Expansion Microscopy of Synaptic Contacts on the Mauthner Cells of Larval Zebrafish.

Because of its genetic tractability and amenability for live imaging, larval zebrafish (Danio rerio) have emerged as a model to study the cellular and synaptic properties underlying behavior. The accessibility of Mauthner cells, a pair of escape-organizing neurons located in the brainstem of teleost fish, along with their associated sensory inputs, enables exploration of the correlation between structural and functional synaptic features. This is the case of the endings of auditory afferents on the lateral dendrite of this cell, known as large myelinated club endings, which provide the excitatory drive for the initiation of auditory-evoked escape responses mediated by the Mauthner cell and its spinal network. Here, we describe the procedures that make it possible to expose the molecular composition of these synapses using protein-retention expansion microscopy (proExM). This method allowed us to generate a map of the distribution of synaptic proteins at these identifiable synapses, which could also be applied to examine the organization of other synaptic contacts in this cell. Key features • This protocol builds upon the method developed by Tillberg et al. [1] • Optimized for the examination of the organization of molecular components at synaptic contacts on the Mauthner cells of larval zebrafish • Requires at least three days to complete and should be preceded by immunostaining. • Results in a linear expansion factor of ~3.9× and an area expansion factor of ~13×.

Required number of blastocysts transferred, and oocytes retrieved to optimize live and cumulative live birth rates in the first complete cycle of IVF for autologous and donated oocytes.

PURPOSE: To investigate live birth rate (LBR) and cumulative live birth rate (CLBR) to achieve the first newborn per blastocyst transferred and oocyte retrieved in the first complete IVF cycle of autologous and donated oocytes and identify the possible success factors. METHODS: This was a retrospective cohort study of a private IVF center. There were 1867 cycles, 1241 of which were fresh transfers and 626, their subsequent thawing transfers. RESULTS: We found significant variables by binary logistic regression. For LBR, female infertility and the day of blastocyst transferred were relevant; however, for CLBR, the numbers of blastocysts available for future transfers, oocyte age, and maternal age were more critical. Oocyte age is a negative factor that begins to affect CLBR gradually beyond 36 years; from that age, there are significant worse results in polycystic ovary syndrome and poor responder patients. CONCLUSION: The LBR and CLBR were optimized for oocyte recipients when eight oocytes were retrieved (63.6%; 87.9%); at most, fourteen oocytes should be assigned to avoid freezing surplus blastocysts. Thirteen autologous oocytes (69.2%; 92.3%) were ideal for optimization. CLBR optimized after three blastocysts in donor oocytes (81.8%) and four for autologous oocyte patients (80.9%). Our outcomes are valuable for doctors and infertile couples, and they give us information on what we can expect from a first complete IVF cycle.

The components of an electrical synapse as revealed by expansion microscopy of a single synaptic contact.

Most nervous systems combine both transmitter-mediated and direct cell-cell communication, known as 'chemical' and 'electrical' synapses, respectively. Chemical synapses can be identified by their multiple structural components. Electrical synapses are, on the other hand, generally defined by the presence of a 'gap junction' (a cluster of intercellular channels) between two neuronal processes. However, while gap junctions provide the communicating mechanism, it is unknown whether electrical transmission requires the contribution of additional cellular structures. We investigated this question at identifiable single synaptic contacts on the zebrafish Mauthner cells, at which gap junctions coexist with specializations for neurotransmitter release and where the contact unequivocally defines the anatomical limits of a synapse. Expansion microscopy of these single contacts revealed a detailed map of the incidence and spatial distribution of proteins pertaining to various synaptic structures. Multiple gap junctions of variable size were identified by the presence of their molecular components. Remarkably, most of the synaptic contact's surface was occupied by interleaving gap junctions and components of adherens junctions, suggesting a close functional association between these two structures. In contrast, glutamate receptors were confined to small peripheral portions of the contact, indicating that most of the synaptic area functions as an electrical synapse. Thus, our results revealed the overarching organization of an electrical synapse that operates with not one, but multiple gap junctions, in close association with structural and signaling molecules known to be components of adherens junctions. The relationship between these intercellular structures will aid in establishing the boundaries of electrical synapses found throughout animal connectomes and provide insight into the structural organization and functional diversity of electrical synapses.

Neurons communicate with each other through specialized structures known as synapses. At chemical synapses, the cells do not physically interact as they rely instead on molecules called neurotransmitters to pass along signals. At electrical synapses, however, neurons are directly connected via gap junctions, which are clusters of intercellular channels that allow ions and other small compounds to move from one cell to another. Both electrical and chemical synapses play critical roles in neural circuits, and both exhibit some amount of plasticity ­ they weaken or strengthen depending on how often they are used, an important feature for the brain to adapt to the needs of the environment. Yet the structure and molecular organization of electrical synapses have remained poorly understood compared to their chemical counterparts. In response, Cárdenas-García, Ijaz and Pereda took advantage of a new approach known as expansion microscopy to examine the electrical synapse that connects neurons bringing sound information to a pair of unusually large neurons in the brain of most bony fish. With this method, a biological sample is prepared in such a way that its size increases, but the relative position of its components is preserved. This allows scientists to better observe structures that would otherwise be too difficult to capture using traditional microscopy techniques. Experiments in larval zebrafish revealed that contrary to previous assumptions, the electrical synapse was formed of not one but multiple gap junctions of various sizes closely associated with a range of structural and signaling molecules typically found in adherens junctions (a type of structure that physically links cells together). The team suggests that these molecular actors could work to ensure that the multiple gap junctions act in concert at the synapse. Overall, these findings offer a new perspective on how electrical synapses are organized and regulated, which refines our understanding of how the nervous system functions both in health and in disease.

Circadian regulation of MGMT expression and promoter methylation underlies daily rhythms in TMZ sensitivity in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite extensive research and clinical trials, median survival post-treatment remains at 15 months. Thus, all opportunities to optimize current treatments and improve patient outcomes should be considered. A recent retrospective clinical study found that taking TMZ in the morning compared to the evening was associated with a 6-month increase in median survival in patients with MGMT-methylated GBM. Here, we hypothesized that TMZ efficacy depends on time-of-day and O6-Methylguanine-DNA Methyltransferase (MGMT) activity in murine and human models of GBM. METHODS AND RESULTS: In vitro recordings using real-time bioluminescence reporters revealed that GBM cells have intrinsic circadian rhythms in the expression of the core circadian clock genes Bmal1 and Per2, as well as in the DNA repair enzyme, MGMT. Independent measures of MGMT transcript levels and promoter methylation also showed daily rhythms intrinsic to GBM cells. These cells were more susceptible to TMZ when delivered at the daily peak of Bmal1 transcription. We found that in vivo morning administration of TMZ also decreased tumor size and increased body weight compared to evening drug delivery in mice bearing GBM xenografts. Finally, inhibition of MGMT activity with O6-Benzylguanine abrogated the daily rhythm in sensitivity to TMZ in vitro by increasing sensitivity at both the peak and trough of Bmal1 expression. CONCLUSION: We conclude that chemotherapy with TMZ can be dramatically enhanced by delivering at the daily maximum of tumor Bmal1 expression and minimum of MGMT activity and that scoring MGMT methylation status requires controlling for time of day of biopsy.

Circadian regulation of MGMT expression and promoter methylation underlies daily rhythms in TMZ sensitivity in glioblastoma.

Background: Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite extensive research and clinical trials, median survival post-treatment remains at 15 months. Thus, all opportunities to optimize current treatments and improve patient outcomes should be considered. A recent retrospective clinical study found that taking TMZ in the morning compared to the evening was associated with a 6-month increase in median survival in patients with MGMT-methylated GBM. Here, we hypothesized that TMZ efficacy depends on time-of-day and O6-Methylguanine-DNA Methyltransferase (MGMT) activity in murine and human models of GBM. Methods and Results: In vitro recordings using real-time bioluminescence reporters revealed that GBM cells have intrinsic circadian rhythms in the expression of the core circadian clock genes Bmal1 and Per2, as well as in the DNA repair enzyme, MGMT. Independent measures of MGMT transcript levels and promoter methylation also showed daily rhythms intrinsic to GBM cells. These cells were more susceptible to TMZ when delivered at the daily peak of Bmal1 transcription. We found that in vivo morning administration of TMZ also decreased tumor size and increased body weight compared to evening drug delivery in mice bearing GBM xenografts. Finally, inhibition of MGMT activity with O6-Benzylguanine abrogated the daily rhythm in sensitivity to TMZ in vitro by increasing sensitivity at both the peak and trough of Bmal1 expression. Conclusion: We conclude that chemotherapy with TMZ can be dramatically enhanced by delivering at the daily maximum of tumor Bmal1 expression and minimum of MGMT activity.

The components of an electrical synapse as revealed by expansion microscopy of a single synaptic contact.

Most nervous systems combine both transmitter-mediated and direct cell-cell communication, known as 'chemical' and 'electrical' synapses, respectively. Chemical synapses can be identified by their multiple structural components. Electrical synapses are, on the other hand, generally defined by the presence of a 'gap junction' (a cluster of intercellular channels) between two neuronal processes. However, while gap junctions provide the communicating mechanism, it is unknown whether electrical transmission requires the contribution of additional cellular structures. We investigated this question at identifiable single synaptic contacts on the zebrafish Mauthner cells, at which gap junctions coexist with specializations for neurotransmitter release and where the contact defines the anatomical limits of a synapse. Expansion microscopy of these contacts revealed a detailed map of the incidence and spatial distribution of proteins pertaining to various synaptic structures. Multiple gap junctions of variable size were identified by the presence of their molecular components. Remarkably, most of the synaptic contact's surface was occupied by interleaving gap junctions and components of adherens junctions, suggesting a close functional association between these two structures. In contrast, glutamate receptors were confined to small peripheral portions of the contact, indicating that most of the synaptic area works as an electrical synapse. Thus, our results revealed the overarching organization of an electrical synapse that operates with not one, but multiple gap junctions, in close association with structural and signaling molecules known to be components of AJs. The relationship between these intercellular structures will aid in establishing the boundaries of electrical synapses found throughout animal connectomes and provide insight into the structural organization and functional diversity of electrical synapses.

The financial and time burden associated with phenylketonuria treatment in the United States.

BACKGROUND: Phenylketonuria (PKU) imposes a substantial burden on people living with the condition and their families. However, little is known about the time cost and financial burden of having PKU or caring for a child with the condition. METHODS AND FINDINGS: Primary data were collected with a detailed cost and utilization survey. Primary outcomes included utilization and out-of-pocket costs of medical services, medical formula, and prescribed low-protein food consumption, as well as the time and perceived effort involved in following the PKU diet. Respondents were people living with PKU or parents of children with PKU identified through a state newborn screening program database. Secondary administrative claims data were also used to calculate mean total, insurer, and out-of-pocket payments in inpatient, outpatient (office visits, emergency room, and laboratory tests), and pharmacy settings for privately insured persons with PKU. Payments were calculated for sapropterin and for PKU formula.In primary data analysis (children n = 32, adults n = 52), annual out-of-pocket costs were highest for low-protein foods (child = $1651; adult = $967) compared with other categories of care. The time burden of PKU care was high; families reported spending more than 300 h per year shopping for and preparing special diet foods.In secondary data analysis, children 12-17 years old had the highest average medical expenditures ($54,147; n = 140) compared to children 0-11 years old ($19,057; n = 396) and adults 18 years and older ($40,705; n = 454). Medication costs were the largest contributor to medical costs, accounting for 61-81% of total costs across age groups. Sapropterin was the largest driver of medication costs, accounting for 85% of child medication costs and 92% of adult medication costs. CONCLUSION: Treatment for PKU incurs a substantial time and cost burden on persons with PKU and their families. Estimated medical expenditures using claims data varied by age group, but sapropterin represented the largest cost for PKU treatment from a payer perspective across age groups.

Incremental Health Care Costs Associated With Food Insecurity and Chronic Conditions Among Older Adults.

INTRODUCTION: The prevalence of food insecurity and chronic health conditions among older adults is a public health concern. However, little is known about associated health care costs. We estimated the incremental health care costs of food insecurity and selected chronic health conditions among older adults, defined as adults aged 50 or older. METHODS: We analyzed 4 years of data (2011-2014) from the National Health Interview Survey and 3 years of data (2013-2015) from the Medical Expenditure Panel Survey; we used 2-part models to estimate the incremental health care costs associated with food insecurity and 9 chronic conditions (hypertension, coronary heart disease, stroke, emphysema, asthma, cancer, chronic bronchitis, arthritis, and diabetes) among older adults. RESULTS: Approximately 14% of older adult respondents (n = 2,150) reported being food insecure. The 3 most common chronic conditions were the same for both food-insecure and food-secure older adults: hypertension, arthritis, and diabetes. The adjusted annual incremental health care costs resulting from food insecurity among older adults were higher in the presence of hypertension, stroke, and arthritis (P ≤ .05) and in the presence of diabetes (P ≤ .10). These findings were also true for the incremental health care costs resulting from food insecurity in the absence of these specific chronic conditions. CONCLUSION: Our findings show that food insecurity interacts with chronic conditions. We observed higher health care costs in the presence of this interaction for those who were food insecure and had poor health than for those who were food secure.

Clinical-Community Partnerships to Identify Patients With Food Insecurity and Address Food Needs.

INTRODUCTION: More than 42 million people in the United States are food insecure. Although some health care entities are addressing food insecurity among patients because of associations with disease risk and management, little is known about the components of these initiatives. METHODS: The Systematic Screening and Assessment Method was used to conduct a landscape assessment of US health care entity-based programs that screen patients for food insecurity and connect them with food resources. A network of food insecurity researchers, experts, and practitioners identified 57 programs, 22 of which met the inclusion criteria of being health care entities that 1) screen patients for food insecurity, 2) link patients to food resources, and 3) target patients including adults aged 50 years or older (a focus of this assessment). Data on key features of each program were abstracted from documentation and telephone interviews. RESULTS: Most programs (n = 13) focus on patients with chronic disease, and most (n = 12) partner with food banks. Common interventions include referrals to or a list of food resources (n = 19), case managers who navigate patients to resources (n = 15), assistance with federal benefit applications (n = 14), patient education and skill building (n = 13), and distribution of fruit and vegetable vouchers redeemable at farmers markets (n = 8). Most programs (n = 14) routinely screen all patients. CONCLUSION: The programs reviewed use various strategies to screen patients, including older adults, for food insecurity and to connect them to food resources. Research is needed on program effectiveness in improving patient outcomes. Such evidence can be used to inform the investments of potential stakeholders, including health care entities, community organizations, and insurers.

Synthesis and optical properties of aqueous soluble indolylfulgimides.

Three novel aqueous soluble fulgimides, trifluoromethyl carboxylic acid indolylfulgimide 4, dicarboxylic acid indolylfulgimide 5, and carboxylic acid indolylfulgimide 6, were synthesized. Both 4 and 5 can switch back and forth between open and closed forms upon illumination with specific wavelengths of light, whereas 6 can only switch from the closed form to the open form. In sodium phosphate buffer (pH 7.4) at 37 degrees C, an unusual hydrolysis of the trifluoromethyl group of the closed form of 4 resulted in 5, which has an additional carboxylic acid group. The closed form of 5 was further decarboxylated to generate 6, which was not photochromic. In buffer, the open form of 4 degraded 20% after 10 days, while the closed form of 4 was converted to 5 rapidly. In buffer, both forms of 5 degraded less than 20% after 21 days at 37 degrees C, and 5 underwent 670 photochemical cycles before degrading by 20%. It is the most robust fulgimide yet reported in aqueous solution.

A prospective duplex Doppler ultrasonographic study in women with sexual arousal disorder to objectively assess genital engorgement induced by EROS therapy.

The EROS therapy device is FDA-approved for the treatment of women with sexual dysfunction and has been shown to improve sexual function and satisfaction. The aim of this study was to obtain objective information regarding the ability of the EROS Therapy device to induce clitoral and corpus spongiosum volumetric and hemodynamic changes following therapeutic use in women with sexual arousal disorder. Seven patients with sexual arousal disorder formed the study population. All seven subjects met inclusion and exclusion criteria, including having normal hormonal values at the time of the study. All seven subjects were able to comfortably operate the device. All seven reported either slight-to-moderate pleasure or orgasm at home with the device. We observed no adverse events. This study shows that EROS therapy is associated with significant increases in clitoral and corpus spongiosum diameter as well as with clitoral and corpus spongiosum peak systolic and end-diastolic velocity values.

Dynamics of the gas-phase reactions of chloride ion with fluoromethane: high excess translational activation energy for an endothermic S(N)2 reaction.

Guided ion beam tandem mass spectrometry techniques are used to examine the competing product channels in the reaction of Cl(-) with CH(3)F in the center-of-mass collision energy range 0.05-27 eV. Four anionic reaction products are detected: F(-), CH(2)Cl(-), FCl(-), and CHCl(-). The endothermic S(N)2 reaction Cl(-) + CH(3)F --> CH(3)Cl + F(-) has an energy threshold of E(0) = 181 +/- 14 kJ/mol, exhibiting a 52 +/- 16 kJ/mol effective barrier in excess of the reaction endothermicity. The potential energy of the S(N)2 transition state is well below the energy of the products. Dynamical impedances to the activation of the S(N)2 reaction are discussed, including angular momentum constraints, orientational effects, and the inefficiency of translational energy in promoting the reaction. The fluorine abstraction reaction to form CH(3) + FCl(-) exhibits a 146 +/- 33 kJ/mol effective barrier above the reaction endothermicity. Direct proton transfer to form HCl is highly inefficient, but HF elimination is observed above 268 +/- 95 kJ/mol. Potential energy surfaces for the reactions are calculated using the CCSD(T)/aug-cc-pVDZ and HF/6-31+G(d) methods and used to interpret the dynamics.