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The authors study the molecular mechanisms that discriminate axonal microtubules from somatodendritic microtubules. They report that amino acid substitutions in the beta loop region of kinsin-1 can change the compartmentalization of kinesin-1 from axons to axons and dendrites. Moreover, tyrosinated tubulins normally prevent kinesin-1 from binding to microtubules, but do not similarly inhibit kinesin-1 that is changed to allow localization to both axons and dendrites. Neurons form distinctive axonal and dendritic compartments that are important for directional signaling, but the mechanisms that discriminate between axons and dendrites remain elusive. Previous studies have demonstrated that the kinesin-1 motor domain is capable of distinguishing the axon from dendrites. Here we found that the amino acid substitutions in the beta5-loop8 region transformed truncated kinesin-1 from a uni-destination (that is, the axon-specific destination) to a bi-destination (that is, axons and dendrites) state. Furthermore, tyrosinated tubulins that are abundant in somatodendrites prevent the wild-type kinesin-1 from binding to microtubules, whereas the bi-destination–type kinesin-1 does not have this inhibition. Consistently, inhibition of tubulin tyrosination in rat hippocampal neurons resulted in the distribution of truncated kinesin-1 in both axons and dendrites. Our study identifies a molecular mechanism that discriminates the axonal microtubules from somatodendritic microtubules, as well as a previously unknown linkage between tubulin modification and polarized trafficking in neurons.

The introduction of small insertion/deletion (indel) mutations in the coding region of genes by the site-specific nucleases such as Cas9 allows researchers to obtain frameshift null mutants. Technically simple and costly reasonable genotyping methods are awaited to efficiently screen the frameshift null mutant candidates. Here, we developed a simple genotyping method called DST-PCR (Double-strand break Site-Targeted PCR) using “face-to-face” primers where the 3’ ends of forward and reverse primers face each other at the position between 3-bp and 4-bp upstream of the PAM sequence, which is generally the Cas9-mediated double-strand break site. Generated amplicons are directly subjected to TBE-High-Resolution PAGE, which contains a high concentration of bis-acrylamide, for mutant clones detection with 1-bp resolution. We present actual cases of screening of CRISPR/Cas9-engineered knockout (KO) cells for six genes, where we screen indels to obtain potential KO cell clones utilizing our approach. This method allowed us to detect 1-bp to 2-bp insertion and 1-bp to 4-bp deletion in one or both alleles of mutant cell clones. In addition, this technique also allowed the identification of heterozygous and homozygous biallelic functional KO candidates. Thus, DST-PCR is a simple and fast method to screen KO candidates generated by the CRISPR/Cas9 system before the final selection of clones with sequencing.

The brain consists of various areas with anatomical features. Neurons communicate with one another via excitatory or inhibitory synaptic transmission. Altered abundance of neurotransmitters, including glutamate and gamma-aminobutyric acid (GABA), in specific brain regions is closely involved in severe neurological diseases, such as schizophrenia and obsessive-compulsive disorder. SCRAPPER, a ubiquitin E3 ligase, regulates synaptic transmission. Scrapper gene deficiency results in defective neurotransmission due to excessive secretion of neurotransmitters. The present study employed matrix-assisted laser desorption/ionization imaging mass spectrometry to analyze the abundance of amino acid neurotransmitters in Scrapper knockout (SCR-KO) mice. SCR-KO mice exhibited significantly increased glutamate levels in the isocortex (CTX), corpus callosum (CC), thalamus (TH), midbrain (MB), cerebellar cortex (CBX), and caudoputamen (CP) and increased GABA levels in the CTX, CC, TH, MB, CBX and hypothalamus (HY) compared with wild-type mice. These findings indicate that Scrapper deficiency leads to upregulated glutamate and GABA levels in multiple regions. Our results show a differential, region-specific effect of Scrapper on the abundance of glutamate and GABA.

Geissoschizine methyl ether (GM) is one of the main active ingredients responsible for ameliorating the behavioral and psychological symptoms of dementia (BPSD) in Kampo medicine yokukansan. GM is mainly metabolized into hydroxylated forms (HM-1/2). However, the brain distributions of GM and HM has not been reported in vivo . In this study, therefore, the plasma concentrations and brain distribution of these compounds were examined in vivo using rats injected intravenously with GM. Plasma concentrations were analyzed using liquid chromatography-tandem mass spectrometry analysis and brain distribution using mass spectrometry imaging analysis. Plasma GM and HM-1 concentrations decreased in the 4 h after injection, whereas the concentration of plasma HM-2 increased at 4 h. In the 0.25 h-brain, GM signals were diffusely observed throughout the brain, including the cerebral cortex, hippocampus, striatum, thalamus, amygdala, cerebellum, and cerebral ventricle. HM signals were detected only in the ventricles of the brain at 4 h. These results suggest that plasma GM enters the brain and distributes in the parenchyma of various brain regions involved in BPSD, while plasma HM does not enter the brain parenchyma. This study is also the first to visually demonstrate the brain distribution of GM and its metabolite in vivo .

Cancer prognosis remains a critical clinical challenge. Lipidomic analysis via mass spectrometry (MS) offers the potential for objective prognostic prediction, leveraging the distinct lipid profiles of cancer patient-derived specimens. This review aims to systematically summarize the application of MS-based lipidomic analysis in prognostic prediction for cancer patients. Our systematic review summarized 38 studies from the past decade that attempted prognostic prediction of cancer patients through lipidomics. Commonly analyzed cancers included colorectal, prostate, and breast cancers. Liquid (serum and urine) and tissue samples were equally used, with liquid chromatography–tandem MS being the most common analytical platform. The most frequently evaluated prognostic outcomes were overall survival, stage, and recurrence. Thirty-eight lipid markers (including phosphatidylcholine, ceramide, triglyceride, lysophosphatidylcholine, sphingomyelin, phosphatidylethanolamine, diacylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidylethanolamine, lysophosphatidic acid, dihydroceramide, prostaglandin, sphingosine-1-phosphate, phosphatidylinosito, fatty acid, glucosylceramide and lactosylceramide) were identified as prognostic factors, demonstrating potential for clinical application. In conclusion, the potential for developing lipidomics in cancer prognostic prediction was demonstrated. However, the field is still nascent, necessitating future studies for validating and establishing lipid markers as reliable prognostic tools in clinical practice.

Ligamentum flavum hypertrophy (HLF) is the most important component of lumbar spinal canal stenosis (LSCS). Analysis of hypertrophied ligamentum flavum (HLF) samples from patients with LSCS can be an important que. The current study analyzed the surgical samples of HLF samples in patients with LCSC using quantitative and qualitative high performance-liquid chromatography and mass spectrometry. We collected ligamentum flavum (LF) tissue from twelve patients with LSCS and from four patients with lumbar disk herniation (LDH). We defined LF from LSCS patients as HLF and that from LDH patients as non-hypertrophied ligamentum flavum (NHLF). Total lipids were extracted from the LF samples and evaluated for quantity and quality using liquid chromatography and mass spectrometry. The total lipid amount of the HLF group was 3.6 times higher than that of the NHLF group. Phosphatidylcholines (PCs), ceramides (Cers), O-acyl-ω-hydroxy fatty acids (OAHFAs), and triglycerides (TGs) in the HLF group were more than 32 times higher than those of the NHLF group. PC(26:0)+H+, PC(25:0)+H+, and PC(23:0)+H+ increased in all patients in the HLF group compared to the NHLF group. The thickness of the LF correlated significantly with PC(26:0)+H+ in HLF. We identified the enriched specific PCs, Cers, OAHFAs, and TGs in HLF.

The identification of cancer biomarkers is critical for target‐linked cancer therapy. The overall level of phosphatidylcholine (PC) is elevated in colorectal cancer (CRC). To investigate which species of PC is overexpressed in colorectal cancer, an imaging mass spectrometry was performed using a panel of non‐neoplastic mucosal and CRC tissues. In the present study, we identified a novel biomarker, PC(16:0/16:1), in CRC using imaging mass spectrometry. Specifically, elevated levels of PC(16:0/16:1) expression were observed in the more advanced stage of CRC. Our data further showed that PC(16:0/16:1) was specifically localized in the cancer region when examined using imaging mass spectrometry. Notably, because the ratio of PC(16:0/16:1) to lyso‐PC(16:0) was higher in CRC, we postulated that lyso‐PC acyltransferase (LPCAT) activity is elevated in CRC. In an in vitro analysis, we showed that LPCAT4 is involved in the deregulation of PC(16:0/16:1) in CRC. In an immunohistochemical analysis, LPCAT4 was shown to be overexpressed in CRC. These data indicate the potential usefulness of PC(16:0/16:1) for the clinical diagnosis of CRC and implicate LPCAT4 in the elevated expression of PC(16:0/16:1) in CRC.

It is important to determine how a candidate drug is distributed and metabolized within the body in early phase of drug discovery. Recently, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS; also referred to as mass spectrometry imaging) has attracted great interest for monitoring drug delivery and metabolism. Since this emerging technique enables simultaneous imaging of many types of metabolite molecules, MALDI-IMS can visualize and distinguish the parent drug and its metabolites. As another important advantage, changes in endogenous metabolites in response to drug administration can be mapped and evaluated in tissue sections. In this review, we discuss the capabilities of current IMS techniques for imaging metabolite molecules and summarize representative studies on imaging of both endogenous and exogenous metabolites. In addition, current limitations and problems with the technique are discussed, and reports of progress toward solving these problems are summarized. With this new tool, the pharmacological research community can begin to map the in situ pharmacometabolome.

Triple-negative breast cancer (TNBC) is one of the breast cancer subtype that displays a high risk of early recurrence and short overall survival. Improvement of the prognosis of patients with TNBC requires identifying a predictive factor of recurrence, which would make it possible to provide beneficial personalized treatment. However, no clinically reliable predictive factor is currently known. In this study, we investigated the predictive factor of recurrence in TNBC using matrix-assisted laser desorption/ionization-imaging mass spectrometry for lipid profiling of breast cancer specimens obtained from three and six patients with recurrent and non-recurrent TNBC, respectively. The signal for phosphatidylcholine (PC) (32:1) at m/z 732.5 was significantly higher in the recurrence group compared to the non-recurrence group (P = 0.024). PC (32:1) was more abundant in the cancer epithelial area than it was in the surrounding stroma, suggesting that abnormal lipid metabolism was associated with malignant transformation. Our results indicate PC (32:1) as a candidate predictive factor of TNBC recurrence. A future prospective study investigating whether personalized therapy based on PC (32:1) intensity improves the prognosis of patients with TNBC is recommended.

Secondary lymphedema often develops after cancer surgery, and over 250 million patients suffer from this complication. A major symptom of secondary lymphedema is swelling with fibrosis, which lowers the patient's quality of life, even if cancer does not recur. Nonetheless, the pathophysiology of secondary lymphedema remains unclear, with therapeutic approaches limited to physical or surgical therapy. There is no effective pharmacological therapy for secondary lymphedema. Notably, the lack of animal models that accurately mimic human secondary lymphedema has hindered pathophysiological investigations of the disease. Here, we developed a novel rat hindlimb model of secondary lymphedema and showed that our rat model mimics human secondary lymphedema from early to late stages in terms of cell proliferation, lymphatic fluid accumulation, and skin fibrosis. Using our animal model, we investigated the disease progression and found that transforming growth factor‐beta 1 (TGFB1) was produced by macrophages in the acute phase and by fibroblasts in the chronic phase of the disease. TGFB1 promoted the transition of fibroblasts into myofibroblasts and accelerated collagen synthesis, resulting in fibrosis, which further indicates that myofibroblasts and TGFB1/Smad signaling play key roles in fibrotic diseases. Furthermore, the presence of myofibroblasts in skin samples from lymphedema patients after cancer surgery emphasizes the role of these cells in promoting fibrosis. Suppression of myofibroblast‐dependent TGFB1 production may therefore represent an effective pharmacological treatment for inhibiting skin fibrosis in human secondary lymphedema after cancer surgery. Secondary lymphedema develops and causes skin fibrosis after cancer surgery. We developed a novel rat hindlimb model that accurately mimics human secondary lymphedema, and investigated disease progression. We found that myofibroblasts and transforming growth factor‐beta 1/Smad signaling play key roles in the skin fibrosis of secondary lymphedema.