HEPES Buffer Research Articles

Synthesis, structural characterization and antitrypanosomal activity of ferrocenyl-based imines with (2 or 4)-nitrophenyl-2-furan group

Two new metallocene complexes with formulation [Fe(η5-C5H5){(η5-C5H4)-NCH-2-C4H2O-(5-C6H4-2-NO2)}] (3) and [Fe(η5-C5H5){(η5-C5H4)-NCH-2-C4H2O-(5-C6H4-4-NO2)}] (4) have been synthesized and their antitrypanosomal activity has been evaluated. Complexes 3 and 4 were obtained by condensation reaction of ferrocenylamine (1) with respective 5-(2-nitrophenyl)-2-furancarboxaldehyde (2a) or 5-(4-nitrophenyl)-2-furancarboxaldehyde (2b). The complexes were fully characterized by spectroscopic techniques (FT-IR, 1H and 13C{1H} NMR, and HRMS), which confirm their correct obtainment. The crystal structures of 3 and 4 were also determined by single crystal X-ray diffraction. Furthermore, UV–visible studies revealed that compounds 3 and 4 exhibited suitable stability in DMSO:HEPES buffer solution (80:20) throughout 24 h. The in vitro antiparasitic activities of ferrocenyl imine derivatives 3 and 4 were assessed against two species of parasites, Trypanosoma cruzi (T. cruzi) and Trypanosoma brucei (T. brucei). The obtained results revealed that compound 3 (EC50 = 0.44 μM) exhibited nearly a 40-fold greater efficacy as an anti-T. brucei agent compared to derivative 4 (EC50 = 16.0 μM). Moreover, compound 3 demonstrated superior potency when compared with its organometallic analogs (EC50 = 2.42–13.3 μM), which were previously reported by our research group. Interestingly, the EC50 value of 3 was found to be 8 times greater than that of nifurtimox (EC50 = 3.56 μM). The cytotoxicity of the ferrocenyl imines was also evaluated on the L6 rat skeletal myoblast cell line.

Transient square pulse-electric field induction of monosex-male reversal of the eggs of Nile tilapia (Oreochromis niloticus L., Chitralada)

We present an alternative technique of monosex-male reversal for Nile tilapia using transient square pulse-electric field inductions to enhance sex reversal rate of tilapia eggs by methyltestosterone (Oreochromis niloticus L., Chitralada). Male tilapia is preferred for aquaculture because of better size and growth rate. The egg membrane was permeabilised by an electric field pulse allowing a minimalized androgen hormone dose of 17alpha-methyltestosterone (MT) (μg l−1 concentration) to achieve monosex sex-male reversal. The technique achieved large-scale inductions of 2000 eggs rather than for individual eggs and was aimed at commercialization (Thailand petty patent no. 13516). The eggs of O. niloticus obtained from our parent breeding stocks were carefully selected to be in the segmentation-pharyngula stage (2–3 dpf - day post fertilization) for electrical inductions in a pulsed-electric field generated between narrow parallel-plate electrodes. The electrodes were powered with an adjustable signal pulse-generator (SPG) developed for on-site operations. The induction medium was prepared using HEPES buffer mixed with a minimized concentration of MT. We experimentally optimized inductions by adjusting the number of square wave pulses, pulse amplitude, pulse durations (period) and a mark-space ratio (pulse width: negative half of period) for the optimum results. A dose of 350 V (electric field intensities of 87.50 kV m−1) of 3 square pulses with 100 μs pulse-period (50:50 mark space ratio) gave the maximum rate of male-sex reversal rate (MSR) of 89.42 ± 2.28 (SD)%, hatching rate (HR) of 93.33 ± 0.58% and survival rate (SR) of 87.49 ± 1.71%, respectively. The off-spring rate (OSR) or sex ratio of the controls were 59.71 ± 3.00% male and 46.33 ± 1.53% female from a hatching rate (HR) of 91.33 ± 1.53% and a survival rate (SR) of 85.08 ± 4.16%, respectively.

An Imidazo[1, 2-a]Pyridine Based Fluorescent Probe for the Continuous "on-off-on" Detection of Fe3+ and F.

An "on-off-on" fluorescent probe LK was synthesized from 2-benzoylpyridine and o-vanillin, which could sequentially detect Fe3+ and F- in DMSO/H2O solutions (v/v = 1/1, HEPES buffer, 1.0 mM, pH 7.0) with large Stokes shift (178nm). LK exhibited not only high selectivity and sensitivity towards Fe3+ and F-, but also strong anti-interference ability to other ions. LK was coordinated with Fe3+ at a ratio of 2:1, with a binding constant (Ka) of 1.3 × 104 M- 1. The detection limits for Fe3+ and F- were 6.9 × 10- 8 M and 3.0 × 10- 7 M, respectively. Due to its excellent sensing performance, LK was successfully applied in actual water samples and test papers for the detection of Fe3+ and F-.

Developing new Hydrazine Carbothioamide Molecules for Selective Colorimetric/Fluorometric Detection of Environmentally Essential Hg2+ and Ag+ Metal Ions in Mixed Aqueous Media.

A novel colorimetric and fluorogenic probe L based on hydrazine carbothioamide and 1,8-naphthalimide moieties has been designed and synthesized for the hypersensitive detection of Hg2+ or Ag+ ions. The observed probe L showed colorimetric and fluorometric responses for these studies when Hg2+ or Ag+ was added to the DMSO - HEPES buffer solution (pH = 7). An interference test with other metal ions was determined, and the high selectivity of Hg2+ and Ag+ did not interfere with other metal ions in colorimetric and fluorogenic methods. The possible mechanism of binding of these metal ions and the probe L 1:1 complex was determined by H1 NMR. Additionally, the reversibility of the affinity of probe L with mercury (Hg2+) and silver (Ag+) ions was investigated by adding Na2EDTA. The naked eye detected the "Off-On" type fluorescence sensor in the presence of Hg2+ and EDTA. The tested test strip kits provided a strong probability of probe L with high response and rapid, sensitive detection with Hg2+ ion, which may be suitable for practical use.

Contribution Of TRPV4 Channels To Calcium Signaling Profiles Of The Lung Microvascular Endothelium Under Acidic pH

Maintenance of lung endothelial barrier integrity is achieved to a great extent through effcient homeostatic endothelial calcium signaling in the lung microvasculature. The transient receptor potential vanilloid 4 (TRPV4), a non-selective calcium permeable channel has been implicated in several pulmonary pathologies involving lung barrier disruption. These channels are activated by various physical forces and chemical stimuli, but their underlying contribution to inherent and distinct microvascular endothelial Ca2+ signaling profiles are unknown. We previously assessed the role of TRPV4 channels in basal Ca2+ signaling dynamics of the pulmonary microcirculation under differing conditions of stretch and temperature. In the current study, we evaluate the impact of acidic pH (7.1, resembling acidosis) on the distinct endothelial Ca2+ signaling events in the mouse lung microcirculation and examine the participation of TRPV4 channels. Lung slices isolated from the Cdh5-GCaMP8 mice (transgenic mice with Ca2+-dependent fluorescent indicator expressed specifically within vascular endothelial cells) were mounted on Sylgard inserts with HEPES buffer (pH 7.45) and imaged for their basal signaling events using spinning disk confocal microscopy. After an initial baseline recording, the bathing solution was replaced with acidic HEPES buffer (pH 7.1) and recordings were made for approximately two minutes at 5 frames per second. These slices were then treated with the TRPV4 channel inhibitor GSK2193874 (3μM) or vehicle. The recorded image sequences were analyzed by using ImageJ and our custom signal tracking software S8, for quantification of the Ca2+ signaling profiles through event frequency, amplitude, duration and spread. Subjecting the lung slices to pH 7.1 increased the amplitude and spatial spread of the signaling events but the duration and frequency of the events remained relatively constant when compared to baseline recorded events. Addition of the channel blocker diminished the signaling event frequency, amplitude and spatial spread of the lung tissues. The normal pH and vehicle control for the above experiments did not show any relative change from the baseline signaling event profiles. To summarize, treating the lung slices to acidic pH increased the event amplitude and event spatial area distributions, indicating widespread activation of Ca2+ entry events. These increased event parameters were reduced upon addition of the antagonist, suggesting higher contribution of TRPV4 channel induced Ca2+ signaling events under acidic pH. We conclude that the channels contribute minimally to basal dynamics under homeostatic conditions but are activated to a greater extent under stimulations resembling pathological conditions like acidosis or acid aspirations. NIH P01 HL066299, R01 HL155288, NIH S10 0D020149. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2096: Role Of Divalent Cations In The Enzymatic Activation Of Coagulation Factor XII By Lipopolysaccharides

Background: Lipopolysaccharide (LPS) is the primary pathogenic factor in Gram-negative sepsis. LPS is a polyanionic molecule that may act as a site of initiation of coagulation by activating factor (F)XII. While the physicochemical properties of LPS are known to be sensitive to interactions with divalent cations, it is unknown whether divalent cations modulate the activation of FXII by LPS. Aims: To determine whether divalent cations regulate the kinetics of FXII activation by select LPS chemotypes from E. coli . Methods: Aggregates of E. Coli LPS chemotypes O26:B6 and Rd2 were prepared in ultrapure water in the presence or absence of calcium (Ca 2+ ). The biophysical characterization of LPS chemotypes were determined by dynamic light scattering (DLS) and measurement of zeta potential (ZP). Chromogenic substrate assays were used to study the effects of LPS on FXII activation. Results: The LPS O26:B6 and Rd2 in the absence of Ca 2+ produced aggregates in form of micelles with hydrodynamic diameters of 190±10 and 200±7 nm and ZP of -25±0.8 and -60±2 mV, respectively. The presence of Ca 2+ produced aggregates in the form of vesicles with hydrodynamic diameters of 400±15 and 1300±60 nm and ZP of –11±2 and –3±0.5 mV, respectively. FXII was incubated in HEPES buffer with the distinct LPS morphologies in varying salt concentrations. We found maximal autoactivation of FXII by O26:B6 and Rd2 chemotypes at lower NaCl concentrations. The presence of Ca 2+ , however, selectively reduced the ability of O26:B6 to activate FXII. Strikingly, the neutralization of the surface net charge of the LPS chemotype Rd2 by Ca 2+ ions abrogated the amidolytic activity of Rd2. Conclusions: The presence of calcium ions induces changes in the physicochemical properties of LPS that result in distinct effects on FXII activation in vitro . We are currently studying whether this phenomena is conserved for other divalent cations including Zn, Mg, Cu, and Mn.

Adenosine washing improves the retention rate of fat grafts under conditions of obesity

Background Fat grafting is a commonly employed aesthetic procedure for contour enhancement. However, outcome prediction is challenging due to the complex regeneration and remodeling processes involved. We investigated whether adenosine improves engraftment and fat graft survival under conditions of obesity.Methods Fat was harvested from mice fed a high-fat diet. This fat was washed with either Krebs-Ringer bicarbonate HEPES buffer (the vehicle group) or a buffer containing 500 nM adenosine (the adenosine wash group). Subsequently, the fat was transplanted into normal mice at 0.2 mL per mouse. In both groups, 50% of the mice were sacrificed at 1 week and the remainder at 4 weeks post-transplantation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was conducted during week 1. In week 4, micro-computed tomography, immunofluorescence staining, and RT-qPCR were performed. A sample of the initially harvested fat was set aside for lipolysis assay.Results Adenosine washing improved fat graft retention volumes by up to 50%. One week post-transplantation, the expression of adipogenic and angiogenic genes was found to be upregulated in the adenosine wash group. After 4 weeks, immunofluorescence staining revealed greater adipocyte integrity and an increased number of vessels. Furthermore, adenosine appeared to modulate inflammation by stabilizing the lipolysis rate.Conclusions Adenosine washing increased the fat graft survival rate under conditions of obesity. Clinically, this suggests a simple, cost-effective adjuvant method for improving fat graft survival in individuals with obesity. Further research is warranted to elucidate the underlying mechanisms and explore the applicability of this technique for autologous transplantation.

Biosynthetic Pathways of Tryptophan Metabolites in Saccharomyces cerevisiae Strain: Insights and Implications.

Tryptophan metabolites, such as 5-hydroxytryptophan (5-HTP), serotonin, and melatonin, hold significant promise as supplements for managing various mood-related disorders, including depression and insomnia. However, their chemical production via chemical synthesis and phytochemical extraction presents drawbacks, such as the generation of toxic byproducts and low yields. In this study, we explore an alternative approach utilizing S. cerevisiae STG S101 for biosynthesis. Through a series of eleven experiments employing different combinations of tryptophan supplementation, Tween 20, and HEPES buffer, we investigated the production of these indolamines. The tryptophan metabolites were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Notably, setups replacing peptone in the YPD media with tryptophan (Run 3) and incorporating tryptophan along with 25 mM HEPES buffer (Run 4) demonstrated successful biosynthesis of 5-HTP and serotonin. The highest 5-HTP and serotonin concentrations were 58.9 ± 16.0 mg L-1 and 0.0650 ± 0.00211 mg L-1, respectively. Melatonin concentrations were undetected in all the setups. These findings underscore the potential of using probiotic yeast strains as a safer and conceivably more cost-effective alternative for indolamine synthesis. The utilization of probiotic strains presents a promising avenue, potentially offering scalability, sustainability, reduced environmental impact, and feasibility for large-scale production.

Conformational Variations of Human C1q in Different Buffers

C1q is a primary activator of Complement system and has a physiological role in maintaining the homeostasis. C1q is now acknowledged as a protein of complex functionality, capable of forming multiple binding sites for ligands of various nature by its C-terminal globular domains (gC1q), composed of the globular fragments of the three types of chains, ghA, ghB and ghC. The conformation of the globular fragments is critical for the formation of different binding sites underlying the functional activity of C1q. We studied the conformational flexibility of C1q and its globular fragments ghA, ghB and ghC in different routinely used buffers by the highly sensitive fluorescent spectroscopy. We compared the conformational state of C1q in PBS, pH 7.2; TBS, pH 7.2; carbonate buffer, pH 9.6; NaHCO3, pH 7.2 and HEPES, pH 7.2. HEPES buffer appeared most favourable solute in terms of conformational stability of C1q. The ghB of all three globular fragments followed most closely the native protein.

Customizable Porphyrin Platform Enables Folate Receptor PET Imaging Using Copper-64.

Folate receptors including folate receptor α (FRα) are overexpressed in up to 90% of ovarian cancers. Ovarian cancers overexpressing FRα often exhibit high degrees of drug resistance and poor outcomes. A porphyrin chassis has been developed that is readily customizable according to the desired targeting properties. Thus, compound O5 includes a free base porphyrin, two water-solubilizing groups that project above and below the macrocycle plane, and a folate targeting moiety. Compound O5 was synthesized (>95% purity) and exhibited aqueous solubility of at least 0.48 mM (1 mg/mL). Radiolabeling of O5 with 64Cu in HEPES buffer at 37 °C gave a molar activity of 1000 μCi/μg (88 MBq/nmol). [64Cu]Cu-O5 was stable in human serum for 24 h. Cell uptake studies showed 535 ± 12% bound/mg [64Cu]Cu-O5 in FRα-positive IGROV1 cells when incubated at 0.04 nM. Subcellular fractionation showed that most radioactivity was associated with the cytoplasmic (39.4 ± 2.7%) and chromatin-bound nuclear (53.0 ± 4.2%) fractions. In mice bearing IGROV1 xenografts, PET imaging studies showed clear tumor uptake of [64Cu]Cu-O5 from 1 to 24 h post injection with a low degree of liver uptake. The tumor standardized uptake value at 24 h post injection was 0.34 ± 0.16 versus 0.06 ± 0.07 in the blocking group. In summary, [64Cu]Cu-O5 was synthesized at high molar activity, was stable in serum, exhibited high binding to FRα-overexpressing cells with high nuclear translocation, and gave uptake that was clearly visible in mouse tumor xenografts.

Dansyl-modified tryptophan fluorescence sensor for highly sensitive and selective detection of Hg2+ in aqueous solutions

A new fluorescent sensor of dansylated tryptophan (DW) was synthesized using solid phase peptide synthesis (SPPS). Upon the addition of different metal ions, only the presence of Hg2+ induced an increase in DW fluorescence intensity with a blue shift from 550 to 496 nm. DW was highly sensitive and selective for detecting Hg2+ with a low detection limit of 6.53 nM in 20 mM HEPES buffer (pH 7.4). The turn-on response could be attributed to the combination of chelation enhanced fluorescence (CHEF) and solvatochromic effects. The binding constant of DW with Hg2+ ion was determined to be 9.4 × 104 M−1/2 with a stoichiometric ratio of 2: 1. The DW + Hg2+ complex can be reversed with EDTA and reused with excellent circulation effect. The fluorescence lifetime and absolute quantum yield of DW were studied before and after the addition of Hg2+. Further characterizations of the interaction between DW and Hg2+ were conducted by IR and NMR.

A thiourea dihydropyridine as a highly selective and sensitive “turn-on” fluorescent chemosensor for mercury(II) ion in water and its application for cell imaging

The existence of extremely toxic mercury contaminants in the environment, even at very low concentrations, poses severe human health issues. Mercury exposure may lead to various diseases, such as prenatal brain damage, cognitive severe, motion disorders, and Minamata diseases. Hence, developing an effective and selective method to detect Hg2+ in the environment and biological samples is of great interest. Amongst various Hg2+ detection techniques, fluorescent chemosensors offer more convenient procedures, affordable cost, high selectivity, and the capability to perform cell imaging for Hg2+ detection. Herein, a novel "turn-on" fluorescent chemosensor, Thiourea-DHP derivative (TUD), highly selective to Hg2+, was successfully developed. TUD was synthesized by cyclotrimerization of β-amino acrylate, followed by the thioacylation of the resulting dihydropyridine (DHP) with dimethylamino thiocarbonyl chloride. The Hg2+ detection by TUD displayed an astonishing naked-eye blue fluorescence signal under blacklight, exhibiting an LOD of 69 nM or 14 ppb at pH 7.4 in the HEPES buffer. Hg2+-induced hydrolytic desulfurization of the low fluorescent TUD to produce the strongly fluorescent urea-DHP (UD) is proposed as a mechanism for this fluorescence enhancement. The formation of UD was confirmed by 1H NMR, 13C NMR, and HRMS. This detecting system of TUD provided excellent percentage recovery (>97 %) for the analysis of Hg2+ contaminants in real-environmental water samples. Besides, the TUD probe showed nontoxic properties in living cells towards RAW264.7 murine macrophage cell line even at high concentrations (100 µM). Our proposed TUD probe demonstrated superior sensitivity in pure aqueous media compared to current mercury-desulfurization fluorescent chemosensors. This sensing system proved effective for analysing Hg2+ contaminants in real-environmental water samples. Additionally, the nontoxic TUD probe successfully revealed the remarkable biocompatibility for Hg2+ detection in living cells.

Nanoscale engineering of gold nanostars for enhanced photoacoustic imaging.

Photoacoustic (PA) imaging is a diagnostic modality that combines the high contrast resolution of optical imaging with the high tissue penetration of ultrasound. While certain endogenous chromophores can be visualized via PA imaging, many diagnostic assessments require the administration of external probes. Anisotropic gold nanoparticles are particularly valued as contrast agents, since they produce strong PA signals and do not photobleach. However, the synthesis of anisotropic nanoparticles typically requires cytotoxic reagents, which can hinder their biological application. In this work, we developed new PA probes based on nanostar cores and polymeric shells. These AuNS were obtained through one-pot synthesis with biocompatible Good's buffers, and were subsequently functionalized with polyethylene glycol, chitosan or melanin, three coatings widely used in (pre)clinical research. Notably, the structural features of the nanostar cores strongly affected the PA signal. For instance, despite displaying similar sizes (i.e. 45nm), AuNS obtained with MOPS buffer generated between 2 and 3-fold greater signal intensities in the region between 700 and 800nm than nanostars obtained with HEPES and EPPS buffers, and up to 25-fold stronger signals than spherical gold nanoparticles. A point source analytical model demonstrated that AuNS synthesized with MOPS displayed greater absorption coefficients than the other particles, corroborating the stronger PA responses. Furthermore, the AuNS shell not only improved the biocompatibility of the nanoconstructs but also affected their performance, with melanin coating enhancing the signal more than 4-fold, due to its own PA capacity, as demonstrated by both in vitro and ex vivo imaging. Taken together, these results highlight the strengths of gold nanoconstructs as PA probes and offer insights into the design rules for the nanoengineering of new nanodiagnostic agents.

Evaluation of acute intravenous toxicity of HEPES: Is Good's buffer good and safe enough for clinical utilization in nuclear medicine?

ObjectiveGood's buffer or HEPES has advantages over other buffers commonly used in radiopharmaceutical preparation as it exhibits significantly lower complexation tendency with metal ions. However, use of HEPES buffer for radiolabeling reactions, meant for clinical applications, has been underrated due to the non-availability of sufficient toxicity data. The objective of the present study is to find the evidences towards safety of intravenous administration of HEPES through systemic toxicological studies in small animal model to support its safe application for clinical exploitation. ExperimentalA pilot study was performed to investigate the lethal dose of HEPES in female Sprague Dawley rats by administering seven different doses of HEPES solution (150 to 2000 mg/kg), through intravenous pathway. Similarly, for determining maximum tolerated dose (MTD), gradually increasing doses of HEPES (50 to 950 mg/kg) were administered in the same species via similar pathway. Various hematological and clinical pathological investigations were carried out in order to find out the safe administration dose of HEPES in rats. ResultsNo mortality was observed up to 2000 mg/kg doses of HEPES. The doses beyond 300 mg/kg resulted few temporary adverse effects, though these were found to disappear within 4–5 days of dosing. ConclusionThe amount of HEPES to be administered during clinical intervention is usually much lower (typically 1–2.5 mg per kg of body weight of healthy adult) than the MTD determined in rat model during present report. Hence, the utilization of this buffer for preparation of radiolabeled drugs for human investigation may be safe. However, further detailed investigations may be warranted for supporting the candidature of Good's buffer for regular clinical exploitation.

Structure of a novel form of phosphopantetheine adenylyltransferase from Klebsiella pneumoniae at 2.59Å resolution.

Phosphopantetheine adenylyltransferase (EC. 2.7.7.3, PPAT) catalyzes the penultimate step of the multistep reaction in the coenzyme A (CoA) biosynthesis pathway. In this step, an adenylyl group from adenosine triphosphate (ATP) is transferred to 4'-phosphopantetheine (PNS) yielding 3'-dephospho-coenzyme A (dpCoA) and pyrophosphate (PPi). PPAT from strain C3 of Klebsiella pneumoniae (KpPPAT) was cloned, expressed and purified. It was crystallized using 0.1M HEPES buffer and PEG10000 at pH 7.5. The crystals belonged to tetragonal space group P41212 with cell dimensions of a = b = 72.82Å and c = 200.37Å. The structure was determined using the molecular replacement method and refined to values of 0.208 and 0.255 for Rcryst and Rfree factors, respectively. The structure determination showed the presence of three crystallographically independent molecules A, B and C in the asymmetric unit. The molecules A and B are observed in the form of a dimer in the asymmetric unit while molecule C belongs to the second dimer whose partner is related by crystallographic twofold symmetry. The polypeptide chain of KpPPAT folds into a β/α structure. The conformations of the side chains of several residues in the substrate binding site in KpPPAT are significantly different from those reported in other PPATs. As a result, the modes of binding of substrates, phosphopantetheine (PNS) and adenosine triphosphate (ATP) differ considerably. The binding studies using fluorescence spectroscopy indicated a KD value of 3.45 × 10-4M for ATP which is significantly lower than the corresponding values reported for PPAT from other species.

Temperature Control of the Self-Assembly Process of 4-Aminoquinoline Amphiphile: Selective Construction of Perforated Vesicles and Nanofibers, and Structural Restoration Capability.

The construction of diverse and distinctive self-assembled structures in water, based on the control of the self-assembly processes of artificial small molecules, has received considerable attention in supramolecular chemistry. Cage-like perforated vesicles are distinctive and interesting self-assembled structures. However, the development of self-assembling molecules that can easily form perforated vesicles remains challenging. This paper reports a lower critical solution temperature (LCST) behavior-triggered self-assembly property of a 4-aminoquinoline (4-AQ)-based amphiphile with a tetra(ethylene glycol) chain, in HEPES buffer (pH 7.4). This property allows to form perforated vesicles after heating at 80 °C (> LCST). The self-assembly process of the 4-AQ amphiphile can be controlled by heating at 80 °C (> LCST) or 60 °C (< LCST). After cooling to room temperature, the selective construction of the perforated vesicles and nanofibers was achieved from the same 4-AQ amphiphile. Furthermore, the perforated vesicles exhibited slow morphological transformation into intertwined-like nanofibers but were easily restored by brief heating above the LCST.

Influx of zwitterionic buffer after intracytoplasmic sperm injection (ICSI) membrane piercing alters the transcriptome of human oocytes.

Does piercing oocyte membranes during ICSI allow the influx of surrounding zwitterionic buffer into human oocytes and result in altered developmental competence? Human oocytes directed to IRB-approved research were used to determine the unrestricted influx of surrounding buffer into the oocyte after piercing of membranes via confocal fluorescence microscopy (n = 80 human MII oocytes) and the influence of the select buffer influx of HEPES, MOPS, and bicarbonate buffer on the oocyte transcriptome using ultra-low input RNA sequencing (n = 40 human MII oocytes). Piercing membranes of human MII oocytes during sham-ICSI resulted in the unrestricted influx of surrounding culture buffer into the oocyte that was beyond technician control. Transcriptome analysis revealed statistically significant decreased cytoskeletal transcripts in the pierced buffer cohorts, higher levels of embryo competency transcripts (IGF2 and G6PD) in the bicarbonate buffer cohort, higher levels of stress-induced transcriptional repressor transcripts (MAF1) in the HEPES and MOPS cohorts, and decreased levels of numerous chromosomal maintenance transcripts (SMC3) in the HEPES buffer cohort. The HEPES buffer cohort also revealed higher levels of transcripts suggesting increased oxidative (GPX1) and lysosomal stress (LAMP1). The influence of zwitterionic buffer on intrinsic cellular mechanisms provides numerous concerns for their use in IVF clinical applications. The primary concern is the ICSI procedure, in which the surrounding buffer is allowed influx into the oocytes after membrane piercing.Selecting a physiological bicarbonate buffer may reduce imposed stress on oocytes, resulting in improved embryo development and clinical results because intracellular MOPS, and especially HEPES, may negatively impact intrinsic biological mechanisms, as revealed by transcriptome changes. These findings further support the utilization of bicarbonate buffer as the oocyte-holding medium during ICSI.

Highly selective and sensitive rhodamine based chemosensors for Al3+ and their applications in living cell imaging

To address limitations like susceptibility to hydrolysis of an imine bond and low selectivity of rhodamine based chemosensors, we report here two rhodamine dyes, namely, L-Et i.e. 3′,6′-bis(ethylamino)-2′,7′-dimethyl-2-(2-morpholinoethyl)spiro[isoindoline-1,9′-xanthen]-3-one and L-Pr i.e. 3′,6′-bis(ethylamino)-2′,7′-dimethyl-2-(3-morpholinopropyl)spiro[isoindoline-1,9′-xanthen]-3-one for detection of Al3+ in 10 mM HEPES buffer in H2O/EtOH = 1:9 (v/v) (pH 7.4). Both the probes detect exclusively Al3+ among 16 different metal ions. The sensing ability has been achieved by noting color change (colorless to pink), increment in absorption intensity at 530 nm and fluorescence intensity at 555 nm. Opening of spirolactam ring of the chemosensors followed by complexation with Al3+ is responsible for such changes. A 1:1 complex is formed by both the probes with high association constant (1.14 × 105 M−1 for L-Et and 1.17 × 105 M−1 for L-Pr). The limit of detection values of L-Et and L-Pr towards Al3+ have been determined as 1.7 nM and 1.1 nM, respectively signifying very high sensitivity. These have been successfully applied in cell imaging studies in C6 cells. Introduction of a methylene group does not influence spectral properties vividly. As both the probes do not contain any imine bond, the possibility of hydrolysis in the presence of Al3+ has been avoided.

A novel fluorescent and colorimetric dual-signals peptide-based probe for sequential recognition of Cu(II) and cimetidine and its multi-functional applications

A novel dual-signals peptide-based probe FDKSH was designed and synthesized based on 5-carboxy fluorescein (5-FAM) labelled tetrapeptide backbone (Asp-Lys-Ser-His-NH2). FDKSH exhibited a highly selective fluorescence “on-off” response and obvious colorimetric recognition to Cu2+ in 10 mM HEPES buffer solution at pH 7.4. Meanwhile, the limit of detection (LOD) was found to be 62.1 nM, which was much lower than that of stated by World Health Organization (WHO) in drinking water (30.0 μM). As a new promising cascade probe, the Cu(II)-assisted peptide ensemble probe (FDKSH-Cu2+ ensemble) has very high selectivity for cimetidine by the displacement method with LOD of 63.5 nM. In addition, FDKSH was successfully used to determine Cu2+ and cimetidine in two actual water samples. Additionally, FDKSH was also successfully used for monitoring Cu2+ and cimetidine in living cells and zebrafish, and showed low toxicity and excellent fluorescence imaging performance. It is worth noting that FDKSH-impregnated test strips exhibited significant fluorescence and colorimetric color changes for Cu2+ and cimetidine directly observed by naked eye. What’s more, smartphone with RGB color recognition function provided a new method for semi-quantitative visual detection of Cu2+ and cimetidine by analyzing different concentrations of Cu2+ and cimetidine without expensive instruments.

Detection of the +3 Oxidation State of Metal Ions with the Aid of a Rhodamine‐Based Dye and Applications in Construction of Complex Logic Circuits and Living Cell Imaging

AbstractDetection of correct oxidation state of a metal ion is important to understand its speciation and redox properties. Some of rhodamine derivatives can identify Al3+, Cr3+ and Fe3+ as the trivalent cations. We have synthesized a rhodamine derivative, HL‐qui, from the reaction of N‐(rhodamine‐6G)lactam‐ethylenediamine with quinoline‐2‐carboxaldehyde for the colorimetric as well as fluorogenic detection of all of metal ions with +3 oxidation state such as Al3+, Cr3+, Fe3+, Ga3+, In3+, Tl3+ and Gd3+. In the presence of these trivalent cations, colorless and nonfluorescent HL‐qui turns pink and strongly fluorescent in HEPES buffer (10 mM) in H2O/ethanol=1/9 (v/v) (pH 7.4) as its spirolactam ring gets open to show the changes in color and fluorescence properties. High sensitivity of the probe towards the cations is reflected from the limit of detection values that are in nM range. To the best of our knowledge, there is no such report where fluorescence sensing has been utilized to determine oxidation state. This multi‐ion sensing feature has been used for fabrication of multi input‐output based logic circuit using Boolean algebraic method. It has been used in imaging of metal ions in living cell (L6 cells).