Chemogenetic activation of mammalian brain neurons expressing insect Ionotropic Receptors by systemic ligand precursor administration.

Chemogenetic approaches employing ligand-gated ion channels are advantageous regarding manipulation of target neuronal population functions independently of endogenous second messenger pathways. Among them, Ionotropic Receptor (IR)-mediated neuronal activation (IRNA) allows stimulation of mammalian neurons that heterologously express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the original protocol, phenylacetic acid, a ligand of the IR84a/IR8a complex, was locally injected into a brain region due to its low permeability of the blood-brain barrier. To circumvent this invasive injection, we sought to develop a strategy of peripheral administration with a precursor of phenylacetic acid, phenylacetic acid methyl ester, which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. This strategy was validated by electrophysiological, biochemical, brain-imaging, and behavioral analyses, demonstrating high utility of systemic IRNA technology in the remote activation of target neurons in the brain.

Japanese General Clinical Oncologists' Knowledge and Real-world Experiences of Cancer Genomic Medicine: A Nationwide Web-based Survey Study.

Introduction: In Japan, insurance began covering two cancer gene panel tests in 2019. However, the availability of these tests remains limited to 247 facilities (as of October 2023). This survey-based study assessed the knowledge and recognition of cancer genomic medicine by physicians involved in cancer treatment. Methods: Written requests for participation in a web-based questionnaire survey were sent to 14,579 affiliated general clinical oncologists certified by the Japanese Board of Cancer Therapy. The survey was conducted from July 1 to 31st, 2021. Data between physicians affiliated with cancer genome hospitals and noncancer genome hospitals and between regions of Japan were compared. Results: In total, 2,402 valid responses were analyzed. Of the respondents, 1,296 and 1,106 were physicians working at cancer and noncancer genome hospitals, respectively. Physicians working at cancer genome hospitals showed significantly higher results for both knowledge of cancer genomic medicine and experience in cancer gene panel test performance compared with those working at noncancer genome hospitals. There were no significant regional differences in the percentage of physicians who reported having performed cancer gene panel tests. Conclusions: The survey results suggest a disparity in the knowledge of cancer genomic medicine between physicians working at cancer genome hospitals and those working at noncancer genome hospitals; this disparity should be addressed by stakeholders. Closer collaboration between these facilities may be necessary to achieve national dissemination of cancer genomic medicine.

In Vitro Metabolism of 2-Methoxy-N-[3-[4-[3-methyl-4-[(6-methyl- 3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) by Aldehyde Oxidase and Predicting Its Percent Contribution Relative to CYP-Mediated Metabolism.

2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) is an anticancer drug that was discontinued due to hepatotoxicity found in clinical studies. Metabolite analysis of CP-724,714 was conducted using human hepatocytes, in which twelve oxidative metabolites and one hydrolyzed metabolite were formed. Among the three mono-oxidative metabolites, the formation of two was inhibited by adding 1-aminobenzotriazole, a pan-CYP inhibitor. In contrast, the remaining one was not affected by this inhibitor but partially inhibited by hydralazine, indicating that aldehyde oxidase (AO) was involved in metabolizing CP-724,714, which contains a quinazoline substructure, a heterocyclic aromatic quinazoline ring, known to be preferably metabolized by AO. One of the oxidative metabolites of CP-724,714 observed in human hepatocytes was also generated in recombinant human AO. Although CP-724,714 is metabolized by both CYPs and AO in human hepatocytes, the contribution level of AO could not be evaluated using its specific inhibitors because of low AO activity in in vitro human materials. Here, we present a metabolic pathway for CP-724,714 in human hepatocytes and the involvement of AO in CP-724,714 metabolism. We showed here a plausible workflow for predicting AO contribution to the metabolism of CP-724,714 based on DMPK screening data. SIGNIFICANCE STATEMENT: 2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) was identified as a substrate of aldehyde oxidase (AO) rather than xanthine oxidase. Since CP-724,714 is also metabolized by cytochrome P450s (CYPs), the contribution levels of AO and CYPs in the metabolism of CP-724,714 were estimated simultaneously based on in vitro drug metabolism screening data.

Possible Significance of a Café-style Event to Introduce Advance Care Planning for General Citizens.

OBJECTIVES: We conducted a survey to determine whether the general public who participated in a café-style event to raise awareness of advance care planning (ACP) actually implemented ACP after attending the event. METHODS: On February 20, 2020, a café-style event (Tokai Blue Café: TBC) was held at the Tokai University Hospital. The TBC consisted of a lecture about ACP, "The Go-Wish Game," and a tea party. A questionnaire-based survey was conducted on ACP implementation after one month of TBC. RESULTS: Of the 14 participants (three males and 11 females), 11 agreed to answer the questionnaire and eight responded. Two respondents were male and six were female. Six of the respondents were aged ≥ 60 years. Seven of the eight respondents implemented ACP with their family members, while none did so with their family doctor, even though all of them indicated that they had a family doctor. Several respondents reported that they were uncomfortable discussing the issue with their doctors. CONCLUSION: The results indicate that a café-style event as an awareness-raising activity may have a significant effect on ACP implementation, although it suggests that there are some challenges in involving family doctors.

Jervell and Lange-Nielsen syndrome with novel KCNQ1 and additional gene mutations.

We encountered a boy with Jervell and Lange-Nielsen syndrome (JLNS) with compound heterozygous KCNQ1 mutations, maternal Trp248Phe and a novel paternal mutation, Leu347Arg. His father showed long QT (LQT) and arrhythmia. His mother was asymptomatic with no ECG abnormalities. The proband and his father had an additional mutation (SNTA1 Thr372Met), which is reportedly related to SIDS. These results suggest that multiple gene mutations influence the phenotype of KCNQ1 mutation-related arrhythmia.

Enhanced Retrieval of Taste Associative Memory by Chemogenetic Activation of Locus Coeruleus Norepinephrine Neurons.

The ability of animals to retrieve memories stored in response to the environment is essential for behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. Here, we developed a novel chemogenetic activation strategy exploiting insect olfactory ionotropic receptors (IRs), termed "IR-mediated neuronal activation," and used it for selective stimulation of NE neurons in the locus coeruleus (LC). Drosophila melanogaster IR84a and IR8a subunits were expressed in LC NE neurons in transgenic mice. Application of phenylacetic acid (a specific ligand for the IR84a/IR8a complex) at appropriate doses induced excitatory responses of NE neurons expressing the receptors in both slice preparations and in vivo electrophysiological conditions, resulting in a marked increase of NE release in the LC nerve terminal regions (male and female). Ligand-induced activation of LC NE neurons enhanced the retrieval process of conditioned taste aversion without affecting taste sensitivity, general arousal state, and locomotor activity. This enhancing effect on taste memory retrieval was mediated, in part, through α1- and ß-adrenergic receptors in the basolateral nucleus of the amygdala (BLA; male). Pharmacological inhibition of LC NE neurons confirmed the facilitative role of these neurons in memory retrieval via adrenergic receptors in the BLA (male). Our findings indicate that the LC NE system, through projections to the BLA, controls the retrieval process of taste associative memory.SIGNIFICANCE STATEMENT Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation, but the role of the NE system in memory retrieval remains unclear. We developed a chemogenetic activation system based on insect olfactory ionotropic receptors and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons enhanced the retrieval of conditioned taste aversion, which was mediated, in part, through adrenoceptors in the basolateral amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC-amygdala pathway plays an important role in the recall of taste associative memory.

Human PZP and common marmoset A2ML1 as pregnancy related proteins.

While pregnancy-related proteins (PRP) are known to contribute to immunotolerance during pregnancy, their significance to development of invasive placenta is unclear. We compared PRP expression in humans and the common marmoset (Callithrix jacchus), a new-world monkey. Invasive placenta was observed at the maternal-foetal interface of marmoset placenta from green fluorescent protein (GFP)-expressing foetus and wild type mother. The pregnancy zone protein (PZP) and alpha-2 macroglobulin-like 1 (A2ML1) proteins exhibited the most prominent increase in expression during the second trimester in humans and marmoset, respectively. In humans, PZP accumulated at the maternal-foetal interface and A2ML1 accumulated in the amnion. Similarly, A2ML1 mRNA was detected in marmoset placenta. These proteins belong to the A2M family of protease inhibitors, and both PZP and A2ML1 share around 90% homology between human and marmoset and have highly conserved structures. However, the protease-reacting bait regions of the proteins had lower homology (56.8-60.7% in proteins) relative to the rest of the sequence. Notably, the cleavage site of a proinflammatory proline-endopeptidase was preserved in human PZP and marmoset A2ML1. These proteins contain multiple sites that are cleaved by proteases involving proline-endopeptidase. Systemic regulation of these A2M family proteins may be important in animals with invasive placenta.

A novel gene (FAM20B encoding glycosaminoglycan xylosylkinase) for neonatal short limb dysplasia resembling Desbuquois dysplasia.

Desbuquois dysplasia (DBQD) is an autosomal recessive heterogeneous disorder characterized by joint laxity and skeletal changes, including a distinctive monkey-wrench appearance of the femora, advanced carpal ossification, and abnormal patterning of the preaxial digits. Two genes for DBQD (CANT1 encoding calcium-activated nucleotidase-1 and XYLT1 encoding xylosyltransferase-1) have been reported. We propose a novel gene for neonatal short limb dysplasia resembling DBQD, based on the phenotype and genotype of two affected siblings. The affected boy and girl died in early infancy and shortly after birth, respectively. The clinical hallmarks included mid-face hypoplasia, thoracic hypoplasia with respiratory failure, very short stature (approximately -7 SD of birth length) with mesomelic shortening of the limbs, and multiple dislocations of the large joints. Radiological examinations showed prominent lesser trochanter, flared metaphyses of the long bones, and joint dislocations. The affected boy had preaxial digital hypoplasia, and the affected girl showed overlapping and syndactyly of the preaxial digits. Molecular analyses of the girl showed compound heterozygous variants in FAM20B (NM_014864: c.174_178delTACCT p.T59Afs*19/c.1038delG p.N347Mfs*4). FAM20B encodes glycosaminoglycan xylosylkinase, which acts downstream of xylosyltransferase-1. Given the fact that FAM20B deficiency causes skeletal phenotypes in mice and zebrafish, these variants are highly probable to be pathogenic.

Exome and copy number variation analyses of Mayer-Rokitansky-Küster- Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by congenital absence of the vagina and uterus. We conducted genome-wide SNP analyses and exome sequencing to detect the causes of MRKH syndrome. We identified de novo variants of MYCBP2, NAV3, and PTPN3 in three families and a variant of MYCBP2 in a sporadic case. Here, we demonstrated the partial genetic makeup of Japanese MRKH syndrome.

Laparoscopic Davydov Procedure for the Creation of a Neovagina in Patients with Mayer-Rokitansky-Kuster-Hauser Syndrome: Analysis of 7 Cases.

OBJECTIVE: Several surgical techniques have been described for creating a neovagina in patients with Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, however as yet there is no standardized treatment. The aim of this report is to assess anatomic and functional outcomes after the laparoscopic Davydov procedure for the creation of a neovagina in patients with MRKH syndrome. METHODS: Seven patients with MRKH syndrome underwent the laparoscopic Davydov technique from January 2005 to August 2010. The anatomic and functional results were evaluated after 3, 6, 12, 24, 36, 48, and 60 months. RESULTS: The surgical procedure was performed with no major complications except in one case in which an intraoperative bladder injury occurred and was successfully corrected. The mean duration of surgery was 162.9 minutes (range, 120-230 min). Mean lengths/widths (cm) of the neovagina were 6.4/2.6, 6.5/2.5, 6.5/2.8, 6.4/2.8, 7.1/2.8, and 7.2/2.8 at 3, 6,12, 24, 36, 48, and 60 postoperative months, respectively. CONCLUSION: The laparoscopic Davydov procedure seems to be a safe and effective surgical treatment for patients with MRKH syndrome if postoperative intermittent self dilation was done.