Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System.

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of <10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

A water-soluble luminescent tris(2,4,6-trichlorophenyl)methyl radical-carbazole dyad.

Organic luminescent radicals are a new class of materials with potential applications not only in light-emitting devices but also in the biochemistry field. New tris(2,4,6-trichlorophenyl)methyl (TTM) radicals with alkoxy-substituted carbazole donors were synthesized and characterized. PEG-substituted carbazole-TTM was found to be water-soluble. The water-soluble TTM radical aqueous solution showed fluorescence at 777 nm and the ability to shorten the longitudinal relaxation time (T1) of water. The concept of water-soluble luminescent radicals is expected to be used to develop a potential fluorescence and MR dual-use imaging moiety.

Exogenous polyserine fibrils change membrane properties of phosphatidylcholine-liposome and red blood cells.

The causative genes for neurodegenerative polyglutamine (polyQ) diseases produce homopolymeric polyglutamine (polyQ), polyserine (polyS), polyalanine (polyA), polycysteine (polyC), and polyleucine (polyL) sequences by repeat-associated non-AUG (RAN) translation. The cytotoxicity of the intracellular polyQ and RAN products has been extensively investigated. However, little is known about the toxicity of the extracellular polyQ and RAN products on the membranes of viable cells. Because polyQ aggregates induce a deflated morphology of a model membrane, we hypothesized that extracellular polyQ and RAN products might affect the membrane properties of viable cells. In this study, we demonstrated that exogenous polyS fibrils but not polyS or polyQ non-fibril aggregates altered the thermal phase transition behavior of a model membrane composed of a phosphatidylcholine bilayer using differential scanning calorimetry. PolyS fibrils induced morphological changes in viable red blood cells (RBCs). However, both polyS and polyQ non-fibril aggregates had no effects on RBCs. These results highlight the possibility that extracellular fibrils generated from RAN products may alter the properties of neuronal cell membranes, which may contribute to changes in the brain pathology.

Age-related ciliopathy: Obesogenic shortening of melanocortin-4 receptor-bearing neuronal primary cilia.

Obesity is often associated with aging. However, the mechanism of age-related obesity is unknown. The melanocortin-4 receptor (MC4R) mediates leptin-melanocortin anti-obesity signaling in the hypothalamus. Here, we discovered that MC4R-bearing primary cilia of hypothalamic neurons progressively shorten with age in rats, correlating with age-dependent metabolic decline and increased adiposity. This "age-related ciliopathy" is promoted by overnutrition-induced upregulation of leptin-melanocortin signaling and inhibited or reversed by dietary restriction or the knockdown of ciliogenesis-associated kinase 1 (CILK1). Forced shortening of MC4R-bearing cilia in hypothalamic neurons by genetic approaches impaired neuronal sensitivity to melanocortin and resulted in decreased brown fat thermogenesis and energy expenditure and increased appetite, finally developing obesity and leptin resistance. Therefore, despite its acute anti-obesity effect, chronic leptin-melanocortin signaling increases susceptibility to obesity by promoting the age-related shortening of MC4R-bearing cilia. This study provides a crucial mechanism for age-related obesity, which increases the risk of metabolic syndrome.

Inner and outer penetrating spinal cord injuries lead to distinct overground walking in mice.

Spinal cord injury (SCI) is a devastating mechanical trauma. Although locomotion of model animals that mimic contusion SCI was actively examined, locomotion after penetrating SCI caused by sharp objects was not extensively studied. Severity of walking difficulty after partial transection of the spinal cord including penetrating SCI likely depends on the regions affected. Therefore, we compared beam walking and overground walking between mice after penetrating SCI at inner spinal cord region and mice with the injury at the outer region. Mice with the both penetrating SCIs did not display changes in beam walking. When appearance and movements of hindlimbs during overground walking was rated using Basso Mouse Scale for locomotion (BMS), however, mice with inner penetrating SCI showed low score shortly after the SCI. However, the score became high at later time points, as seen in contusion SCI mice. By contrast, BMS score did not decrease shortly after the outer penetrating SCI. However, the score became low 3 weeks after the SCI. As quantitative values during overground walking, movement duration in an open field were shorter at 1 day after the two penetrating SCIs. However, slower moving speed and fewer number of movement at 1 day were specific to mice with inner and outer penetrating SCIs, respectively. Moreover, BMS score was correlated with walking distance in open field only in mice with inner penetrating SCI. Thus, inner and outer penetrating SCI cause difficulty in overground walking with different severity and progress.

Non-fibril form but not fibril form of human islet amyloid polypeptide 8-20 changes brain functions in mice.

Whether fibril formation increases or decreases cytotoxicity remains unclear. Aggregation of human islet amyloid polypeptide (hIAPP), a pivotal regulator of glucose homeostasis, impairs the function and viability of pancreatic ß cells. Evidence suggests that low-order oligomers of hIAPP are more toxic to ß cells than fibril. However, it remains unclear whether non-fibril form of hIAPP specifically alters brain functions. This study produced fibril and non-fibril forms from a single hIAPP 8-20 peptide. The non-fibril form-injected mice showed changes in spontaneous motor activities, preference for location in the open field and social behavior. In contrast, the fibril-injected mice showed no changes in these behavioral tests. In line with the behavioral changes, the non-fibril form led to impaired neurite outgrowth of cultured neuron-like cells and the loss of neurons in the mouse hippocampus. These findings suggest that non-fibril form but not fibril form of hIAPP changes brain functions.

Effects of halogen atom substitution on luminescent radicals: a case study on tris(2,4,6-trichlorophenyl)methyl radical-carbazole dyads.

A series of halogen-substitute carbazole TTM radicals was synthesized. The effect of halogen substituents on radical luminescence was systematically evaluated. It was found that the well-known heavy atom effect does not work in the emission of radicals and that halogen substitution of the donor carbazole can change the HOMO and alter the absorption and emission wavelengths. In addition, the photostability was found to be improved with respect to TTM but not significantly different from that of closed-shell fluorescent molecules.

Tracheostoma Closure Technique Using Three Local Flaps.

When a tracheostoma is no longer needed, the opening normally closes spontaneously after cannula removal, but some cases require tracheostoma closure. This procedure has been well described, but must be performed in such a way as to minimize its invasiveness and complications while securing a high closure rate. Our procedure for conducting tracheostoma closure technique involves the creation of two hinge flaps and one cover flap to close the tracheostomy opening. We reviewed the medical records of 23 patients (12 men, 11 women; mean age 60.0 SD19.7 years) who underwent tracheostoma closure technique between 2001 and 2019. Surgery was indicated for patients in whom closure had not occurred after conservative monitoring for ≥ 2 months following cannula removal. The surgical procedure began by raising two hinge flaps on either side of the tracheostomy opening, turning the skin surface to the luminal side to form the anterior tracheal wall. Rather than a single layer of skin, multiple skin layers were sutured together to prevent air leakage from between hinge flaps. A further cover flap was produced to cover the anterior tracheal wall, closing the tracheostomy opening. Postoperatively, the tracheal lumen was observed via fiberscopy. No stenosis of the tracheal lumen occurred in any patients, and the tracheocutaneous fistula was successfully closed in all cases. Tracheostoma closure technique using hinge flaps to reconstruct the anterior tracheal wall and a cover flap as a skin flap to cover the skin defect appears useful for patients with failure of spontaneous tracheocutaneous fistula closure.

Dynamic Analysis Of Male "Extra-high Voice" Using Multi-row Detector Computed Tomography.

OBJECTIVES/HYPOTHESIS: Some people who practice singing on a daily basis may be able to produce a voice higher than the upper limit of the normal range (extra high voice), but there is much regarding the movement of the larynx that remains unknown. We have been conducting dynamic analysis of the larynx using multi-row detection computed tomography (MD-CT) at our university and report herein an analysis of the extra high voice. STUDY DESIGN: Observational. METHODS: Images of a normal male participant capable of extremely high-frequency speech (the highest speech range is C7 [2093 Hz] and the singing application range is up to B5 [988 Hz]) during speech were captured by MD-CT. The acquisition time was 2 seconds, and the rise of the voice from low to high and then to very high tones was recorded. Ten frames per second were analyzed as three-dimensional images. RESULTS: In the fundamental frequency range from A3 to D5 (220-587 Hz), laryngeal elevation movements were observed as the voice rose in pitch. However, posterior upward displacement of the laryngeal cartilage was observed as the frequency range increased from E5 to B5 (659-988 Hz). CONCLUSIONS: In the E5-B5 range, laryngeal movements were different from those observed in the previous range. MD-CT analysis is useful in the study of this range because it allows visualization of laryngeal movements that are unclear using endoscopy or external examination.

Dissociation process of polyalanine aggregates by free electron laser irradiation.

Polyalanine (polyA) disease-causative proteins with an expansion of alanine repeats can be aggregated. Although curative treatments for polyA diseases have not been explored, the dissociation of polyA aggregates likely reduces the cytotoxicity of polyA. Mid-infrared free electron laser (FEL) successfully dissociated multiple aggregates. However, whether the FEL dissociates polyA aggregates like other aggregates has not been tested. Here, we show that FEL at 6.1 µm experimentally weakened the extent of aggregation of a peptide with 13 alanine repeats (13A), and the irradiated 13A exerted lesser cytotoxicity to neuron-like cells than non-irradiated 13A. Then, we applied molecular dynamics (MD) simulation to follow the dissociation process by FEL. We successfully observed how the intermolecular ß-sheet of polyA aggregates was dissociated and separated into monomers with helix structures upon FEL irradiation. After the dissociation by FEL, water molecules inhibited the reformation of polyA aggregates. We recently verified the same dissociation process using FEL-treated amyloid-ß aggregates. Thus, a common mechanism underlies the dissociation of different protein aggregates that cause different diseases, polyA disease and Alzheimer's disease. However, MD simulation indicated that polyA aggregates are less easily dissociated than amyloid-ß aggregates and require longer laser irradiation due to hydrophobic alanine repeats.

Auto-Lectin Dotcoding by Two Octopuses: Rapid Analysis of Fluorescence-Labeled Glycoproteins by an 8-channel Fully-Automatic Bead Array Scanner with a Rolling-Circle Detector.

Protein glycosylation is a crucial factor that must be evaluated in biological pharmaceuticals. The glycoform profile of a protein can vary depending on the conditions of the cultivation, purification process, and the selection of a host cell. Lectin microarrays are reliable bioanalytical methods used in the early phases of bioprocesses for the detection of glycosylation. The concept of a fully automated glycan detection with a bead array has been previously reported; however, no simple system has been constructed on fluorescence-based detection using a microarray. Here, we present a fully automated detection system equipped with a novel fluorescence detector for a 13-lectin bead array with a single tip. The lattice-like arrangement of a set of fibers proximate to the tip of the light emitting diode and photomultiplier tube detector minimized the noise caused by the reflection of incident light on the plastic capillary tip and bead. A unique rolling-circle fiber unit with quadruple lattices stacked in two layers realizes the 8-parallel automeasurement with a drastic reduction in scanning time and machine size. The 8-glycan profiles obtained automatically within 25 min were identical with those obtained with the conventional lectin microarray after overnight incubation. The signals obtained were represented as lectin dotcodes. Therefore, autolectin dotcoding assisted by the twin 8 legs named as "detection and irradiation octopuses" may be a rapid glyco-evaluation system during the production and development of biopharmaceuticals.

Significance of perilesional T1 hyperintense areas in the differential diagnosis of primary adult-type diffuse glioma: A case report.

Perilesional T1 hyperintensity on magnetic resonance imaging (MRI) of intra-axial brain masses is an unusual feature of the perilesional area, characteristic of cavernous malformations (CMs) and metastatic brain tumors (METs). Here, we report a case of primary diffuse glioma with a perilesional T1 hyperintense area (HIA) on MRI. A 61-year-old woman with transient aphasia visited our hospital. Radiological examination revealed an intra-axial mass with acute/subacute hemorrhaging and calcification in the left frontal lobe. It was presumed to be a CM because of the perilesional T1 HIA. Gross total resection of the tumor was performed, and the pathological diagnosis was anaplastic oligodendroglioma, not otherwise specified by World Health Organization 2016 classification. Histopathological findings in the perilesional T1 HIA indicated hemorrhage involvement in the surrounding white matter. No recurrence appeared after radio-chemotherapy. Perilesional T1 HIAs, characteristic of CMs and METs, are also seen in primary diffuse gliomas. Therefore, caution should be taken when using this sign for the differential diagnosis of intracranial masses.

Two Ascending Thermosensory Pathways from the Lateral Parabrachial Nucleus That Mediate Behavioral and Autonomous Thermoregulation.

Thermoregulatory behavior in homeothermic animals is an innate behavior to defend body core temperature from environmental thermal challenges in coordination with autonomous thermoregulatory responses. In contrast to the progress in understanding the central mechanisms of autonomous thermoregulation, those of behavioral thermoregulation remain poorly understood. We have previously shown that the lateral parabrachial nucleus (LPB) mediates cutaneous thermosensory afferent signaling for thermoregulation. To understand the thermosensory neural network for behavioral thermoregulation, in the present study, we investigated the roles of ascending thermosensory pathways from the LPB in avoidance behavior from innocuous heat and cold in male rats. Neuronal tracing revealed two segregated groups of LPB neurons projecting to the median preoptic nucleus (MnPO), a thermoregulatory center (LPB→MnPO neurons), and those projecting to the central amygdaloid nucleus (CeA), a limbic emotion center (LPB→CeA neurons). While LPB→MnPO neurons include separate subgroups activated by heat or cold exposure of rats, LPB→CeA neurons were only activated by cold exposure. By selectively inhibiting LPB→MnPO or LPB→CeA neurons using tetanus toxin light chain or chemogenetic or optogenetic techniques, we found that LPB→MnPO transmission mediates heat avoidance, whereas LPB→CeA transmission contributes to cold avoidance. In vivo electrophysiological experiments showed that skin cooling-evoked thermogenesis in brown adipose tissue requires not only LPB→MnPO neurons but also LPB→CeA neurons, providing a novel insight into the central mechanism of autonomous thermoregulation. Our findings reveal an important framework of central thermosensory afferent pathways to coordinate behavioral and autonomous thermoregulation and to generate the emotions of thermal comfort and discomfort that drive thermoregulatory behavior.SIGNIFICANCE STATEMENT Coordination of behavioral and autonomous thermoregulation is important for maintaining thermal homeostasis in homeothermic animals. However, the central mechanism of thermoregulatory behaviors remains poorly understood. We have previously shown that the lateral parabrachial nucleus (LPB) mediates ascending thermosensory signaling that drives thermoregulatory behavior. In this study, we found that one pathway from the LPB to the median preoptic nucleus mediates heat avoidance, whereas the other pathway from the LPB to the central amygdaloid nucleus is required for cold avoidance. Surprisingly, both pathways are required for skin cooling-evoked thermogenesis in brown adipose tissue, an autonomous thermoregulatory response. This study provides a central thermosensory network that coordinates behavioral and autonomous thermoregulation and generates thermal comfort and discomfort that drive thermoregulatory behavior.

Natural variations of wheat EARLY FLOWERING 3 highlight their contributions to local adaptation through fine-tuning of heading time.

KEY MESSAGE: We identified a large chromosomal deletion containing TaELF-B3 that confers early flowering in wheat. This allele has been preferred in recent wheat breeding in Japan to adapt to the environment. Heading at the appropriate time in each cultivation region can greatly contribute to stabilizing and maximizing yield. Vrn-1 and Ppd-1 are known as the major genes for vernalization requirement and photoperiod sensitivity in wheat. Genotype combinations of Vrn-1 and Ppd-1 can explain the variation in heading time. However, the genes that can explain the remaining variations in heading time are largely unknown. In this study, we aimed to identify the genes conferring early heading using doubled haploid lines derived from Japanese wheat varieties. Quantitative trait locus (QTL) analysis revealed a significant QTL on the long arm of chromosome 1B in multiple growing seasons. Genome sequencing using Illumina short reads and Pacbio HiFi reads revealed a large deletion of a ~ 500 kb region containing TaELF-B3, an orthologue of Arabidopsis clock gene EARLY FLOWERING 3 (ELF3). Plants with the deleted allele of TaELF-B3 (ΔTaELF-B3 allele) headed earlier only under short-day vernalization conditions. Higher expression levels of clock- and clock-output genes, such as Ppd-1 and TaGI, were observed in plants with the ΔTaELF-B3 allele. These results suggest that the deletion of TaELF-B3 causes early heading. Of the TaELF-3 homoeoalleles conferring early heading, the ΔTaELF-B3 allele showed the greatest effect on the early heading phenotype in Japan. The higher allele frequency of the ΔTaELF-B3 allele in western Japan suggests that the ΔTaELF-B3 allele was preferred during recent breeding to adapt to the environment. TaELF-3 homoeologs will help to expand the cultivated area by fine-tuning the optimal timing of heading in each environment.

The neural pathway of the hyperthermic response to antagonists of the transient receptor potential vanilloid-1 channel.

We identified the neural pathway of the hyperthermic response to TRPV1 antagonists. We showed that hyperthermia induced by i.v. AMG0347, AMG 517, or AMG8163 did not occur in rats with abdominal sensory nerves desensitized by pretreatment with a low i.p. dose of resiniferatoxin (RTX, TRPV1 agonist). However, neither bilateral vagotomy nor bilateral transection of the greater splanchnic nerve attenuated AMG0347-induced hyperthermia. Yet, this hyperthermia was attenuated by bilateral high cervical transection of the spinal dorsolateral funiculus (DLF). To explain the extra-splanchnic, spinal mediation of TRPV1 antagonist-induced hyperthermia, we proposed that abdominal signals that drive this hyperthermia originate in skeletal muscles - not viscera. If so, in order to prevent TRPV1 antagonist-induced hyperthermia, the desensitization caused by i.p. RTX should spread into the abdominal-wall muscles. Indeed, we found that the local hypoperfusion response to capsaicin (TRPV1 agonist) in the abdominal-wall muscles was absent in i.p. RTX-desensitized rats. We then showed that the most upstream (lateral parabrachial, LPB) and the most downstream (rostral raphe pallidus) nuclei of the intrabrain pathway that controls autonomic cold defenses are also required for the hyperthermic response to i.v. AMG0347. Injection of muscimol (inhibitor of neuronal activity) into the LPB or injection of glycine (inhibitory neurotransmitter) into the raphe blocked the hyperthermic response to i.v. AMG0347, whereas i.v. AMG0347 increased the number of c-Fos cells in the raphe. We conclude that the neural pathway of TRPV1 antagonist-induced hyperthermia involves TRPV1-expressing sensory nerves in trunk muscles, the DLF, and the same LPB-raphe pathway that controls autonomic cold defenses.

Fenestrated Anterior Communicating Artery Complex Mimicking an Unruptured Aneurysm: Diagnostic Pitfall.

Anatomical variations often occur in the anterior communicating artery (AComA) complex, and a careful preoperative evaluation is required before repair of this lesion. We report a case of a fenestrated AComA complex mimicking an unruptured cerebral aneurysm. A 49-year-old woman was referred to our hospital under suspicion of unruptured aneurysms of the AComA and the left middle cerebral artery on magnetic resonance angiography (MRA). Additional three-dimensional computed tomographic angiography (CTA) showed the lesion arising from the AComA complex with a maximum diameter of 4.2 mm. Intraoperative findings showed that the putative aneurysm was actually a fenestrated AComA complex as the blood vessels that formed the AComA complex were dilated and meandering. After the operation, MRA and CTA three-dimensional images were reviewed again but we could still not diagnose the lesion as a fenestrated AComA complex rather than an aneurysm. However, in the MRA source image, a secant line in the lesion was the only finding suggestive of a fenestration. The AComA complex is often associated with various vascular malformations, and it is essential to consider this association in the preoperative evaluation. The interpretation of source images may be helpful for accurate diagnosis and surgical planning.

Sleep-wake patterns are altered with age, Prdm13 signaling in the DMH, and diet restriction in mice.

Old animals display significant alterations in sleep-wake patterns such as increases in sleep fragmentation and sleep propensity. Here, we demonstrated that PR-domain containing protein 13 (Prdm13)+ neurons in the dorsomedial hypothalamus (DMH) are activated during sleep deprivation (SD) in young mice but not in old mice. Chemogenetic inhibition of Prdm13+ neurons in the DMH in young mice promotes increase in sleep attempts during SD, suggesting its involvement in sleep control. Furthermore, DMH-specific Prdm13-knockout (DMH-Prdm13-KO) mice recapitulated age-associated sleep alterations such as sleep fragmentation and increased sleep attempts during SD. These phenotypes were further exacerbated during aging, with increased adiposity and decreased physical activity, resulting in shortened lifespan. Dietary restriction (DR), a well-known anti-aging intervention in diverse organisms, ameliorated age-associated sleep fragmentation and increased sleep attempts during SD, whereas these effects of DR were abrogated in DMH-Prdm13-KO mice. Moreover, overexpression of Prdm13 in the DMH ameliorated increased sleep attempts during SD in old mice. Therefore, maintaining Prdm13 signaling in the DMH might play an important role to control sleep-wake patterns during aging.

Carbazole-Dendronized Luminescent Radicals.

A series of carbazole-dendronized tris(2,4,6-trichlorophenyl)methyl (TTM) radicals have been synthesized. The photophysical properties of dendronized radicals up to the fourth generation were compared systematically to understand how structure-property relationships evolve with generation. The photoluminescence quantum yield (PLQY) was found to increase with the increasing generation, and the fourth generation (G4TTM) in cyclohexane solution showed a PLQY as high as 63 % at a wavelength of 627 nm (in the deep-red region) from the doublet state. The dendron modification strategy also showed a blue-shift of the emission on increasing the generation number, and the photostability was also increased compared to the bare TTM radical.

Continuous Epidural Saline Infusion for the Treatment of Spontaneous Intracranial Hypotension.

OBJECTIVE: Spontaneous intracranial hypotension (SIH) is an increasingly recognized cause of orthostatic headache, but treatment strategies remain controversial. The epidural blood patch is a well-known and widely used treatment in patients with conservative treatment-resistant SIH, but symptoms may not improve even after multiple epidural blood patches, and resistant patients suffer from a lack of appropriate treatment options. Therefore, this study assessed the safety and efficacy of continuous epidural saline infusion (CESI) for SIH treatment. METHODS: CESI was performed in 11 consecutive patients affected by conservative treatment-resistant SIH. Patient characteristics were obtained by reviewing medical records retrospectively. Headache intensity was assessed using the numerical rating scale (NRS), and changes in NRS before and after treatment were recorded. RESULTS: The average treatment period for CESI was 21.3 ± 9.6 days, and the average follow-up period was 35.0 ± 30.2 months. CESI was without major complications or mortality, and no infections occurred, even without prophylactic antibiotics. The median NRS score before treatment was 10 points, improving to 5 points 1 day after infusion (P < 0.05), 2 points 1 week after infusion (P < 0.05), and 0 points at 3 months after infusion and the final follow-up. No patients have since experienced recurrence of orthostatic headaches. CONCLUSIONS: CESI appears to be a safe and well-tolerated procedure for SIH. Further experience may demonstrate this technique to be a viable treatment option for SIH.

Probing protein misfolding and dissociation with an infrared free-electron laser.

Misfolding is observed in the mutant proteins that are causative for neurodegenerative disorders such as polyglutamine diseases. These proteins are prone to aggregate in the cytoplasm and nucleus of cells. To reproduce cells with the aggregated proteins, gene expression system is usually applied, in which the expression construct having the mutated DNA sequence of the interest is transfected into cells. The transfected DNA is finally converted into the mutant protein, which is gradually aggregated in the cells. In addition, a simple method to prepare the cells having aggregates inside has been recently applied. Peptides were first aggregated by incubating them in water. The aggregates are spontaneously taken up by cells because aggregated proteins generally transfer between cells. Peptides with different degrees of aggregation can be made by changing the incubation times and temperatures, which enables to examine contribution of aggregation to the toxicity to the recipient cells. Moreover, such cells can be used for therapeutic researches of diseases in which aggregates are involved. In this chapter, we show methods to induce aggregation of peptides. The functional analyses of the cells with aggregates are also described. Then, experimental dissociation of the aggregates produced using this method by mid infrared free electron laser irradiation and its theoretical support by molecular dynamics simulation are introduced as the therapeutic research for neurodegenerative disorders.