Synthetic Gene Circuit-Mediated Monitoring of Endogenous Metabolites: Identification of <i>GAL11</i> as a Novel Multicopy Enhancer of <i>S</i>‑Adenosylmethionine Level in Yeast

Monitoring levels of key metabolites in living cells comprises a critical step in various investigations. The simplest approach to this goal is a fluorescent reporter gene using an endogenous promoter responsive to the metabolite. However, such a promoter is often not identified or even present in the species of interest. An alternative can be a synthetic gene circuit based on a heterologous pair consisting of a promoter and a transcription factor known to respond to the metabolite. We exploited the <i>met</i> operator and MetJ repressor of <i>Escherichia coli</i>, the interaction between which depends on <i>S</i>-adenosylmethionine (SAM), to construct synthetic gene circuits that report SAM levels in <i>Saccharomyces cerevisiae</i>. Using a dual-input circuit that outputs selection marker genes in a doxycycline-tunable manner, we screened a genomic library to identify <i>GAL11</i> as a novel multicopy enhancer of SAM levels. These results demonstrate the potential and utility of synthetic gene circuit-mediated metabolite monitoring

Synthetic Gene Circuit-Mediated Monitoring of Endogenous Metabolites: Identification of <i>GAL11</i> as a Novel Multicopy Enhancer of <i>S</i>‑Adenosylmethionine Level in Yeast

Monitoring levels of key metabolites in living cells comprises a critical step in various investigations. The simplest approach to this goal is a fluorescent reporter gene using an endogenous promoter responsive to the metabolite. However, such a promoter is often not identified or even present in the species of interest. An alternative can be a synthetic gene circuit based on a heterologous pair consisting of a promoter and a transcription factor known to respond to the metabolite. We exploited the <i>met</i> operator and MetJ repressor of <i>Escherichia coli</i>, the interaction between which depends on <i>S</i>-adenosylmethionine (SAM), to construct synthetic gene circuits that report SAM levels in <i>Saccharomyces cerevisiae</i>. Using a dual-input circuit that outputs selection marker genes in a doxycycline-tunable manner, we screened a genomic library to identify <i>GAL11</i> as a novel multicopy enhancer of SAM levels. These results demonstrate the potential and utility of synthetic gene circuit-mediated metabolite monitoring

Direct Microscopic Analysis of Individual C₆₀ Dimerization Events: Kinetics and Mechanisms

Modern transition state theory states that the statistical behavior of a chemical reaction is the sum of individual chemical events that occur randomly. Statistical analysis of each event for individual molecules in a three-dimensional space however is practically impossible. We report here that kinetics and mechanisms of chemical reactions can be investigated by using a one-dimensional system where reaction events can be observed in situ and counted one by one using variable-temperature (VT) atomic-resolution transmission electron microscopy (TEM). We thereby provide direct proof that the ensemble behavior of random events conforms to the Rice–Ramsperger–Kassel–Marcus theory, as illustrated for [2 + 2] cycloaddition of C₆₀ molecules in carbon nanotubes (CNTs). This method gives kinetic and structural information for different types of reactions occurring simultaneously in the microscopic view field, suggesting that the VT-TEM opens a new dimension of chemical kinetics research on molecules and their assemblies in their excited and ionized states. The study carried out at 393–493 K showed that pristine CNT primarily acts as a singlet sensitizer of the cycloaddition reaction that takes place with an activation energy of 33.5 ± 6.8 kJ/mol. On the other hand, CNT suffers electron damage of the conjugated system at 103–203 K and promotes a reactive radical cation path that takes place with an activation energy of only 1.9 ± 0.7 kJ/mol. The pre-exponential factor of the Arrhenius plot gave us further mechanistic insights

Direct Microscopic Analysis of Individual C₆₀ Dimerization Events: Kinetics and Mechanisms

Modern transition state theory states that the statistical behavior of a chemical reaction is the sum of individual chemical events that occur randomly. Statistical analysis of each event for individual molecules in a three-dimensional space however is practically impossible. We report here that kinetics and mechanisms of chemical reactions can be investigated by using a one-dimensional system where reaction events can be observed in situ and counted one by one using variable-temperature (VT) atomic-resolution transmission electron microscopy (TEM). We thereby provide direct proof that the ensemble behavior of random events conforms to the Rice–Ramsperger–Kassel–Marcus theory, as illustrated for [2 + 2] cycloaddition of C₆₀ molecules in carbon nanotubes (CNTs). This method gives kinetic and structural information for different types of reactions occurring simultaneously in the microscopic view field, suggesting that the VT-TEM opens a new dimension of chemical kinetics research on molecules and their assemblies in their excited and ionized states. The study carried out at 393–493 K showed that pristine CNT primarily acts as a singlet sensitizer of the cycloaddition reaction that takes place with an activation energy of 33.5 ± 6.8 kJ/mol. On the other hand, CNT suffers electron damage of the conjugated system at 103–203 K and promotes a reactive radical cation path that takes place with an activation energy of only 1.9 ± 0.7 kJ/mol. The pre-exponential factor of the Arrhenius plot gave us further mechanistic insights

Direct Microscopic Analysis of Individual C₆₀ Dimerization Events: Kinetics and Mechanisms

Modern transition state theory states that the statistical behavior of a chemical reaction is the sum of individual chemical events that occur randomly. Statistical analysis of each event for individual molecules in a three-dimensional space however is practically impossible. We report here that kinetics and mechanisms of chemical reactions can be investigated by using a one-dimensional system where reaction events can be observed in situ and counted one by one using variable-temperature (VT) atomic-resolution transmission electron microscopy (TEM). We thereby provide direct proof that the ensemble behavior of random events conforms to the Rice–Ramsperger–Kassel–Marcus theory, as illustrated for [2 + 2] cycloaddition of C₆₀ molecules in carbon nanotubes (CNTs). This method gives kinetic and structural information for different types of reactions occurring simultaneously in the microscopic view field, suggesting that the VT-TEM opens a new dimension of chemical kinetics research on molecules and their assemblies in their excited and ionized states. The study carried out at 393–493 K showed that pristine CNT primarily acts as a singlet sensitizer of the cycloaddition reaction that takes place with an activation energy of 33.5 ± 6.8 kJ/mol. On the other hand, CNT suffers electron damage of the conjugated system at 103–203 K and promotes a reactive radical cation path that takes place with an activation energy of only 1.9 ± 0.7 kJ/mol. The pre-exponential factor of the Arrhenius plot gave us further mechanistic insights

DataSheet_1_Patient-reported outcomes in patients with primary immunodeficiency diseases in Japan: baseline results from a prospective observational study.docx

IntroductionPrimary immunodeficiency diseases (PIDs) are rare inherited diseases resulting in impaired immunity. People with PID experience lower health-related quality of life (HR-QOL) and disease-related burdens in daily activities. This ongoing, prospective observational study aims to evaluate disease activity, treatment status, treatment-related burden, daily activities, and HR-QOL in patients with PID in Japan over a 1-year period. In this interim report (database lock: July 29, 2022), we present baseline results.MethodsParticipants were enrolled between November 2021 and May 2022; data were collected four times/year per participant until May 2023 using an online electronic patient-reported outcomes system. Patients with PID and healthy volunteers aged ≥12 years, residing in Japan, and with access to a smartphone were eligible. HR-QOL (primary endpoint) was assessed by the EuroQol-5 Dimensions-5 Levels (EQ-5D-5L) and the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36). Work productivity was assessed by the Work Productivity and Activity Impairment (WPAI) Questionnaire. Other aspects of PID and burden were assessed with a new questionnaire developed in-house. The study is registered at the University hospital Medical Information Network clinical trials registry (UMIN000045622).ResultsThe full interim analysis set comprised 71 patients with PID and 47 healthy volunteers. The most common International Union of Immunological Societies PID category was primary antibody deficiency (56.3% of patients). Complications were common, especially recurrent respiratory tract infections (63.4%). Most patients with PID were treated with immunoglobulin replacement therapy (73.2%); 22.4% of these patients had serum immunoglobulin levels DiscussionThis interim analysis confirms that patients with PID in Japan have lower HR-QOL and work productivity compared with healthy individuals and experience substantial limitations and burdens in their daily lives.</p

Typical MRI of G0 (right panel) and G2 (left panel; arrows indicate leukoaraiosis).

<p>Typical MRI of G0 (right panel) and G2 (left panel; arrows indicate leukoaraiosis).</p

Differences in critical operation errors only for temporary stop with and without PASAT (one-way ANOVA analysis: * <i>F</i>(1, 18) = 4.91 and <i>p</i><0.05; ** <i>F</i>(1, 22) = 4.34 and <i>p</i><0.05; and *** <i>F</i>(1, 20) = 17.54 and <i>p</i><0.001).

<p>Differences in critical operation errors only for temporary stop with and without PASAT (one-way ANOVA analysis: * <i>F</i>(1, 18) = 4.91 and <i>p</i><0.05; ** <i>F</i>(1, 22) = 4.34 and <i>p</i><0.05; and *** <i>F</i>(1, 20) = 17.54 and <i>p</i><0.001).</p

Steering entropy values with and without PASAT for left turns.

<p>Steering entropy values with and without PASAT for left turns.</p

Positions of the 6-axis accelerometers in the car.

<p>Positions of the 6-axis accelerometers in the car.</p