent low oxygen

This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.

Analysis of Microcystis aeruginosa physiology by spectral flow cytometry: Impact of chemical and light exposure.

M. aeruginosa fluorescent changes were observed using a Cytek Aurora spectral flow cytometer that contains 5 lasers and 64 narrow band detectors located between 365 and 829 nm. Cyanobacteria were treated with different concentrations of H2O2 and then monitored after exposure between 1 and 8 days. The red fluorescence emission derived from the excitation of cyanobacteria with a yellow green laser (550 nm) was measured in the 652-669 nm detector while green fluorescence from excitation with a violet laser (405 nm) was measured in the 532-550 nm detector. The changes in these parameters were measured after the addition of H2O2. There was an initial increase in red fluorescence intensity at 24 hours. This was followed by a daily decrease in red fluorescence intensity. In contrast, green fluorescence increased at 24 hours and remained higher than the control for the duration of the 8-day study. A similar fluorescence intensity effect as H2O2 on M. aeruginosa fluorescence emissions was observed after exposure to acetylacetone, diuron (DCMU), peracetic acid, and tryptoline. Minimal growth was also observed in H2O2 treated cyanobacteria during exposure of H2O2 for 24 days. In another experiment, H2O2-treated cyanobacteria were exposed to high-intensity blue (14 mW) and UV (1 mW) lights to assess the effects of light stress on fluorescence emissions. The combination of blue and UV light with H2O2 had a synergistic effect on M. aeruginosa that induced greater fluorescent differences between control and treated samples than exposure to either stimulus individually. These experiments suggest that the early increase in red and green fluorescence may be due to an inhibition in the ability of photosynthesis to process photons. Further research into the mechanisms driving these increases in fluorescence is necessary.

Investigating two structurally different penta-coordinated trigonal bipyramidal Co(II) non-symmetric salamo-type complexes

A new non-symmetric salamo-type multidentate chelating ligand H2L (4-Formyl-6ʹ-methoxyl-2,2ʹ-[ethylenedioxybis(nitrilomethylidyne)]diphenol) was designed and prepared. Two Co(II) complexes with different structures, [Co(L)(H2O)] (1) and [Co2(L)2]2·3CH3CN·2CH3OH (2) were prepared by the reactions of H2L with two different Co(II) salts (Co(OAc)2·4H2O/CoCl2·6H2O) in various organic solvents at an ambient temperature, respectively. Due to varying degrees of distortion of the ligand during the coordination reactions, the bonding modes of the ligand in complexes 1 and 2 are different. Complex 1 forms a penta-coordinated mononuclear, and complex 2 forms a penta-coordinated binuclear structure. By comparing UV–Vis and infrared spectroscopy of H2L with those of the corresponding complexes further characterize coordination between the ligand H2L and Co(II) ions. Theoretical calculations of DFT, BVS (Valence Bonding and Calculation), and IRI (Interaction Region Indicator) were used to show the different forces present in complexes 1 and 2, and BVS calculations confirmed that Co is ‘+2′ valence in the complexes. Fluorescence property studies indicated that the possible mechanism for the decrease in fluorescence intensity upon binding of H2L to Co(II) ions is chelation fluorescence quenching.

Silencing essential gene expression in Mycobacterium abscessus during infection.

Mycobacterium abscessus is a multi-drug-resistant non-tuberculous mycobacterial species causing tuberculosis-like lung infections. The functional characterization of the extensive repertoire of genes encoding virulence factors and drug targets has been largely restricted by the lack of powerful genetic tools. In this study, we evaluated the performances of a Tet-OFF system, previously optimized for M. tuberculosis and M. smegmatis. Fluorescence reporter M. abscessus strains were developed where mCherry was under the control of the Tet-OFF regulatory system on an integrative plasmid. The addition of anhydrotetracycline (ATc) correlated with a decrease in fluorescence intensity not only in solid and broth medium but also in colony biofilms, for both smooth and rough variants of M. abscessus. Next, unmarked mmpL3 conditional knockdown mutants were engineered in both smooth and rough M. abscessus. Biochemical studies indicated that the addition of ATc was associated with a dose-dependent decrease in (i) the production of the MmpL3 protein, (ii) the biosynthesis of trehalose dimycolate and mycolylated arabinogalactan, (iii) bacterial viability on agar and liquid medium as well as in biofilms. Importantly, intravenous injection of ATc in zebrafish embryos infected with the rough mmpL3 conditional mutant impeded bacterial growth and correlated with a significant gain in embryo survival. These results suggest that mmpL3 is essential for M. abscessus growth in vitro and in the infected host, thus validating MmpL3 as an attractive drug target. Together, this study demonstrates the potential of ATc-dependent repression to modulate the expression of M. abscessus genes in the infected host. IMPORTANCE Mycobacterium abscessus represents the most common rapidly growing mycobacterial pathogen in cystic fibrosis and is extremely difficult to eradicate. Essential genes are required for growth, often participate in pathogenesis, and encode valid drug targets for further chemotherapeutic developments. However, assessing the function of essential genes in M. abscessus remains challenging due to the limited spectrum of efficient genetic tools. Herein, we generated a Tet-OFF-based system allowing to knock down the expression of mmpL3, encoding the mycolic acid transporter in mycobacteria. Using this conditional mutant, we confirm the essentiality of mmpL3 in planktonic cultures, in biofilms, and during infection in zebrafish embryos. Thus, in this study, we developed a robust and reliable method to silence the expression of any M. abscessus gene during host infection.

MicroRNA-30a inhibits cell proliferation in a sepsis-induced acute kidney injury model by targeting the YAP-TEAD complex

BackgroundAcute kidney injury (AKI) is a primary feature of renal complications in patients with sepsis. MicroRNA (miRNA/miR)-30a is an essential regulator of cardiovascular diseases, tumors, phagocytosis, and other physical processes, but whether it participates in sepsis-induced AKI (sepsis-AKI) is unknown. We aimed to elucidate the functions and molecular mechanism underlying miR-30a activity in sepsis-AKI. MethodsThe classical cecal ligation and puncture (CLP) method and lipopolysaccharide (LPS)-induced Human Kidney 2 (HK-2) cells were used to establish in vivo and in vitro sepsis-AKI models. Specific pathogen-free and mature male Sprague-Dawley (SD) rats, aged 6–8 weeks (weight 200–250 g), were randomly divided into five-time phase subgroups. Fluid resuscitation with 30 mL/kg 37 °C saline was administered after the operation, without antibiotics. Formalin-fixed, paraffin-embedded kidney sections were stained with hematoxylin and eosin. SD rat kidney tissue samples were collected for analysis by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. HK-2 cells were transfected with hsa-miR-30a-3p mimics or inhibitors, and compared with untreated normal controls. RNA, protein, and cell viability were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot, and cell counting kit-8 methods. A Dual-Luciferase Assay Kit (Promega) was used to measure luciferase activity 48 h after transfection with miR-30a-3p mimics. ResultsExpression levels of miR-30a-3p and miR-30a-5p in renal tissues of the sepsis group were significantly reduced at 12 h and 24 h (P <0.05). Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly increased in renal tissue 3 h after the operation in rats (P <0.05), and gradually decreased 6 h, 12 h, and 24 h after CLP. Levels of miR-30a-5p and miR-30a-3p were significantly down-regulated at 3 h after LPS treatment (P <0.05), and gradually decreased in HK-2 cells. One hour after LPS (10 µg/mL) treatment, TNF-α and IL-1β levels in HK-2 cells were significantly up-regulated (P < 0.05), and they were markedly down-regulated after 3 h (P <0.05). IL-6 expression levels began to rise after LPS treatment of cells, peaked at 6 h (P <0.05), and then decreased to the initial level within a few hours. Stimulation with 10 µg/mL LPS promoted HK-2 cells proliferation, which was inhibited after miR-30a-3p-mimic transfection. Bioinformatics prediction identified 37 potential miR-30a-3p target genes, including transcriptional enhanced associate domain 1 (TEAD1). After transfection of HK-2 cells with miR-30a-3p mimics and miR-30a-3p inhibitor, TEAD1 transcript was significantly up- and down-regulated, respectively (both P <0.05). After LPS treatment (24 h), expression of TEAD1 in the inhibitors group was significantly increased (P <0.01), while that in the mimics group was significantly suppressed (P <0.01). In the dual luciferase reporter experiment, miR-30a-3p overexpression decreased fluorescence intensity (P <0.01) from TEAD1-wt-containing plasmids, but did not influence fluorescence intensity from TEAD1-muta-containing plasmids. LPS may promote HK-2 cells proliferation through the miR-30a-3p/TEAD1 pathway. ConclusionIn a background of expression of inflammatory factors, including TNF-α, IL-1β, and IL-6, which were transiently increased in the sepsis-AKI model, miR-30a was down-regulated. Down-regulated miR-30a-3p may promote cell proliferation by targeting TEAD1 in LPS-induced HK-2 cells, demonstrating its potential as a biomarker for early sepsis-AKI diagnosis.