Stabilization of adalimumab Fab through the introduction of disulfide bonds between the variable and constant domains.

Fab is a promising format for antibody drug. Therefore, efforts have been made to improve its thermal stability for therapeutic and commercial use. So far, we have attempted to introduce a disulfide bond into the Fab fragment to improve its thermal stability and demonstrated that it is possible to do this without sacrificing its biochemical function. In this study, to develop a novel stabilization strategy for Fab, we attempted to introduce a disulfide bond between the variable and constant domains and prepared three variants of Fab; H:G10C + H:P210C, L:P40C + L:E165C, and H:G10C + H:P210C + L:P40C + L:E165C. Differential scanning calorimetry measurements showed that each of these variants had improved thermal stability. In addition, the variants with two disulfide bonds demonstrated a 6.5 °C increase in their denaturation temperatures compared to wild-type Fab. The introduction of disulfide bonds was confirmed by X-ray crystallography, and the variants retained their antigen-binding activity. The variants were also found to be less aggregative than the wild type. Our results demonstrate that the introduction of a disulfide bond between the variable and constant domains significantly improves the thermal stability of Fab.

Analysis of thermostability for seven Phe to Ala and six Pro to Gly mutants in the Fab constant region of adalimumab.

To identify amino acids that play important roles in the structural stability of Fab, seven phenylalanine residues in the Fab constant region of the therapeutic antibody adalimumab were subjected to alanine mutagenesis. Six Fab mutants, H:F130A, H:F154A, H:F174A, L:F118A, L:F139A and L:F209A, showed decreased thermostability compared with wild-type Fab. In contrast, the Tm for the L:F116A mutant was 1.7°C higher than that of wild-type Fab, indicating that the F116 residue was unfavorable for Fab thermostability. Six proline mutants, H:P131G, H:P155G, H:P175G, L:P119G, L:P120G and L:P141G, were also prepared to investigate the effect of proline residues adjacent to mutated phenylalanine residues. The thermostability of the H:P155G and L:P141G mutants in particular was significantly reduced, with decreases in Tm of 5.0 and 3.0°C, respectively, compared with wild-type Fab. The H:P155 and L:P141 residues have a cis conformation, whereas the other mutated proline residues have a trans conformation. H:P155 and L:P141 had stacking interactions with the H:F154 and L:Y140, respectively, at the interface between the variable and constant regions. It is suggested that the interactions of the aromatic ring with a cis-form proline at the interface between the variable and constant regions is important for stability of Fab.

Peritoneal dialysis-related peritonitis caused by Rhodococcus corynebacterioides.

A 57-year-old Japanese man on peritoneal dialysis developed peritoneal dialysis-associated peritonitis caused by Rhodococcus corynebacterioides. After the introduction of peritoneal dialysis, he had experienced four episodes of peritonitis, but the causative organism was not identified in any of episode. When he was hospitalized for the fifth episode of peritonitis, Rhodococcus corynebacterioides was detected in the ascitic fluid. He improved after an intraperitoneal administration of vancomycin (VCM) that was used based on the treatment of peritonitis caused by Corynebacterium spp. However, he then had repeated flare-ups and eventually required the removal of the peritoneal dialysis catheter due to recurrent peritonitis. 16S rRNA gene sequencing is generally needed to positively identify Rhodococcus corynebacterioides. In this case, we were able to rapidly identify the organism by using mass spectrometry and then apply this knowledge to the patient's treatment. To the best of our knowledge, this is the first reported case of peritoneal dialysis-associated peritonitis caused by Rhodococcus corynebacterioides.

Application of machine learning in the diagnosis of vestibular disease.

Machine learning is considered a potential aid to support human decision making in disease prediction. In this study, we determined the utility of various machine learning algorithms in classifying peripheral vestibular (PV) and non-PV diseases based on the results of equilibrium function tests. A total of 1009 patients who had undergone our standardized neuro-otological examinations were recruited. We applied five supervised machine learning algorithms (random forest, adaboost, gradient boosting, support vector machine, and logistic regression). After preprocessing the data, optimizing the hyperparameters using GridSearchCV, and performing a final evaluation on the test set using scikit-learn, we evaluated the predictive capability using various performance metrics, namely, accuracy, F1-score, area under the receiver operating characteristic curve, precision, recall, and Matthews correlation coefficient (MCC). All five machine learning algorithms yielded satisfactory results; the accuracy of the algorithms ranged from 76 to 79%, with the support vector machine classifier having the highest accuracy. In cases where the predictions of the five models were consistent, the accuracy of the PV diagnostic results was improved to 83%, whereas it increased to 85% for the non-PV diagnostic results. Future research should increase the number of patients and optimize the classification methods to obtain the highest diagnostic accuracy.

12-month effect of middle ear pressure therapy with the EFET01 device for intractable definite Meniere's disease and delayed endolymphatic hydrops after certification by the public health insurance system in Japan.

BACKGROUND: Middle ear pressure therapy (MEPT) is effective for intractable vertigo in patients with definite Meniere's disease (MD) and treatment-refractory delayed endolymphatic hydrops (DEH). Four-month MEPT with the EFET01®, an MEPT device developed in Japan and covered by national health insurance since September 2018, has shown efficacy. However, efficacy and safety after 12 months of treatment, which is appropriate for determining the therapeutic effect of MEPT devices, is unclear. OBJECTIVES: Examine the therapeutic effect of 12-month MEPT using the ETET01®. MATERIAL AND METHODS: Patients underwent MEPT using the EFET01® from September 2018 to July 2021. Thirty-three patients followed for >12 months were enrolled in this retrospective study. Clinical data were evaluated in the first and second 6-month treatment periods. Data from the second 6-month period were compared with data from an MEPT study using a different device. RESULTS: MEPT with the EFET01® significantly improved vertigo in the first period, with further improvement in the second period. The efficacy and safety were comparable to MEPT with other devices. CONCLUSIONS: MEPT with the EFET01® is effective for intractable vertigo in patients with definite MD and DEH, and 12-month follow-up is recommended. SIGNIFICANCE: The efficacy of 12-month MEPT with the EFET01® was demonstrated.

Efficiency of a novel middle ear pressure device for intractable definite Meniere's disease and delayed endolymphatic hydrops after certification by the public health insurance system in Japan.

BACKGROUND: Middle ear pressure therapy (MEPT) is effective in treating intractable vertigo in patients with definite Meniere's disease (MD) and delayed endolymphatic hydrops (DEH) refractory to conservative treatment. A novel middle ear pressure device, the EFET01®, which requires no transtympanic ventilation tubes, was developed in Japan, approved by the Japanese Ministry of Health, Labour and Welfare, and has been used under Japanese national health insurance since September 2018. OBJECTIVES: To examine short-term therapeutic effect of MEPT using the ETET01® compared with previous clinical trial results. METHODS: Patients selected according to Japan Society for Equilibrium Research (JSER) guidelines underwent MEPT using the EFET01 from September 2018 to July 2021, and 44 patients were enrolled in this retrospective study. Clinical data analysed at 4 months after the start of MEPT were compared with those of the previous clinical trial for the EFET01. RESULTS: MEPT using the EFET01 showed the same therapeutic efficacy as that of the previous clinical trial, i.e. improvement in the intensity and frequency of vertigo with no effect on hearing, even under JSER guidelines for proper use of MEPT. CONCLUSION: MEPT using the EFET01 provided an effective treatment option for intractable vertigo in patients with definite MD and DEH.

Effect of an intermolecular disulfide bond introduced into the first loop of CH1 domain of Adalimumab Fab on thermal stability and antigen-binding activity.

The introduction of intermolecular disulfide bonds by amino acid mutations is an effective method for stabilizing dimeric proteins. X-ray crystal structure of Fab of a therapeutic antibody, adalimumab, revealed the first loop of the CH1 domain to be partially unsolved at position 135-141. To find new sites for the introduction of intermolecular disulfide bonds in adalimumab Fab, Fab mutants targeting the unsolved region were predicted using molecular simulation software. Four Fab mutants, H:K137C-L:I117C, H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C, were expressed in the methylotrophic yeast Pichia pastoris. SDS-PAGE analysis of these mutants indicated that H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C mutants mostly formed intermolecular disulfide bonds, whereas some H:K137C-L:I117C mutants formed intermolecular disulfide bonds and some did not. Differential scanning calorimetry measurements showed increased thermal stability in all Fab mutants with engineered disulfide bonds. The bio-layer interferometry measurements, for binding of the antigen tumor necrotic factor α, indicated that Fab mutants had less antigen-binding activity than wild-type Fab. In particular, the KD value of H:K137C-L:F209C was ~17 times higher than that of wild-type Fab. Thus, we successfully introduced intermolecular disulfide bonds between the first loop region of the CH1 and CL domains and observed that it increases the thermostability of Fab and affects the antigen-binding activity.

Long-term effect of transtympanic intermittent pressure therapy using a tympanic membrane massage device for intractable meniere's disease and delayed endolymphatic hydrops.

BACKGROUND: A 12-month follow-up study showed that middle ear pressure treatment with a transtympanic membrane massage (TMM) device had a similar effect to a Meniett device. OBJECTIVES: The effects of pressure treatment with a TMM device were retrospectively compared to the effects of treatment with a Meniett device in patients with Meniere's disease (MD) and delayed endolymphatic hydrops (DEH) who were followed for a minimum of 24 months. MATERIALS AND METHODS: Twenty-seven patients were treated with the TMM device and 14 patients were treated with a Meniett device. The insertion of a transtympanic ventilation tube was necessary for the Meniett device but not for the TMM device. RESULTS: In patients treated with the TMM and Meniett devices, the frequency of vertigo significantly improved at 19-24 months after treatment. The distribution of vertigo at 19-24 months after treatment did not differ between the patients treated with the two types of devices. Pressure treatment for 8 months or more was suitable to achieve remission. CONCLUSIONS AND SIGNIFICANCE: Middle ear pressure treatment for 8 months or more with a TMM or Meniett device was equally effective and provided minimally invasive treatment options for intractable MD and DEH.

A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab.

Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab's constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and ß-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab's constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

Nivolumab-associated glomerular endothelial injury in a patient with gastric cancer.

A 68-year-old male with gastric cancer was treated with tegafur/gimeracil/oteracil and oxaliplatin for 6 months. Thereafter, he was treated with paclitaxel and ramucirumab for 3 months. However, neither regimen had much effect. Thus, he was treated with nivolumab for 2 months, but he developed proteinuria, microhematuria, and an acute kidney injury. A kidney biopsy revealed occasional swollen endothelial cells and proliferating mesangial cells. Few abnormal findings were seen in the tubules or interstitial tissue. Immunofluorescent staining showed segmental immunoglobulin A and complement component 3 deposition, in the mesangial area. Electron microscopy showed a small amount of electron-dense deposits in the paramesangial area and swollen endothelial cells. Mesangial interposition, the loss of endothelial cell fenestration, and subendothelial edema were also observed. Furthermore, foot process effacement and villous transformation of epithelial cells were noted. After the discontinuation of nivolumab, the patient's renal function gradually improved, and his proteinuria disappeared. Nivolumab treatment was restarted at that time because of cancer progression; however, it was ineffective. No occult blood was detected from 7 months after the administration of the last dose of nivolumab. This is a unique case, in which a kidney biopsy revealed evidence of nivolumab-associated glomerular endothelial injury.

Glycosylation decreases aggregation and immunogenicity of adalimumab Fab secreted from Pichia pastoris.

Glycoengineering of therapeutic proteins has been applied to improve the clinical efficacy of several therapeutics. Here, we examined the effect of glycosylation on the properties of the Fab of the therapeutic antibody, adalimumab. An N-glycosylation site was introduced at position 178 of the H chain constant region of adalimumab Fab through site-directed mutagenesis (H:L178N Fab), and the H:L178N Fab was produced in Pichia pastoris. Expressed mutant Fab contained long and short glycan chains (L-glyco Fab and S-glyco Fab, respectively). Under the condition of aggregation of Fab upon pH shift-induced stress, both of L-glyco Fab and S-glyco Fab were less prone to aggregation, with L-glyco Fab suppressing aggregation more effectively than the S-glyco Fab. Moreover, the comparison of the antigenicity of glycosylated and wild-type Fabs in mice revealed that glycosylation resulted in the suppression of antigenicity. Analysis of the pharmacokinetic behaviour of the Fab, L-glyco Fab and S-glyco Fab indicated that the half-lives of glycosylated Fabs in the rats were shorter than that of wild-type Fab, with L-glyco Fab having a shorter half-life than S-glyco Fab. Thus, we demonstrated that the glycan chain influences Fab aggregation and immunogenicity, and glycosylation reduces the elimination half-life in vivo.

Cerebral Hemodynamic Responses to the Sensory Conflict Between Visual and Rotary Vestibular Stimuli: An Analysis With a Multichannel Near-Infrared Spectroscopy (NIRS) System.

Sensory conflict among visual, vestibular, and somatosensory information induces vertiginous sensation and postural instability. To elucidate the cognitive mechanisms of the integration between the visual and vestibular cues in humans, we analyzed the cortical hemodynamic responses during sensory conflict between visual and horizontal rotatory vestibular stimulation using a multichannel near-infrared spectroscopy (NIRS) system. The subjects sat on a rotatory chair that was accelerated at 3°/s2 for 20 s to the right or left, kept rotating at 60°/s for 80 s, and then decelerated at 3°/s2 for 20 s. The subjects were instructed to watch white stripes projected on a screen surrounding the chair during the acceleration and deceleration periods. The white stripes moved in two ways; in the "congruent" condition, the stripes moved in the opposite direction of chair rotation at 3°/s2 (i.e., natural visual stimulation), whereas in the "incongruent" condition, the stripes moved in the same direction of chair rotation at 3°/s2 (i.e., conflicted visual stimulation). The cortical hemodynamic activity was recorded from the bilateral temporoparietal regions. Statistical analyses using NIRS-SPM software indicated that hemodynamic activity increased in the bilateral temporoparietal junctions (TPJs) and human MT+ complex, including the medial temporal (MT) area and medial superior temporal (MST) area in the incongruent condition. Furthermore, the subjective strength of the vertiginous sensation was negatively correlated with hemodynamic activity in the dorsal part of the supramarginal gyrus (SMG) in and around the intraparietal sulcus (IPS). These results suggest that sensory conflict between the visual and vestibular stimuli promotes cortical cognitive processes in the cortical network consisting of the TPJ, the medial temporal gyrus (MTG), and IPS, which might contribute to self-motion perception to maintain a sense of balance or equilibrioception during sensory conflict.

Discovery of the Gene Encoding a Novel Small Serum Protein (SSP) of Protobothrops flavoviridis and the Evolution of SSPs.

Small serum proteins (SSPs) are low-molecular-weight proteins in snake serum with affinities for various venom proteins. Five SSPs, PfSSP-1 through PfSSP-5, have been reported in Protobothrops flavoviridis ("habu", Pf) serum so far. Recently, we reported that the five genes encoding these PfSSPs are arranged in tandem on a single chromosome. However, the physiological functions and evolutionary origins of the five SSPs remain poorly understood. In a detailed analysis of the habu draft genome, we found a gene encoding a novel SSP, SSP-6. Structural analysis of the genes encoding SSPs and their genomic arrangement revealed the following: (1) SSP-6 forms a third SSP subgroup; (2) SSP-5 and SSP-6 were present in all snake genomes before the divergence of non-venomous and venomous snakes, while SSP-4 was acquired only by venomous snakes; (3) the composition of paralogous SSP genes in snake genomes seems to reflect snake habitat differences; and (4) the evolutionary emergence of SSP genes is probably related to the physiological functions of SSPs, with an initial snake repertoire of SSP-6 and SSP-5. SSP-4 and its derivative, SSP-3, as well as SSP-1 and SSP-2, appear to be venom-related and were acquired later.

C-Terminal Cysteine PEGylation of Adalimumab Fab with an Engineered Interchain SS Bond.

Conjugation with polyethylene glycol (PEG) is performed to increase serum half-life of the Fab for clinical applications. However, current designs for recombinant Fab only allow PEGylation at the interchain SS bond (disulfide bond) at the C-terminal end of the heavy chain and light chain of the Fab, which the decrease of thermostability occurred by partial reduction of the interchain SS bond. An adalimumab Fab mutant with a novel interchain SS bond (CH1 : C177-CL : C160) and one cysteine at the C-terminal end (mutSS FabSH) was designed to maintain Fab thermostability and for site-specific PEGylation. MutSS FabSH was expressed in Pichia pastoris and purified mutSS FabSH was conjugated with 20-kDa PEG targeted at the free cysteine. Based on enzyme-linked immunosorbent assay (ELISA), PEGylation did not affect the binding capacity of the mutSS FabSH. To confirm the influence of PEGylation on the pharmacokinetic behavior of the Fab, PEGylated mutSS FabSH was administered to rats via tail vein injection. Analysis of the mean serum concentration of the PEGylated mutSS FabSH versus time through ELISA indicated an increase in half-life compared to that of non-PEGylated wild-type Fab. Consequently, we have successfully demonstrated that a Fab mutant with a novel interchain SS bond and one free cysteine at the C-terminal end can be PEGylated without changes in functionality. This design can potentially be used as a platform for modification of other recombinant Fabs.

Alternative mRNA Splicing in Three Venom Families Underlying a Possible Production of Divergent Venom Proteins of the Habu Snake, Protobothrops flavoviridis.

Snake venoms are complex mixtures of toxic proteins encoded by various gene families that function synergistically to incapacitate prey. A huge repertoire of snake venom genes and proteins have been reported, and alternative splicing is suggested to be involved in the production of divergent gene transcripts. However, a genome-wide survey of the transcript repertoire and the extent of alternative splicing still remains to be determined. In this study, the comprehensive analysis of transcriptomes in the venom gland was achieved by using PacBio sequencing. Extensive alternative splicing was observed in three venom protein gene families, metalloproteinase (MP), serine protease (SP), and vascular endothelial growth factors (VEGF). Eleven MP and SP genes and a VEGF gene are expressed as a total of 81, 61, and 8 transcript variants, respectively. In the MP gene family, individual genes are transcribed into different classes of MPs by alternative splicing. We also observed trans-splicing among the clustered SP genes. No other venom genes as well as non-venom counterpart genes exhibited alternative splicing. Our results thus indicate a potential contribution of mRNA alternative and trans-splicing in the production of highly variable transcripts of venom genes in the habu snake.

[Difficulties in distinguishing abnormal intensities associated with convulsion from tumor on MRI: a case report].

A 48-year-old man was admitted to our department with generalized convulsive seizures followed by recurrent partial clonic convulsions in the left face and arm. Convulsions stopped temporarily after administration of diazepam, fosphenytoin, and levetiracetam. However, frequent partial seizures occurred repeatedly and general anesthesia was required to control seizures. Diffusion-weighted and T2-weighted images revealed a high-intensity lesion in the right frontal lobe. A tumor-like area in the white matter showed high intensity on T2-weighted images with ring enhancement on gadolinium-enhanced T1-weighted images. An area of frontal cortex near the tumor was also enhanced. Brain surgery was performed for the purposes of diagnosis, seizure control and tumor resection. Histological findings demonstrated oligodendroglioma in the ring-enhancing area, but not in the frontal cortex. This fact indicated that contrast enhancement of the frontal cortex was caused by status epilepticus. It is important to recognize that status epilepticus could cause contrast enhancement on magnetic resonance imaging.

Unique structure (construction and configuration) and evolution of the array of small serum protein genes of Protobothrops flavoviridis snake.

The nucleotide sequence of Protobothrops flavoviridis (Pf) 30534 bp genome segment which contains genes encoding small serum proteins (SSPs) was deciphered. The genome segment contained five SSP genes (PfSSPs), PfSSP-4, PfSSP-5, PfSSP-1, PfSSP-2, and PfSSP-3 in this order and had characteristic configuration and constructions of the particular nucleotide sequences inserted. Comparison between the configurations of the inserted chicken repeat-1 (CR1) fragments of P. flavoviridis and Ophiophagus hannah (Oh) showed that the nucleotide segment encompassing from PfSSP-1 to PfSSP-2 was inverted. The inactive form of PfSSP-1, named PfSSP-1δ(Ψ), found in the intergenic region (I-Reg) between PfSSP-5 and PfSSP-1 had also been destroyed by insertions of the plural long interspersed nuclear elements (LINEs) and DNA transposons. The L2 LINE inserted into the third intron or the particular repetitive sequences inserted into the second intron structurally divided five PfSSPs into two subgroups, the Long SSP subgroup of PfSSP-1, PfSSP-2 and PfSSP-5 or the Short SSP subgroup of PfSSP-3 and PfSSP-4 The mathematical analysis also showed that PfSSPs of the Long SSP subgroup evolved alternately in an accelerated and neutral manner, whereas those of the Short SSP subgroup evolved in an accelerated manner. Moreover, the ortholog analysis of SSPs of various snakes showed that the evolutionary emerging order of SSPs was as follows: SSP-5, SSP-4, SSP-2, SSP-1, and SSP-3 The unique interpretation about accelerated evolution and the novel idea that the transposable elements such as LINEs and DNA transposons are involved in maintaining the host genome besides its own transposition natures were proposed.

SDS-induced oligomerization of Lys49-phospholipase A2 from snake venom.

Phospholipase A2 (PLA2) is one of the representative toxic components of snake venom. PLA2s are categorized into several subgroups according to the amino acid at position 49, which comprises either Asp49, Lys49, Arg49 or Ser49. Previous studies suggested that the Lys49-PLA2 assembles into an extremely stable dimer. Although the behavior on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions suggested the presence of intermolecular disulfide bonds, these bonds were not observed in the crystal structure of Lys49-PLA2. The reason for this discrepancy between the crystal structure and SDS-PAGE of Lys49-PLA2 remains unknown. In this study, we analyzed a Lys49-PLA2 homologue from Protobothrops flavoviridis (PflLys49-PLA2 BPII), by biophysical analyses including X-ray crystallography, SDS-PAGE, native-mass spectrometry, and analytical ultracentrifugation. The results demonstrated that PflLys49-PLA2 BPII spontaneously oligomerized in the presence of SDS, which is one of the strongest protein denaturants.

Therapy-related acute myeloid leukemia after chemotherapy in extensive disease-small cell lung cancer.

We experienced therapy-related acute myeloid leukemia (t-AML) in a patient with extensive disease-small cell lung cancer (ED-SCLC). This case is rare and has educational message because ED-SCLC has a poor prognosis and often cannot survive until developing therapy related hematological malignancy. Furthermore this case had unique chromosomal abnormalities. With recent advances in chemotherapy and radiotherapy, the prognosis of lung cancer has improved, while t-AML has been increasing in frequency.

A morphology-based assay platform for neuroepithelial-like cells differentiated from human pluripotent stem cells.

Cell morphology is recognized as an important hallmark of neural cells. During the differentiation of human pluripotent stem cells (hPSCs) into neural cells, cell morphology changes dynamically. Therefore, characterization of the morphology of cells during this period is important to improve our understanding of the differentiation and development of neural cells. General methods for the directed induction of hPSCs include the steps of multi-cellular aggregation or high-density cell culture, particularly at the early phase of neural differentiation, and therefore, the morphology of each differentiating cell is difficult to recognize. Here, we have developed a new method for the directed differentiation of neuroepithelial-like cells (NELCs) from hPSCs at a low cell density in an adherent monolayer culture, as well as an image-processing algorithm to evaluate the cell morphology of differentiating NELCs, in order to follow cell morphology during the differentiation of hPSCs into NELCs. Using these methods, the morphological transition of differentiating cells was observed in real time using phase contrast imaging and then quantified. Because cell morphology is also considered an inherent biological marker of neural cells cultured in vitro, this method is potentially useful to study the mechanisms underlying neural cell differentiation.