Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm.

Acrosome exocytosis (AE), in which the sperm's single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process essential for fertilization. However, our understanding of how calcium signaling regulates AE is still incomplete. In particular, the interplay between intra-acrosomal calcium dynamics and the intermediate steps leading to AE is not well-defined. Here, we describe a method that provides spatial and temporal insights into acrosomal calcium dynamics and their relationship to membrane fusion and subsequent exocytosis of the acrosome vesicle. The method utilizes a novel transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE). The sensor combines a genetically encoded calcium indicator (GCaMP) fused with mCherry. This fusion protein was specifically designed to enable the concurrent observation of acrosomal calcium dynamics and membrane fusion events. Real-time monitoring of acrosomal calcium dynamics and AE in live AcroSensE sperm is achieved using a combination of high frame-rate imaging and a stimulant delivery system that can target single sperm. This protocol also provides several examples of basic methods to quantify and analyze the raw data. Because the AcroSensE model is genetically encoded, its scientific significance can be augmented by using readily available genetic tools, such as crossbreeding with other mouse genetic models or gene-editing (CRISPR) based methods. With this strategy, the roles of additional signaling pathways in sperm capacitation and fertilization can be resolved. In summary, the method described here provides a convenient and effective tool to study calcium dynamics in a specific subcellular compartment-the sperm acrosome-and how those dynamics regulate the intermediate steps leading to membrane fusion and acrosome exocytosis.

Segmental differentiation of the murine epididymis: identification of segment-specific, GM1-enriched vesicles and regulation by luminal fluid factors†.

The murine epididymis has 10 distinct segments that provide the opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis. Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wild-type mice after efferent duct ligation. These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. The RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to efferent duct ligation. Additionally, GM1 localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis.

Fluctuations in Serum Creatinine Levels During Hospitalization and Long-Term End-Stage Kidney Disease and Mortality.

Importance: Acute kidney injury is associated with poor outcomes, but the clinical implication of reversible serum creatinine level fluctuations during hospitalization not necessarily defined as acute kidney injury is poorly understood. Objective: To investigate the long-term outcomes of patients without previously diagnosed kidney disease who present with decreased kidney function and are subsequently discharged with apparently normal kidney function. Design, Setting, and Participants: A retrospective cohort study was conducted of patients hospitalized in a large tertiary hospital in Israel between September 1, 2007, and July 31, 2022. The study included patients admitted to an internal medicine ward. Patients had not undergone dialysis during the index hospitalization, had at least 3 creatinine tests performed during hospitalization, and had a discharge estimated glomerular filtration rate (eGFR) exceeding 60 mL/min/1.73 m2. Patients with preexisting chronic kidney disease were excluded. Exposure: Glomerular filtration rate was estimated from serum creatinine values using the updated 2022 Chronic Kidney Disease Epidemiology Collaboration formula, and eGFR greater than 60 mL/min/1.73 m2 was regarded as normal. Exposure was defined based on the association between the first and last values determined during hospitalization. Main Outcomes and Measures: All-cause mortality in the year following the index hospitalization and end-stage kidney disease (ESKD) in the 10 years following the index hospitalization. Results: A total of 40 558 patients were included. Median age was 69 (IQR, 56-80) years, with 18 004 women (44%) and 22 554 men (56%). A total of 34 332 patients (85%) were admitted with a normal eGFR and 6226 (15%) with decreased eGFR. Patients with decreased eGFR on presentation had an 18% increased mortality in the year following hospitalization (adjusted hazard ratio [AHR], 1.18; 95% CI, 1.11-1.24) and a 267% increased risk of ESKD in the 10 years following hospitalization (AHR, 3.67; 95% CI, 2.43-5.54), despite having been discharged with apparently normal kidney function. The highest risk was noted in patients who presented to the hospital with an eGFR of 0 to 45 mL/min/1.73 m2. Conclusions and Relevance: The findings of this cohort study suggest that patients who present with decreased kidney function and are discharged without clinically evident residual kidney disease may be at increased long-term risk for ESKD and mortality.

Biotic-abiotic hybrids for bioanalytics and biocatalysis.

The advances in biotic-abiotic interfaced systems open new directions toward bioanalytics and biocatalysis applications. Conjugating the unique electronic and optic properties of nanoelements with the high selectivity and extraordinary catalytic abilities of biotic materials holds great promise to gain superior new features. Herein, we present a wide scope of biotic-abiotic research, with key examples for its utilization in bioanalytics applications as well as in biocatalysis. The described configurations feature methodologies that enable extending the known scientific toolbox to gain synergy. These new nanobiohybrids may contribute to major global challenges, for example, developing alternative energy utilization or new affordable biodiagnostics and therapeutics tools.

Postexposure-vaccine-prophylaxis against COVID-19.

During the COVID-19 pandemic, postexposure-vaccine-prophylaxis is not a practice. Following exposure, only patient isolation is imposed. Moreover, no therapeutic prevention approach is applied. We asked whether evidence exists for reduced mortality rate following postexposure-vaccine-prophylaxis. To estimate the effectiveness of postexposure-vaccine-prophylaxis, we obtained data from the Israeli Ministry of Health registry. The study population consisted of Israeli residents aged 12 years and older, identified for the first time as PCR-positive for SARS-CoV-2, between December 20th, 2020 (the beginning of the vaccination campaign) and October 7th, 2021. We compared "recently injected" patients-that proved PCR-positive on the same day or on 1 of the 5 consecutive days after first vaccination (representing an unintended postexposure-vaccine-prophylaxis)s-to unvaccinated control group. Among Israeli residents identified PCR-positive for SARS-CoV-2, 11 687 were found positive on the day they received their first vaccine injection (BNT162b2) or on 1 of the 5 days thereafter. In patients over 65 years, 143 deaths occurred among 1412 recently injected (10.13%) compared to 255 deaths among the 1412 unvaccinated (18.06%), odd ratio (OR) 0.51 (95% confidence interval [CI]: 0.41-0.64; p < 0.001). A significant reduction in the death toll was observed among the 55-64 age group, with 8 deaths occurring among the 1320 recently injected (0.61%) compared to 24 deaths among the 1320 unvaccinated control (1.82%), OR 0.33 (95% CI: 0.13-0.76; p = 0.007). Postexposure-vaccine-prophylaxis is effective against death in COVID-19 infection.

An Oxygen-Insensitive biosensor and a biofuel cell device based on FMN l-lactate dehydrogenase.

Lactate sensing has high importance for metabolic disease diagnostics, food spoilage, sports medicine, or the construction of biofuel cell devices. Therefore, continuous lactate sensing devices which enable accurate detection should be developed. Here we present the overexpression and utilization of FMN-lactate dehydrogenase from Saccharomyces cerevisiae for oxygen-insensitive, continuous amperometric lactate biosensing. The developed sensors exhibit a high signal-to-noise ratio, low interference effect, and a wide range of linear responses using both direct and mediated electron transfer configurations. The thionine-based mediated electron transfer configuration was stable for 8 h of continuous activity and two weeks of periodic activity with storage at 4 °C. We further grafted the redox mediators on multiwall carbon nanotubes to lower the redox mediator leaching effect. The developed grafting technique improved the biosensor stability and allowed continuous operation for at least 20 h. Both the mediator-entrapped and the grafted bioanodes were further coupled with a bilirubin oxidase-based biocathode to construct a biofuel cell device. The various biofuel cells have generated a maximal power output of 110 µW/cm2 under atmospheric conditions and 200 µW/cm2 under oxygen saturation.

A Machine Learning Approach for Continuous Mining of Nonidentifiable Smartphone Data to Create a Novel Digital Biomarker Detecting Generalized Anxiety Disorder: Prospective Cohort Study.

BACKGROUND: Anxiety is one of the leading causes of mental health disability around the world. Currently, a majority of the population who experience anxiety go undiagnosed or untreated. New and innovative ways of diagnosing and monitoring anxiety have emerged using smartphone sensor-based monitoring as a metric for the management of anxiety. This is a novel study as it adds to the field of research through the use of nonidentifiable smartphone usage to help detect and monitor anxiety remotely and in a continuous and passive manner. OBJECTIVE: This study aims to evaluate the accuracy of a novel mental behavioral profiling metric derived from smartphone usage for the identification and tracking of generalized anxiety disorder (GAD). METHODS: Smartphone data and self-reported 7-item GAD anxiety assessments were collected from 229 participants using an Android operating system smartphone in an observational study over an average of 14 days (SD 29.8). A total of 34 features were mined to be constructed as a potential digital phenotyping marker from continuous smartphone usage data. We further analyzed the correlation of these digital behavioral markers against each item of the 7-item Generalized Anxiety Disorder Scale (GAD-7) and its influence on the predictions of machine learning algorithms. RESULTS: A total of 229 participants were recruited in this study who had completed the GAD-7 assessment and had at least one set of passive digital data collected within a 24-hour period. The mean GAD-7 score was 11.8 (SD 5.7). Regression modeling was tested against classification modeling and the highest prediction accuracy was achieved from a binary XGBoost classification model (precision of 73%-81%; recall of 68%-87%; F1-score of 71%-79%; accuracy of 76%; area under the curve of 80%). Nonparametric permutation testing with Pearson correlation results indicated that the proposed metric (Mental Health Similarity Score [MHSS]) had a colinear relationship between GAD-7 Items 1, 3 and 7. CONCLUSIONS: The proposed MHSS metric demonstrates the feasibility of using passively collected nonintrusive smartphone data and machine learning-based data mining techniques to track an individuals' daily anxiety levels with a 76% accuracy that directly relates to the GAD-7 scale.

First-Principle Colloidal Gate for Controlling Liquid and Molecule Flow Using 2D Claylike Nanoparticles.

Herein, we exploit the natural tendency of two-dimensional (2D) clay nanoparticles to self-assemble and restrict water permeability in soils to fabricate a first of its kind synthetic, pH-activated, reversible, and tunable colloidal flow gate. To realize this, we studied the effect of the pH level of a suspension of claylike layered double hydroxide (LDH) nanoparticles on the LDH coagulation process. We then packed the LDH into a fixed-bed column and examined the effect of pH on mass transport through the column. We found that the 2D platelike LDH particles coagulate in an edge-to-edge configuration, which renders highly nonisotropic aggregates, pivotal for obstructing the transport of liquid and molecules therein. We showed that the coagulation and flow through the column may be regulated by imposing various pH levels as an external stimulus to affect LDH zeta potential. Hence, this work shows that the flow through a column comprising a 2D particle bed can be regulated in a reversible manner by simply alternating the pH of the wash solution, equilibration time, or gate dimensions. Furthermore, we show that, subject to pH treatment, we may open and close the colloidal gate for the transport of large molecules and provide selective transport thereof.

A Machine Learning Approach for Detecting Digital Behavioral Patterns of Depression Using Nonintrusive Smartphone Data (Complementary Path to Patient Health Questionnaire-9 Assessment): Prospective Observational Study.

BACKGROUND: Depression is a major global cause of morbidity, an economic burden, and the greatest health challenge leading to chronic disability. Mobile monitoring of mental conditions has long been a sought-after metric to overcome the problems associated with the screening, diagnosis, and monitoring of depression and its heterogeneous presentation. The widespread availability of smartphones has made it possible to use their data to generate digital behavioral models that can be used for both clinical and remote screening and monitoring purposes. This study is novel as it adds to the field by conducting a trial using private and nonintrusive sensors that can help detect and monitor depression in a continuous, passive manner. OBJECTIVE: This study demonstrates a novel mental behavioral profiling metric (the Mental Health Similarity Score), derived from analyzing passively monitored, private, and nonintrusive smartphone use data, to identify and track depressive behavior and its progression. METHODS: Smartphone data sets and self-reported Patient Health Questionnaire-9 (PHQ-9) depression assessments were collected from 558 smartphone users on the Android operating system in an observational study over an average of 10.7 (SD 23.7) days. We quantified 37 digital behavioral markers from the passive smartphone data set and explored the relationship between the digital behavioral markers and depression using correlation coefficients and random forest models. We leveraged 4 supervised machine learning classification algorithms to predict depression and its severity using PHQ-9 scores as the ground truth. We also quantified an additional 3 digital markers from gyroscope sensors and explored their feasibility in improving the model's accuracy in detecting depression. RESULTS: The PHQ-9 2-class model (none vs severe) achieved the following metrics: precision of 85% to 89%, recall of 85% to 89%, F1 of 87%, and accuracy of 87%. The PHQ-9 3-class model (none vs mild vs severe) achieved the following metrics: precision of 74% to 86%, recall of 76% to 83%, F1 of 75% to 84%, and accuracy of 78%. A significant positive Pearson correlation was found between PHQ-9 questions 2, 6, and 9 within the severely depressed users and the mental behavioral profiling metric (r=0.73). The PHQ-9 question-specific model achieved the following metrics: precision of 76% to 80%, recall of 75% to 81%, F1 of 78% to 89%, and accuracy of 78%. When a gyroscope sensor was added as a feature, the Pearson correlation among questions 2, 6, and 9 decreased from 0.73 to 0.46. The PHQ-9 2-class model+gyro features achieved the following metrics: precision of 74% to 78%, recall of 67% to 83%, F1 of 72% to 78%, and accuracy of 76%. CONCLUSIONS: Our results demonstrate that the Mental Health Similarity Score can be used to identify and track depressive behavior and its progression with high accuracy.

A genetically targeted sensor reveals spatial and temporal dynamics of acrosomal calcium and sperm acrosome exocytosis.

Secretion of the acrosome, a single vesicle located rostrally in the head of a mammalian sperm, through a process known as "acrosome exocytosis" (AE), is essential for fertilization. However, the mechanisms leading to and regulating this complex process are controversial. In particular, poor understanding of Ca2+ dynamics between sperm subcellular compartments and regulation of membrane fusion mechanisms have led to competing models of AE. Here, we developed a transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE) to investigate the spatial and temporal Ca2+ dynamics in AE in live sperm. AcroSensE combines a genetically encoded Ca2+ indicator (GCaMP) fused with an mCherry indicator to spatiotemporally resolve acrosomal Ca2+ rise (ACR) and membrane fusion events, enabling real-time study of AE. We found that ACR is dependent on extracellular Ca2+ and that ACR precedes AE. In addition, we show that there are intermediate steps in ACR and that AE correlates better with the ACR rate rather than absolute Ca2+ amount. Finally, we demonstrate that ACR and membrane fusion progression kinetics and spatial patterns differ with different stimuli and that sites of initiation of ACR and sites of membrane fusion do not always correspond. These findings support a model involving functionally redundant pathways that enable a highly regulated, multistep AE in heterogeneous sperm populations, unlike the previously proposed "acrosome reaction" model.

Utilization of FAD-Glucose Dehydrogenase from T. emersonii for Amperometric Biosensing and Biofuel Cell Devices.

Flavin-dependent glucose dehydrogenases (FAD-GDH) are oxygen-independent enzymes with high potential to be used as biocatalysts in glucose biosensing applications. Here, we present the construction of an amperometric biosensor and a biofuel cell device, which are based on a thermophilic variant of the enzyme originated from Talaromyces emersonii. The enzyme overexpression in Escherichia coli and its isolation and performance in terms of maximal bioelectrocatalytic currents were evaluated. We examined the biosensor's bioelectrocatalytic activity in 2,6-dichlorophenolindophenol-, thionine-, and dichloro-naphthoquinone-mediated electron transfer configurations or in a direct electron transfer one. We showed a negligible interference effect and good stability for at least 20 h for the dichloro-naphthoquinone configuration. The constructed biosensor was also tested in interstitial fluid-like solutions to show high bioelectrocatalytic current responses. The bioanode was coupled with a bilirubin oxidase-based biocathode to generate 270 µW/cm2 in a biofuel cell device.

DARPin_9-29-Targeted Mini Gold Nanorods Specifically Eliminate HER2-Overexpressing Cancer Cells.

We have demonstrated that designed ankyrin repeat protein (DARPin) _9-29, which specifically targets human epidermal growth factor receptor 2 (HER2), binds tightly to gold mini nanorods (GNRs). Molecular dynamic simulations showed that a single layer of DARPin_9-29 molecules is formed on the surface of the nanorod and that conjugation with the nanorod does not involve the protein's domain responsible for specific binding to HER2. The nanorod-DARPin (DARPin-GNR) conjugate is specifically bound (in nanomolar concentrations) to human breast adenocarcinoma SK-BR-3 cells overexpressing HER2. Illumination by near-infrared light (850 nm) led to almost complete eradication of the conjugate-treated SK-BR-3 cells; the viability of epithelial human breast cancer cells expressing normal amounts of the receptor was much less affected by the illumination. The results reported here pave the way toward application of DARPin-GNR conjugates in phototherapy of cancer.

Biomimicry Promotes the Efficiency of a 10-Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate.

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo.

Lipid Regulation of Acrosome Exocytosis.

Lipids are critical regulators of mammalian sperm function, first helping prevent premature acrosome exocytosis, then enabling sperm to become competent to fertilize at the right place/time through the process of capacitation, and ultimately triggering acrosome exocytosis. Yet because they do not fit neatly into the "DNA--RNA-protein" synthetic pathway, they are understudied and poorly understood. Here, we focus on three lipids or lipid classes-cholesterol, phospholipids, and the ganglioside G(M1)--in context of the modern paradigm of acrosome exocytosis. We describe how these various- species are precisely segregated into membrane macrodomains and microdomains, simultaneously preventing premature exocytosis while acting as foci for organizing regulatory and effector molecules that will enable exocytosis. Although the mechanisms responsible for these domains are poorly defined, there is substantial evidence for their composition and functions. We present diverse ways that lipids and lipid modifications regulate capacitation and acrosome exocytosis, describing in more detail how removal of cholesterol plays a master regulatory role in enabling exocytosis through at least two complementary pathways. First, cholesterol efflux leads to proteolytic activation of phospholipase B, which cleaves both phospholipid tails. The resultant changes in membrane curvature provide a mechanism for the point fusions now known to occur far before a sperm physically interacts with the zona pellucida. Cholesterol efflux also enables G(M1) to regulate the voltage-dependent cation channel, Ca(V)2.3, triggering focal calcium transients required for acrosome exocytosis in response to subsequent whole-cell calcium rises. We close with a model integrating functions for lipids in regulating acrosome exocytosis.

Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis.

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo(-/-)) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.

Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection.

BACKGROUND: Rapid diagnosis for time-sensitive illnesses such as stroke, cardiac arrest, and septic shock is essential for successful treatment. Much attention has therefore focused on new strategies for rapid and objective diagnosis, such as Point-of-Care Tests (PoCT) for blood biomarkers. Here we use a biomimicry-based approach to demonstrate a new diagnostic platform, based on enzymes tethered to nanoparticles (NPs). As proof of principle, we use oriented immobilization of pyruvate kinase (PK) and luciferase (Luc) on silica NPs to achieve rapid and sensitive detection of neuron-specific enolase (NSE), a clinically relevant biomarker for multiple diseases ranging from acute brain injuries to lung cancer. We hypothesize that an approach capitalizing on the speed and catalytic nature of enzymatic reactions would enable fast and sensitive biomarker detection, suitable for PoCT devices. METHODS AND FINDINGS: We performed in-vitro, animal model, and human subject studies. First, the efficiency of coupled enzyme activities when tethered to NPs versus when in solution was tested, demonstrating a highly sensitive and rapid detection of physiological and pathological concentrations of NSE. Next, in rat stroke models the enzyme-based assay was able in minutes to show a statistically significant increase in NSE levels in samples taken 1 hour before and 0, 1, 3 and 6 hours after occlusion of the distal middle cerebral artery. Finally, using the tethered enzyme assay for detection of NSE in samples from 20 geriatric human patients, we show that our data match well (r = 0.815) with the current gold standard for biomarker detection, ELISA-with a major difference being that we achieve detection in 10 minutes as opposed to the several hours required for traditional ELISA. CONCLUSIONS: Oriented enzyme immobilization conferred more efficient coupled activity, and thus higher assay sensitivity, than non-tethered enzymes. Together, our findings provide proof of concept for using oriented immobilization of active enzymes on NPs as the basis for a highly rapid and sensitive biomarker detection platform. This addresses a key challenge in developing a PoCT platform for time sensitive and difficult to diagnose pathologies.

Effects of Nanoparticle Size on Multilayer Formation and Kinetics of Tethered Enzymes.

Despite numerous applications, we lack fundamental understanding of how variables such as nanoparticle (NP) size influence the activity of tethered enzymes. Previously, we showed that biomimetic oriented immobilization yielded higher specific activities versus nonoriented adsorption or carboxyl-amine binding. Here, we standardize NP attachment strategy (oriented immobilization via hexahistidine tags) and composition (Ni-NTA coated gold NPs), to test the impact of NP size (⌀5, 10, 20, and 50 nm) on multilayer formation, activity, and kinetic parameters (kcat, KM, kcat/KM) of enzymes representing three different classes: glucose-6-phosphate isomerase (GPI), an isomerase; Glyceraldehyde-3-phosphate dehydrogenase S (GAPDHS), an oxidoreductase; and pyruvate kinase (PK), a transferase. Contrary to other reports, we observed no trend in kinetic parameters for individual enzymes when found in monolayers (<100% enzyme coverage), suggesting an advantage for oriented immobilization versus other attachment strategies. Saturating the NPs to maximize activity per NP resulted in enzyme multilayer formation. Under these conditions, total activity per NP increased with increasing NP size. Conversely, specific activity for all three enzymes was highest when tethered to the smallest NPs, retaining a remarkable 73-94% of the activity of free/untethered enzymes. Multilayer formations caused a clear trend of kcat decreasing with increasing NP size, yet negligible change in KM. Understanding the fundamental relationships between NP size and tethered enzyme activity enables optimized design of various applications, maximizing activity per NP or activity per enzyme molecule.

Real-time imaging of Ca(2+) mobilization and degranulation in mast cells.

Mast cells play a key role in allergy and inflammation processes as part of the immune response. The activation of mast cells via antigen binding and cross-linking of IgE receptors initiates the onset of dramatic calcium (Ca(2+)) mobilization dynamics that promote the release of mediators of inflammation and allergy. Ca(2+) signaling in mast cells has been studied extensively using a variety of research tools and techniques. In these studies, a large number of proteins have been identified to participate in various stages of these processes. Here we describe single-cell imaging as an important approach for examining Ca(2+) signaling and exocytosis in mast cells. Single-cell imaging tools have advanced significantly over the last 10 years, in part due to improvements in microscope technology and in part due to the development of a new generation of Ca(2+) indicators and genetically encoded Ca(2+) sensors. The single-cell imaging techniques described here provide the spatial and temporal resolution required to decipher the signaling events that are critical for mast cell functions.

Lipid modulation of calcium flux through CaV2.3 regulates acrosome exocytosis and fertilization.

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3's pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1's lipid and sugar components and CaV2.3's α1E and α2δ subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.

The role of MATER in endoplasmic reticulum distribution and calcium homeostasis in mouse oocytes.

Ca(2+) oscillations are a hallmark of mammalian fertilization and play a central role in the activation of development. The calcium required for these oscillations is primarily derived from the endoplasmic reticulum (ER), which accumulates in clusters at the microvillar subcortex during oocyte maturation. The migration of the ER to the cortex during maturation is thought to play an important role in rendering the ER competent to generate the calcium transients, and the redistribution of ER is believed to be primarily mediated by microtubules and microfilaments. We have previously shown that the oocyte- and early embryo-restricted maternal effect gene Mater (Nlrp5) localizes to, and is required for, formation of the oocyte cytoplasmic lattices, a tubulin-containing structure that appears to play an important role in organelle positioning and distribution during oocyte maturation. Given these observations, we hypothesized that Mater may also be required for ER redistribution and Ca(2+) homeostasis in oocytes. To test this hypothesis, we first investigated ER localization in metaphase-II Mater(tm/tm) (hypomorph) oocytes and found ER clusters to be less abundant at the microvillar cortex when compared to wild type oocytes. To examine the potential mechanisms by which MATER mediates ER redistribution, we tested whether tubulin expression levels and localization were affected in the mutant oocytes and found that the Triton-insoluble fraction of tubulin was significantly decreased in Mater(tm/tm) oocytes. To identify potential functional defects associated with these ER abnormalities, we next set out to investigate if the pattern of Ca(2+) oscillations was altered in Mater(tm/tm) oocytes after fertilization in vitro. Intriguingly, Ca(2+) oscillations in Mater(tm/tm) oocytes exhibited a significantly lower first peak amplitude and a higher frequency when compared to wild type oocytes. We then found that the Ca(2+) oscillation defect in Mater(tm/tm) oocytes was likely caused by a reduced amount of Ca(2+) in the ER stores. Taken together, these observations support the hypothesis that MATER is required for ER distribution and Ca(2+) homeostasis in oocytes, likely due to defects in lattice-mediated ER positioning and/or redistribution.