Ankyrin-R Links Kv3.3 to the Spectrin Cytoskeleton and Is Required for Purkinje Neuron Survival.

Ankyrin scaffolding proteins are critical for membrane domain organization and protein stabilization in many different cell types including neurons. In the cerebellum, Ankyrin-R (AnkR) is highly enriched in Purkinje neurons, granule cells, and in the cerebellar nuclei (CN). Using male and female mice with a floxed allele for Ank1 in combination with Nestin-Cre and Pcp2-Cre mice, we found that ablation of AnkR from Purkinje neurons caused ataxia, regional and progressive neurodegeneration, and altered cerebellar output. We show that AnkR interacts with the cytoskeletal protein ß3 spectrin and the potassium channel Kv3.3. Loss of AnkR reduced somatic membrane levels of ß3 spectrin and Kv3.3 in Purkinje neurons. Thus, AnkR links Kv3.3 channels to the ß3 spectrin-based cytoskeleton. Our results may help explain why mutations in ß3 spectrin and Kv3.3 both cause spinocerebellar ataxia.SIGNIFICANCE STATEMENT Ankyrin scaffolding proteins localize and stabilize ion channels in the membrane by linking them to the spectrin-based cytoskeleton. Here, we show that Ankyrin-R (AnkR) links Kv3.3 K+ channels to the ß3 spectrin-based cytoskeleton in Purkinje neurons. Loss of AnkR causes Purkinje neuron degeneration, altered cerebellar physiology, and ataxia, which is consistent with mutations in Kv3.3 and ß3 spectrin causing spinocerebellar ataxia.

Endogenously expressed Ranbp2 is not at the axon initial segment.

Ranbp2 (also known as Nup358) is a member of the nucleoporin family, which constitutes the nuclear pore complex. Ranbp2 localizes at the nuclear membrane and was recently reported at the axon initial segment (AIS). However, we show that the anti-Ranbp2 antibody used in previous studies is not specific for Ranbp2. We mapped the antibody binding site to the amino acid sequence KPLQG, which is present in both Ranbp2 and neurofascin (Nfasc), a well-known AIS protein. After silencing neurofascin expression in neurons, the AIS was not stained by the antibody. Surprisingly, an exogenously expressed N-terminal fragment of Ranbp2 localizes at the AIS. We show that this fragment interacts with stable microtubules. Finally, using CRISPR/Cas9 in primary cultured neurons, we inserted an HA-epitope tag at N-terminal, C-terminal or internal sites of the endogenously expressed Ranbp2. No matter the location of the HA-epitope, endogenous Ranbp2 was found at the nuclear membrane but not the AIS. These results show that endogenously expressed Ranbp2 is not found at AISs.This article has an associated First Person interview with the first author of the paper.

Motor-evoked potential monitoring from urinary sphincter muscle during pediatric untethering surgery: a case series.

PURPOSE: Postoperative urinary dysfunction following untethering surgery for spinal lipoma is devastating. To assess urinary function, we invented a pediatric urinary catheter equipped with electrodes for the direct transurethral recording of myogenic potential from the external urethral sphincter (EUS). This paper presents two cases in which urinary function was monitored intraoperatively by recording of motor-evoked potential (MEP) from EUS during untethering surgery in children. METHODS: Two children (aged 2 and 6 years) were included in this study. One patient had no preoperative neurological dysfunction, while the other had frequent urination and urinary incontinence. A pair of surface electrodes was attached to a silicone rubber urethral catheter (6 or 8 Fr; diameter, 2 or 2.6 mm). The MEP from the EUS was recorded to assess the function of the centrifugal tract from the motor cortex to the pudendal nerve. RESULTS: Baseline MEP waveforms from the EUS were successfully recorded with latency and amplitude of 39.5 ms and 66 µV in patient 1 and 39.0 ms and 113 µV in patient 2, respectively. A significant decrease in amplitude was not observed during surgery in the two cases. No new urinary dysfunction and complications associated with the urinary catheter-equipped electrodes developed postoperatively. CONCLUSION: Using an electrode-equipped urinary catheter, monitoring of MEP from the EUS could be applicable during untethering surgery in pediatric patients.

Microenvironment for spermatogenesis and sperm maturation.

The male reproductive system consists of testes, a series of ducts connecting the testes to the external urethral orifice, accessory sex glands, and the penis. Spermatogonial stem cells differentiate and mature in testes and epididymides, and spermatozoa are ejaculated with exocrine fluids secreted by accessory sex glands. Many studies have clarified the detailed structure and function of the male reproductive system, and have shown that various biologic controls, including genomics, epigenetics, and the neuroendocrine-immune system regulate proliferation, differentiation, and maturation of germ cells. In other words (1) genetic deletion or abnormalities, (2) aberration of DNA methylation and histone modifications, as well as small RNA dysfunction, and (3) neuroendocrine-immune disorders are involved in functional failure of the male reproductive system. In this article, we review these three factors for germ cell microcircumstance, especially focused on the immunoendocrine environment. In particular, the relation between factors protecting germ cells with strong auto-immunogenicity and opposite factors compromising this protection are discussed. Reductions in sperm count, concentration, and semen quality are serious problems in developed countries, although the causes are complex and remain unclear. The accumulation of basic knowledge regarding the structure, function, and regulation of the male reproductive system under various experimental conditions will be important to resolve these problems.

Rheumatoid arthritis is a risk factor for refracture in patients with fragility fractures.

OBJECTIVES: To determine whether patients with rheumatoid arthritis (RA) who have had fragility fractures are at an increased risk of refractures. METHODS: Patients with fragility fractures who were treated surgically at 10 hospitals from 2008 to 2017 and who underwent follow-up for >24 months were either categorized into a group comprising patients with RA or a group comprising patients without RA (controls). The groups were matched 1:1 by propensity score matching. Accordingly, 240 matched participants were included in this study. The primary outcome was the refracture rate in patients with RA as compared to in the controls. Multivariable analyses were also conducted on patients with RA to evaluate the odds ratios (ORs) for the refracture rates. RESULTS: Patients with RA were significantly associated with increased rates of refractures during the first 24 months (OR: 2.714, 95% confidence interval [95% CI]: 1.015-7.255; p = 0.040). Multivariable analyses revealed a significant association between increased refracture rates and long-term RA (OR: 6.308, 95% CI: 1.195-33.292; p = 0.030). CONCLUSIONS: Patients with RA who have experienced fragility fractures are at an increased risk of refractures. Long-term RA is a substantial risk factor for refractures.

Molecular Targeted Therapy for the Bone Loss Secondary to Pyogenic Spondylodiscitis Using Medications for Osteoporosis: A Literature Review.

Pyogenic spondylodiscitis can cause severe osteolytic and destructive lesions in the spine. Elderly or immunocompromised individuals are particularly susceptible to infectious diseases; specifically, infections in the spine can impair the ability of the spine to support the trunk, causing patients to be bedridden, which can also severely affect the physical condition of patients. Although treatments for osteoporosis have been well studied, treatments for bone loss secondary to infection remain to be elucidated because they have pathological manifestations that are similar to but distinct from those of osteoporosis. Recently, we encountered a patient with severely osteolytic pyogenic spondylodiscitis who was treated with romosozumab and exhibited enhanced bone formation. Romosozumab stimulated canonical Wnt/ß-catenin signaling, causing robust bone formation and the inhibition of bone resorption, which exceeded the bone loss secondary to infection. Bone loss due to infections involves the suppression of osteoblastogenesis by osteoblast apoptosis, which is induced by the nuclear factor-κB and mitogen-activated protein kinase pathways, and osteoclastogenesis with the receptor activator of the nuclear factor-κB ligand-receptor combination and subsequent activation of the nuclear factor of activated T cells cytoplasmic 1 and c-Fos. In this study, we review and discuss the molecular mechanisms of bone loss secondary to infection and analyze the efficacy of the medications for osteoporosis, focusing on romosozumab, teriparatide, denosumab, and bisphosphonates, in treating this pathological condition.

Neonatal maternal separation causes decreased numbers of sertoli cell, spermatogenic cells, and sperm in mice.

Neonatal maternal separation is an experimental model used to evaluate the effects of toxic stress in neonates, or early life stress. Although various physiological and psychological stresses during childhood have been reported, the effects of neonatal maternal separation on the male reproductive system remain unclear. Therefore, the present study evaluated the effects of neonatal maternal separation on the male reproductive system. In neonatal male ICR mice, maternal separation was performed for 0.5, 1, 2, and 4 hours/day, from postnatal day 1 to 10. At 10 weeks of age, the neonatal maternal separation mice exhibited decreases in both testicular weight and epididymal sperm number, along with various testicular morphological changes involving germ cells, Sertoli cells, and interstitial cells. Notably, neonatal maternal separation mice showed decreased numbers of Sertoli cells. Animals subjected to 0.5-, 1-, and 2-h/day neonatal maternal separation exhibited decreases in serum levels of testosterone but not in those of gonadotropin (luteinizing hormone and follicle-stimulating hormone). Together, these data showed that neonatal maternal separation in male mice causes decreased Sertoli cell numbers following puberty, resulting in subsequent decreased spermatogenic activity.

A cross-sectional study of relationships between periodontal disease and general health: The Hitachi Oral Healthcare Survey.

BACKGROUND: This cross-sectional study performed to clarify the relationship between periodontal disease and non-communicable diseases (NCDs), such as obesity, diabetes mellitus, impaired glucose tolerance (IGT), chronic obstructive pulmonary disease (COPD), and atherosclerotic cardiovascular disease (ASCVD) by introducing dental examinations into the annual health examinations conducted by Japanese companies, and to highlights the importance of a medical system that connects dental and medical professionals. METHODS: A total of 1.022 Hitachi Ltd. employees were enrolled in this cross-sectional study. We examined correlations and odds ratios (ORs) between the dental and overall health of employees using stratification and multiple logistic regression analyses based on the periodontal health indicators, general health indicators, and occlusal force. RESULTS: The adjusted OR of PPD for obesity (OR, 1.42; 95% confidence interval [CI], 1.09-1.84; p = 0.009), IGT (OR, 1.48; 95% CI, 1.00-2.20; p = 0.049), and COPD (OR, 1.38; 95% CI, 1.02-1.88; p = 0.038) significantly differed. The adjusted OR of body mass index (OR, 1.28; 95% CI 1.15-1.42; p < 0.001), haemoglobin A1C (HbA1c) (OR, 4.34; 95% CI, 1.89-9.98; p < 0.001), fasting blood glucose (FBG) levels (OR, 1.08; 95% CI 1.04-1.11; p < 0.001), postbronchodilator forced expiratory volume in one second/forced vital capacity ratio (%FEV1) (OR, 0.95; 95% CI 0.91-1.00; p = 0.031) and smoking (OR, 2.32; 95% CI 1.62-3.33; p < 0.001) for severe periodontal disease also significantly differed. Occlusal force was significantly reduced in employees aged 50-59 years compared to those aged 40-49 years. Both PPD, HbA1c, FBG levels were significantly associated with occlusal force among employees with moderate/severe periodontitis. PPD was significantly associated with occlusal force among employees with and moderate COPD, and ASCVD. %FEV1 was significantly associated with occlusal force among employees with IGT. CONCLUSIONS: This cross-sectional study revealed mutual relationships among periodontal disease, NCDs, and occlusal force on Japanese corporate workers. We demonstrated that a comprehensive, regional healthcare system centred on annual integrated dental and physical health examinations in the workplace will benefit employees and positively impact corporate health insurance.

QUANTIFICATION OF CHOROIDAL VASCULATURE BY HIGH-QUALITY STRUCTURE EN FACE SWEPT-SOURCE OPTICAL COHERENCE TOMOGRAPHY IMAGES IN EYES WITH CENTRAL SEROUS CHORIORETINOPATHY.

PURPOSE: To determine the density of the choroidal vasculature by high-quality structure en face optical coherence tomography (OCT) scanned at the same time as OCT angiography in eyes with central serous chorioretinopathy (CSC). METHODS: Thirty-five eyes of 30 patients with CSC (20 men and 10 women; average age, 48.4 years) were studied. Volume scans (12 × 12 mm-square) were obtained at the same time as OCT angiographic scans (Plex Elite 9000; Swept-Source OCT, Zeiss). High-quality structure en face images were flattened at Bruch membrane and binarized to identify and quantify the choroidal vascular density by the Bernsen method of the segmentation slab of one-half of the choroidal thickness. Similarly, high-quality structure en face choroidal images of 35 healthy eyes of 29 patients (18 men and 11 women; average age, 51.7 years) were binarized and analyzed as controls. The en face images were cropped to exclude the optic disk. RESULTS: The mean cropped image size was 9.57 mm × 9.57 mm in the eyes with CSC and 9.48 mm × 9.48 mm in the healthy eyes (P = 0.41). In the eyes with CSC, the choroidal vascular density was 61.6 ± 7.5% of the choroid, which was significantly greater than that in the healthy eyes at 49.4 ± 7.5% (P < 0.01). CONCLUSION: High-quality structure en face OCT can be used to assess the density of the choroidal vessels quantitatively and noninvasively in eyes with CSC.

Co-Administration of the Traditional Medicines Hachimi-Jio-Gan and Hochu-Ekki-To Can Reverse Busulfan-Induced Aspermatogenesis.

Busulfan is used as a chemotherapeutic drug to treat childhood and adult chronic myelogenous leukemia, and as an immunosuppressive agent before bone marrow transplantation. A key side effect of busulfan is the alteration of male reproductive function. Infertility caused by anti-cancer treatments has become a significant concern, but there are currently limited treatments for this condition. Recently, we demonstrated that Gosha-jinki-gan, a traditional Japanese medicine, completely reversed the spermatogenesis defects caused by cancer treatment in mice. Hochu-ekki-to and Hachimi-jio-gan are commonly used to treat male infertility, and Hachimi-jio-gan shares herbal ingredients with Gosha-jinki-gan. Therefore, in the present study, we administered Hachimi-jio-gan and Hochu-ekki-to alone or in combination to mice with severe aspermatogenesis caused by busulfan treatment. We performed testis weight measurements, quantitative histological assessments of the testes and the epididymis, and evaluated sperm counts and morphology. We also assessed the expression of immune mediators and macrophage markers. Treatment with a combination of both the medicines significantly reduced busulfan-induced testicular toxicity when compared to the lone treatment with either medicine. We demonstrated that treatment efficacy was related to a differential impact on testicular inflammation, and that the synergistic effect of co-administration completely reversed the busulfan-induced damage to the reproductive functions.

Immunological microenvironment in the testis.

BACKGROUND: The testis is specific in that it produces haploid germ cells of which autoantigens newly appear long after the neonatal immune tolerance. Under normal condition, these autoantigens are protected by the blood-testis barrier formed by Sertoli cells. Thus, the testis is an immunologically privileged site where haploid cells are protected from autoimmune attack. METHODS: The immunological microenvironment in the testis was experimentally investigated using mice and rats. MAIN FINDINGS: Not only the blood-testis barrier but also various immuno-suppressive factors are involved in the immune-privileged testis. Indeed, germ cells transplanted into the xenogeneic seminiferous tubules could proliferate and differentiate with no aid of artificial immunosuppression. On the other hand, autoimmune orchitis could be experimentally produced by various methods of immunization with syngeneic or xenogeneic germ cell antigens. CONCLUSION: Our results indicate that the testis is immunologically privileged but also immunologically fragile organ. Therefore, the dual nature is critical for immunoregulation of testicular function.

The energetic brain - A review from students to students.

The past 20 years have resulted in unprecedented progress in understanding brain energy metabolism and its role in health and disease. In this review, which was initiated at the 14th International Society for Neurochemistry Advanced School, we address the basic concepts of brain energy metabolism and approach the question of why the brain has high energy expenditure. Our review illustrates that the vertebrate brain has a high need for energy because of the high number of neurons and the need to maintain a delicate interplay between energy metabolism, neurotransmission, and plasticity. Disturbances to the energetic balance, to mitochondria quality control or to glia-neuron metabolic interaction may lead to brain circuit malfunction or even severe disorders of the CNS. We cover neuronal energy consumption in neural transmission and basic ('housekeeping') cellular processes. Additionally, we describe the most common (glucose) and alternative sources of energy namely glutamate, lactate, ketone bodies, and medium chain fatty acids. We discuss the multifaceted role of non-neuronal cells in the transport of energy substrates from circulation (pericytes and astrocytes) and in the supply (astrocytes and microglia) and usage of different energy fuels. Finally, we address pathological consequences of disrupted energy homeostasis in the CNS.

Matrin 3 Is a Component of Neuronal Cytoplasmic Inclusions of Motor Neurons in Sporadic Amyotrophic Lateral Sclerosis.

Mutations in the MATR3 gene have been identified as a cause of familial amyotrophic lateral sclerosis, but involvement of the matrin 3 (MATR3) protein in sporadic amyotrophic lateral sclerosis (SALS) pathology has not been fully assessed. We immunohistochemically analyzed MATR3 pathology in the spinal cords of SALS and control autopsy specimens. MATR3 immunostaining of the motor neuron nuclei revealed two distinct patterns: mild and strong staining. There were no differences in the ratio of mild versus strong nuclear staining between the SALS and control cases. MATR3-containing neuronal cytoplasmic inclusions (NCIs) were observed in 60% of SALS cases. Most motor neurons with MATR3-positive NCIs exhibited a mild nuclear staining pattern. Although 16.8% of NCIs positive for transactivating response region DNA-binding protein 43 (TDP-43) were estimated as double-labeled by MATR3, no MATR3-positive or TDP-43-negative NCIs were observed. Although a previous study found that MATR3-positive NCIs are present only in cases with C9orf72 hexanucleotide repeat expansion, ubiquitin-positive granular NCIs were not observed in the cerebellum, which have been reported as specific to C9orf72-related ALS. Six ALS cases were confirmed to be negative for the GGGGCC hexanucleotide. Our results reveal that MATR3 is a component of TDP-43-positive NCIs in motor neurons, even in SALS, and indicate the broader involvement of MATR3 in ALS pathology and the heterogeneity of TDP-43-positive NCIs.

Interaction potential surface between Raman scattering enhancing nanoparticles conjugated with a functional copolymer.

A novel Raman scattering enhancement was discovered using colloid nanoparticles conjugated with an amine-based copolymer. The interaction potential surface between Raman scattering enhancing nanoparticles was clarified by combining a small-angle scattering method and a model-potential-free liquid-state theory as an in situ observation in the solution state. The potential surface indicates that the most stable position is located around 0.9 nm from the particle surface, suggesting the existence of a nanogap structure between the nanocomposites. The change in Raman scattering enhancement was also acquired during the dispersion process of the aggregated nanocomposites through a glutathione-triggered nanosensing reaction.

The effectiveness of traditional Japanese medicine Goshajinkigan in irradiation-induced aspermatogenesis in mice.

BACKGROUND: Infertility and gonadal dysfunction are well known side-effects by cancer treatment in males. In particularly, chemotherapy and radiotherapy induced testicular damage, resulting in prolonged azoospermia. However, information regarding therapeutics to treat spermatogenesis disturbance after cancer treatment is scarce. Recently, we demonstrated that Goshajinkigan, a traditional Japanese medicine, can completely rescue severe busulfan-induced aspermatogenesis in mice. In this study, we aimed to detect the effects of Goshajinkigan on aspermatogenesis after irradiation. METHODS: This is animal research about the effects of traditional Japanese medicine on infertility after cancer treatment. C57BL/6 J male mice received total body irradiation (TBI: a single dose of 6Gy) at 4 weeks of age and after 60 days were reared a Goshajinkigan (TJ107)-containing or TJ107-free control diet from day 60 to day 120. Then, two untreated females were mated with a single male from each experimental group. On day 60, 120 and 150, respectively, the sets of testes and epididymis of the mice in each group after deep anesthetization were removed for histological and cytological examinations. RESULTS: Histological and histopathological data showed that 6Gy TBI treatment decreased the fertility rate (4/10) in the control diet group; in contrast, in the TJ107-diet group, the fertility rate was 10/10 (p < 0.05 vs. 6Gy group). Supplementation with TJ107 was found to rescue the disrupted inter-Sertoli tight junctions via the normalization of claudin11, occludin, and ZO-1 expression and reduce serum anti-germ cell autoantibodies. CONCLUSIONS: These findings show the therapeutic effect on TBI-induced aspermatogenesis and the recovering disrupted gonadal functions by supplementation with TJ107.

Gosha-Jinki-Gan Recovers Spermatogenesis in Mice with Busulfan-Induced Aspermatogenesis.

Busulfan is an anti-cancer chemotherapeutic drug and is often used as conditioning regimens prior to bone marrow transplant for treatment of chronic myelogenous leukemia. Male infertility, including spermatogenesis disturbance, is known to be one of the side effects of anticancer drugs. While hormone preparations and vitamin preparations are used for spermatogenesis disturbance, their therapeutic effects are low. Some traditional herbal medicines have been administered to improve spermatogenesis. In the present study, we administered Gosha-jinki-gan (TJ107; Tsumura Co., Ltd., Tokyo, Japan) to mice suffering from severe aspermatogenesis after busulfan treatment to determine whether TJ107 can recover spermatogenesis. Male 4-week-old C57BL/6J mice were administered a single intraperitoneal injection of busulfan, and they were then fed a normal diet for 60 days and then a TJ107 diet or TJ107-free normal diet for another 60 days. After busulfan treatment, the weight of the testes and the epididymal sperm count progressively decreased in the normal diet group. On the other hand, in the TJ107 group, these variables dramatically recovered at 120 days. These results suggest that busulfan-induced aspermatogenesis is irreversible if appropriate treatment is not administered. Supplementation of TJ107 can completely recover the injured seminiferous epithelium via normalization of the macrophage migration and reduction of the expressions of Tool-like receptor (TLR) 2 and TLR4, suggesting that TJ107 has a therapeutic effect on busulfan-induced aspermatogenesis.

Population pharmacokinetics of doxapram in low-birth-weight Japanese infants with apnea.

UNLABELLED: This study aimed to determine the population pharmacokinetics of doxapram in low-birth-weight (LBW) infants. A total of 92 serum concentration measurements that were obtained from 34 Japanese neonates were analyzed using nonlinear mixed-effect modeling (NONMEM). Estimates generated by NONMEM indicated that clearance of doxapram (CL; L/kg/h) was affected by postmenstrual age (PMA; weeks), body weight (BW; g), and aspartate aminotransferase (AST; IU/L). In addition, the volume of distribution (Vd; L/kg) was affected by gestational age (GA; weeks). The final pharmacokinetic model was as follows: CL = BW / PMA × 0.0453 × serum AST(-0.373); Vd = 2.54 (if GA >28 weeks) and Vd = 2.54 × 2.11 (if GA ≤28 weeks). The interindividual variabilities in CL and Vd were 39.9 and 83.0 %, respectively, and the residual variability was 20.9 %. To clarify the reasons for large interindividual variations, the enzymes involved in the metabolic pathway of doxapram were also determined. We found that doxapram was metabolized by CYP3A4/5. CONCLUSION: We report the population pharmacokinetics of doxapram in neonates and the involvement of CYP3A4/5 in its metabolism. The final model of population pharmacokinetics may be useful for formulating a safe and effective dosage regimen and for predicting serum doxapram concentrations in neonates.

Combination of thermographic and ultrasonographic assessments for early detection of deep tissue injury.

Early detection and intervention of deep tissue injury are important to lead good outcome. Although the efficiency of ultrasonographic assessment of deep tissue injury has been reported previously, it requires a certain level of skill for accurate assessment. In this study, we present an investigation of the combination of thermographic and ultrasonographic assessments for early detection of deep tissue injury. We retrospectively reviewed 28 early-stage pressure ulcers (21 patients) presenting at the University of Tokyo Hospital between April 2009 and February 2010, surveying the associated thermographic and ultrasonographic findings. The wound temperature patterns were divided into low, even and high compared with the surrounding skin. Ultrasonographic findings were classified into unclear layer structure, hypoechoic lesion, discontinuous fascia and heterogeneous hypoechoic area. All 13 ulcers that were associated with low temperature showed good outcome; three ulcers had even temperatures and 12 ulcers showed high temperature on thermographic assessment. The two deep tissue injuries were rated high on thermographic assessment and showed heterogeneous hypoechoic area findings on ultrasonographic assessment. No non-deep tissue injury lesion was associated with these two findings simultaneously. The combination of thermographic and ultrasonographic assessments is expected to increase the accuracy of the early detection of deep tissue injuries.

TRIM46 is not required for axon specification or axon initial segment formation in vivo.

Vertebrate nervous systems use the axon initial segment (AIS) to initiate action potentials and maintain neuronal polarity. The microtubule-associated protein tripartite motif containing 46 (TRIM46) was reported to regulate axon specification, AIS assembly, and neuronal polarity through the bundling of microtubules in the proximal axon. However, these claims are based on TRIM46 knockdown in cultured neurons. To investigate TRIM46 function in vivo , we examined TRIM46 knockout mice. Contrary to previous reports, we find that TRIM46 is dispensable for AIS formation and maintenance, and axon specification. TRIM46 knockout mice are viable, have normal behavior, and have normal brain structure. Thus, TRIM46 is not required for AIS formation, axon specification, or nervous system function. We also show TRIM46 enrichment in the first ∼100 µm of axon occurs independently of ankyrinG (AnkG), although AnkG is required to restrict TRIM46 only to the AIS. Our results suggest an unidentified protein may compensate for loss of TRIM46 in vivo and highlight the need for further investigation of the mechanisms by which the AIS and microtubules interact to shape neuronal structure and function. SIGNIFICANCE STATEMENT: A healthy nervous system requires the polarization of neurons into structurally and functionally distinct compartments, which depends on both the axon initial segment (AIS) and the microtubule cytoskeleton. In contrast to previous reports, we show that the microtubule-associated protein TRIM46 is not required for axon specification or AIS formation in mice. Our results emphasize the need for further investigation of the mechanisms by which the AIS and microtubules interact to shape neuronal structure and function.

Metabolic and enzymatic engineering approach for the production of 2-phenylethanol in engineered Escherichia coli.

2-Phenylethanol, known for its rose-like odor and antibacterial activity, is synthesized via exogenous phenylpyruvate by the sequential reaction of phenylpyruvate decarboxylase (PDC) and aldehyde reductase. We first targeted ARO10, a phenylpyruvate decarboxylase gene from Saccharomyces cerevisiae, and identified a suitable aldehyde reductase gene. Co-expression of ARO10 and yahK in E. coli transformants yielded 1.1 g/L of 2-phenylethanol in batch culture. We hypothesized that there might be a bottleneck in PDC activity. The computer-based enzyme evolution was utilized to enhance production. The introduction of an amino acid substitution in ARO10 (ARO10 I544W) stabilized the aromatic ring of the phenylpyruvate substrate, increasing 2-phenylethanol yield 4.1-fold compared to wild-type ARO10. Cultivation of ARO10 I544W-expressing E. coli produced 2.5 g/L of 2-phenylethanol with a yield from glucose of 0.16 g/g after 72 h. This approach represents a significant advancement, achieving the highest yield of 2-phenylethanol from glucose using microbes to date.