Hepatocytes differentiate into intestinal epithelial cells through a hybrid epithelial/mesenchymal cell state in culture.

Hepatocytes play important roles in the liver, but in culture, they immediately lose function and dedifferentiate into progenitor-like cells. Although this unique feature is well-known, the dynamics and mechanisms of hepatocyte dedifferentiation and the differentiation potential of dedifferentiated hepatocytes (dediHeps) require further investigation. Here, we employ a culture system specifically established for hepatic progenitor cells to study hepatocyte dedifferentiation. We found that hepatocytes dedifferentiate with a hybrid epithelial/mesenchymal phenotype, which is required for the induction and maintenance of dediHeps, and exhibit Vimentin-dependent propagation, upon inhibition of the Hippo signaling pathway. The dediHeps re-differentiate into mature hepatocytes by forming aggregates, enabling reconstitution of hepatic tissues in vivo. Moreover, dediHeps have an unexpected differentiation potential into intestinal epithelial cells that can form organoids in three-dimensional culture and reconstitute colonic epithelia after transplantation. This remarkable plasticity will be useful in the study and treatment of intestinal metaplasia and related diseases in the liver.

Structure-specific DNA endonuclease T7 endonuclease I cleaves DNA containing UV-induced DNA lesions.

The T7 gene 3 product, T7 endonuclease I, acts on various substrates with DNA structures, including Holliday junctions, heteroduplex DNAs, and single-mismatch DNAs. Genetic analyses have suggested the occurrence of DNA recombination, replication, and repair in E.coli. In this study, T7 endonuclease I digested UV-irradiated covalently closed circular plasmid DNA into linear and nicked plasmid DNA, suggesting that the enzyme generates single- and double-strand breaks (SSB and DSB). To further investigate the biochemical functions of T7 endonuclease I, we have analyzed endonuclease activity in UV-induced DNA substrates containing a single lesion, cyclobutane pyrimidine dimers (CPD), and 6-4 photoproducts (6-4PP). Interestingly, the leading cleavage site for CPD by T7 endonuclease I is at the second and fifth phosphodiester bonds that are 5' to the lesion of CPD on the lesion strand. However, in the case of 6-4PP, the cleavage pattern on the lesion strand resembled that of CPD, and T7 endonuclease I could also cleave the second phosphodiester bond that is 5' to the adenine-adenine residues opposite the lesion, indicating that the enzyme produces DSB in DNA containing 6-4PP. These findings suggest that T7 endonuclease I accomplished successful UV damage repair by SSB in CPD and DSB in 6-4PP.

Anamorelin Induced Acute Hyperglycemia in a Patient with Advanced Pancreatic Cancer and Diabetes: A Case Report.

Anamorelin (ANAM) is a novel ghrelin receptor agonist for the treatment of cancer cachexia. In clinical trials of ANAM, glucose metabolism disorders as adverse effects were relatively frequent, however, when and how they occur remains unclear. Moreover, the safety in patients with pancreatic cancer and/or diabetes has not been clarified because most previous studies focused on patients with non-small cell lung cancer and had excluded patients with poorly controlled diabetes. Herein, a 66-year-old man with advanced pancreatic cancer and diabetes was administered ANAM, and acute hyperglycemia was developed and could be monitored by the self-monitoring of blood glucose (SMBG). Increasing the insulin dose failed to control hyperglycemia adequately, but the hyperglycemia ameliorated quickly after ANAM discontinuation. The continuous glucose monitoring (CGM) revealed that the sensor glucose levels had remained in the high range throughout the day during ANAM administration despite using 1.5 times more insulin. Our report is one of the few that describe the details of ANAM-induced hyperglycemia and provides important information for the safe and effective use of ANAM.

Frequency of EMG abnormalities in idiopathic inflammatory myopathies under the EULAR/ACR classification criteria.

The European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) classification criteria for idiopathic inflammatory myopathies (IIM) have been widely used in recent times. However, no studies have focused on electromyography (EMG) findings of IIM, considering the criteria. This study aimed to elucidate the frequency of EMG abnormalities, particularly fibrillation potentials and positive sharp waves (Fib/PSW), the most objective EMG findings of IIM. Clinical and EMG records of adult patients who were clinically diagnosed with polymyositis (PM), dermatomyositis (DM), amyopathic DM (ADM), or inclusion body myositis (IBM) were retrospectively reviewed and classified according to the EULAR/ACR classification criteria. The frequency of Fib/PSW in EMG was investigated in the recruited cases. Seventy-nine patients with clinically diagnosed IIM (44 with PM, 17 with DM, 7 with ADM, and 11 with IBM) were recruited. After classification using EULAR/ACR, 75 satisfied definite or probable IIM (61 and 14, respectively), and the frequency of Fib/PSW in this group was 95%. Furthermore, the remaining 4 patients with insufficient IIM probability also showed Fib/PSW. Fib/PSW may also be seen in cases with insufficient IIM probability not satisfying the criteria. EMG may help detect muscle involvement in these cases through Fib/PSW.

Insulin Requirements During Severe COVID-19 Were Relatively Low in Japanese Patients With Type 2 Diabetes: Two Case Reports.

The global coronavirus disease 2019 (COVID-19) pandemic has caused myriad adverse effects on the pathology of other diseases. Numerous studies on COVID-19 have reported that, in patients with type 2 diabetes mellitus (T2DM) who have contracted severe COVID-19, glucose metabolism is exacerbated by multiple factors, such as severe inflammation, beta-cell dysfunction caused by the SARS-CoV-2 infection itself, corticosteroid therapy, vasopressor administration, and enteral or parenteral nutrition. Very high doses of insulin are often required in the acute phase of such patients; however, the factors that affect insulin requirements and to what extent remain unclear. A 50-year-old Japanese woman and a 67-year-old Japanese man, both with T2DM and obesity, were admitted to our hospital with severe COVID-19. Both patients required mechanical ventilation and were treated with dexamethasone and tocilizumab, an interleukin-6 (IL-6) receptor monoclonal antibody. Subcutaneous insulin injections failed to control the patients' hyperglycemia, requiring up to 1.83 and 1.81 units/kg/day of intravenous insulin, respectively. Insulin requirements were rapidly decreased with improvement of the respiratory condition, termination of dexamethasone, and discontinuation of tube feeding. Both patients were discharged with oral antidiabetic agents alone. We experienced two Japanese patients who achieved satisfactory glycemic control with a lower intravenous insulin dose than previous reports. Comparing the clinical factors with the previous literature, ethnic differences in insulin sensitivity and the administration of IL-6 receptor antibodies may have been related to the relatively low insulin requirements.

Optogenetic stimulation of vagal nerves for enhanced glucose-stimulated insulin secretion and ß cell proliferation.

The enhancement of insulin secretion and of the proliferation of pancreatic ß cells are promising therapeutic options for diabetes. Signals from the vagal nerve regulate both processes, yet the effectiveness of stimulating the nerve is unclear, owing to a lack of techniques for doing it so selectively and prolongedly. Here we report two optogenetic methods for vagal-nerve stimulation that led to enhanced glucose-stimulated insulin secretion and to ß cell proliferation in mice expressing choline acetyltransferase-channelrhodopsin 2. One method involves subdiaphragmatic implantation of an optical fibre for the photostimulation of cholinergic neurons expressing a blue-light-sensitive opsin. The other method, which suppressed streptozotocin-induced hyperglycaemia in the mice, involves the selective activation of vagal fibres by placing blue-light-emitting lanthanide microparticles in the pancreatic ducts of opsin-expressing mice, followed by near-infrared illumination. The two methods show that signals from the vagal nerve, especially from nerve fibres innervating the pancreas, are sufficient to regulate insulin secretion and ß cell proliferation.

A deazariboflavin chromophore kinetically stabilizes reduced FAD state in a bifunctional cryptochrome.

An animal-like cryptochrome derived from Chlamydomonas reinhardtii (CraCRY) is a bifunctional flavoenzyme harboring flavin adenine dinucleotide (FAD) as a photoreceptive/catalytic center and functions both in the regulation of gene transcription and the repair of UV-induced DNA lesions in a light-dependent manner, using different FAD redox states. To address how CraCRY stabilizes the physiologically relevant redox state of FAD, we investigated the thermodynamic and kinetic stability of the two-electron reduced anionic FAD state (FADH-) in CraCRY and related (6-4) photolyases. The thermodynamic stability of FADH- remained almost the same compared to that of all tested proteins. However, the kinetic stability of FADH- varied remarkably depending on the local structure of the secondary pocket, where an auxiliary chromophore, 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF), can be accommodated. The observed effect of 8-HDF uptake on the enhancement of the kinetic stability of FADH- suggests an essential role of 8-HDF in the bifunctionality of CraCRY.

[Mitral Valve Plasty for Complicated Lesions with Barlow-like Anterior Leaflet].

A 66 year-old male was admitted to our clinic suffering from dyspnea on effort. Cardio thoracic ratio (CTR) was 62%. Electrocardiogram showed atrial fibrillation. Echocardiogram showed severe mitral regurgitation (MR), Barlow like billowing and thickened A2 and A3, and loss of P2 and P3. Operation was performed through median sternotomy and right sided left atrial incision. Left atrial appendage was closed with running suture. Maze operation was done. Triangular resection of A2 and A3 was done. P2 and P3 were adhered to the left ventricular wall. First we cut the adhered posterior leaflet in a shape of inverted T. And the adhered leaflet was dissected from the left ventricle by the scissors. The detached annulus was mattress-sutured with a pledgetted suture. The leaflets were sutured together, then a new posterior leaflet was remade using mitral valve leaflet tissue and the shape became higher and round. Post operatively, MR was none, and posterior leaflet functioned well. Sinus rhythm was recovered. Eleven years later, no MR and sinus rhythm were shown.

Repair of topoisomerase 1-induced DNA damage by tyrosyl-DNA phosphodiesterase 2 (TDP2) is dependent on its magnesium binding.

Topoisomerases are enzymes that relax DNA supercoiling during replication and transcription. Camptothecin, a topoisomerase 1 (TOP1) inhibitor, and its analogs trap TOP1 at the 3'-end of DNA as a DNA-bound intermediate, resulting in DNA damage that can kill cells. Drugs with this mechanism of action are widely used to treat cancers. It has previously been shown that tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs TOP1-induced DNA damage generated by camptothecin. In addition, tyrosyl-DNA phosphodiesterase 2 (TDP2) plays critical roles in repairing topoisomerase 2 (TOP2)-induced DNA damage at the 5'-end of DNA and in promoting the repair of TOP1-induced DNA damage in the absence of TDP1. However, the catalytic mechanism by which TDP2 processes TOP1-induced DNA damage has not been elucidated. In this study, we found that a similar catalytic mechanism underlies the repair of TOP1- and TOP2-induced DNA damage by TDP2, with Mg2+-TDP2 binding playing a role in both repair mechanisms. We show chain-terminating nucleoside analogs are incorporated into DNA at the 3'-end and abort DNA replication to kill cells. Furthermore, we found that Mg2+-TDP2 binding also contributes to the repair of incorporated chain-terminating nucleoside analogs. Overall, these findings reveal the role played by Mg2+-TDP2 binding in the repair of both 3'- and 5'-blocking DNA damage.

[How to Repair the Bicuspid Aortic Valve Accompanying Regurgitation?]

We repaired the bicuspid aoric valve( BAV) with aortic regurgitation( AR) by bicuspidization. However, repaired fused cusp does not open full, and shows doming. Between 1997 and 2023 we repaired 30 BAV with AR. Mean Age was 44( 15-79) years old. Male gender was 26/30. Between 1997 and 2017, we repaired by triangular resection and cusp suspension or central plication and the commissural positions remained as it was, in 17 cases. Between 2018 and 2023, we repaired by triangular resection and aortic root remodeling to make the commissure angle 180 degree in 13 cases. One patient died because of compression occlusion of left main trunk by Schaefer's annulplasty suture post-operatively. Postoperative aortic valve pressure gradient was 12.2±5.4 mmHg in natural commissure position group, 14.7±6.8 mmHg in the 180 degree commissure position group( p=0.37). And in the 180 degree commissure position group, the fused cusp did not show doming. In the 180 degree commissure position group, the fused cusp did not show doming. However, trans aortic valve pressure gradient did not decrease. On the other hand, in the natural commissure group, the fused cusp showed doming. However, the valves well functioned up to 25 years without aortic stenosis.

A highly sensitive strategy for monitoring real-time proliferation of targeted cell types in vivo.

Cell proliferation processes play pivotal roles in timely adaptation to many biological situations. Herein, we establish a highly sensitive and simple strategy by which time-series showing the proliferation of a targeted cell type can be quantitatively monitored in vivo in the same individuals. We generate mice expressing a secreted type of luciferase only in cells producing Cre under the control of the Ki67 promoter. Crossing these with tissue-specific Cre-expressing mice allows us to monitor the proliferation time course of pancreatic ß-cells, which are few in number and weakly proliferative, by measuring plasma luciferase activity. Physiological time courses, during obesity development, pregnancy and juvenile growth, as well as diurnal variation, of ß-cell proliferation, are clearly detected. Moreover, this strategy can be utilized for highly sensitive ex vivo screening for proliferative factors for targeted cells. Thus, these technologies may contribute to advancements in broad areas of biological and medical research.

Sarcopenia and decline in appendicular skeletal muscle mass are associated with hypoperfusion in key hubs of central autonomic network on 3DSRT in older adults with progression of normal cognition to Alzheimer's disease.

AIM: Although sarcopenia is common in patients with Alzheimer's disease (AD), the neural substrates involved remain unclear. We investigated the relationship between sarcopenia, as well as its definition components, and regional cerebral blood flow (rCBF) in older adults with progression of normal cognition to AD. METHODS: 99m Tc-ethyl-cysteinate-dimer single-photon emission computed tomography was carried out in 95 older adults with progression of normal cognition to AD (40 men and 55 women, mean ± SD age 80.9 ± 6.8 years). The associations of rCBF determined by 3-D stereotactic region of interest template software, with sarcopenia and its definition components, slower gait speed, weaker grip strength, and decline in appendicular skeletal muscle mass index (ASMI) were analyzed. RESULTS: Logistic regression analysis adjusted by age, sex, mini-mental state examination score and education showed that sarcopenia as well as ASMI less than the cut-off (men 7.0 kg/m2 , women 5.7 kg/m2 ) were associated with significantly reduced rCBF in the key hub of the central autonomic network, including the insula, anterior cingulate cortex, subcallosal area, rectal gyrus, hypothalamus, amygdala and caudate head. Sarcopenia and ASMI decline were associated with hypoperfusion in the aforementioned cortical hubs of the central autonomic network in men, but with hypoperfusion of the hypothalamus in women. Linear regression analysis showed significant correlations of ASMI/cut-off with rCBF in the bilateral medial frontal cortex, as well as rCBF in the aforementioned key hubs. CONCLUSIONS: Hypoperfusion in key hubs of central autonomic network is implicated in the emergence of sarcopenia, probably through ASMI decline in vulnerable older adults. Geriatr Gerontol Int 2023; 23: 16-24.

Validity and reproducibility of the intake of trans-fatty acids estimated using a FFQ and characteristics of trans-fatty acid intake of the Japanese population: the JPHC FFQ Validation Study.

We aimed to validate a method for assessing trans-fatty acid (TFA) intake in the Japanese population using the FFQ developed in the 1990s from a prospective study that was based on the Japan Public Health Center-based Prospective Cohort Study. For FFQ validation, we included 565 participants (Cohort I: n 215, Cohort II: n 350) aged 40-69 years. We used a 28-d dietary record (DR) over 1 year and two FFQ administered before and after DR assessment. We calculated total TFA intake, TFA from industrial oils (i-TFA) and TFA from ruminants (r-TFA) considering a database of measurements obtained mainly from Japan. Spearman's rank correlation coefficients (CC) were computed for validity and reproducibility. Energy adjustments were applied using two methods considering the TFA measurement: density method for TFA % of total energy and residual method for TFA g/d. The total TFA intake (% of the total energy intake) was 0·08-0·76 % (median, 0·27-0·37 %) in DR of both cohorts and was 0·00-1·13 % (median, 0·30-0·40 %) in FFQ. The i-TFA accounted for approximately 50 % of the total TFA intake in DR and approximately 40 % in FFQ. For total TFA (% of the total energy intake), CC were 0·54-0·69, and weighted κ coefficients were 0·88-0·92 for both cohorts. The de-attenuated CC was 0·46-0·62 for i-TFA (g/d) and 0·57-0·68 for r-TFA (g/d). Our study showed that the validity and reproducibility of TFA intake estimation using the FFQ were reasonable, suggesting its suitability among the Japanese population with low-TFA intake.

Roles of FoxM1-driven basal ß-cell proliferation in maintenance of ß-cell mass and glucose tolerance during adulthood.

AIMS/INTRODUCTION: Whether basal ß-cell proliferation during adulthood is involved in maintaining sufficient ß-cell mass, and if so, the molecular mechanism(s) underlying basal ß-cell proliferation remain unclear. FoxM1 is a critical transcription factor which is known to play roles in 'adaptive' ß-cell proliferation, which facilitates rapid increases in ß-cell mass in response to increased insulin demands. Therefore, herein we focused on the roles of ß-cell FoxM1 in 'basal' ß-cell proliferation under normal conditions and in the maintenance of sufficient ß-cell mass as well as glucose homeostasis during adulthood. MATERIALS AND METHODS: FoxM1 deficiency was induced specifically in ß-cells of 8-week-old mice, followed by analyzing its short- (2 weeks) and long- (10 months) term effects on ß-cell proliferation, ß-cell mass, and glucose tolerance. RESULTS: FoxM1 deficiency suppressed ß-cell proliferation at both ages, indicating critical roles of FoxM1 in basal ß-cell proliferation throughout adulthood. While short-term FoxM1 deficiency affected neither ß-cell mass nor glucose tolerance, long-term FoxM1 deficiency suppressed ß-cell mass increases with impaired insulin secretion, thereby worsening glucose tolerance. In contrast, the insulin secretory function was not impaired in islets isolated from mice subjected to long-term ß-cell FoxM1 deficiency. Therefore, ß-cell mass reduction is the primary cause of impaired insulin secretion and deterioration of glucose tolerance due to long-term ß-cell FoxM1 deficiency. CONCLUSIONS: Basal low-level proliferation of ß-cells during adulthood is important for maintaining sufficient ß-cell mass and good glucose tolerance and ß-cell FoxM1 underlies this mechanism. Preserving ß-cell FoxM1 activity may prevent the impairment of glucose tolerance with advancing age.

Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme.

Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD•- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FAD•-. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.

Limited solvation of an electron donating tryptophan stabilizes a photoinduced charge-separated state in plant (6-4) photolyase.

(6-4) Photolyases ((6-4) PLs) are ubiquitous photoenzymes that use the energy of sunlight to catalyze the repair of carcinogenic UV-induced DNA lesions, pyrimidine(6-4)pyrimidone photoproducts. To repair DNA, (6-4) PLs must first undergo so-called photoactivation, in which their excited flavin adenine dinucleotide (FAD) cofactor is reduced in one or two steps to catalytically active FADH- via a chain of three or four conserved tryptophan residues, transiently forming FAD•-/FADH- ⋯ TrpH•+ pairs separated by distances of 15 to 20 Å. Photolyases and related photoreceptors cryptochromes use a plethora of tricks to prevent charge recombination of photoinduced donor-acceptor pairs, such as chain branching and elongation, rapid deprotonation of TrpH•+ or protonation of FAD•-. Here, we address Arabidopsis thaliana (6-4) PL (At64) photoactivation by combining molecular biology, in vivo survival assays, static and time-resolved spectroscopy and computational methods. We conclude that At64 photoactivation is astonishingly efficient compared to related proteins-due to two factors: exceptionally low losses of photoinduced radical pairs through ultrafast recombination and prevention of solvent access to the terminal Trp3H•+, which significantly extends its lifetime. We propose that a highly conserved histidine residue adjacent to the 3rd Trp plays a key role in Trp3H•+ stabilization.

A clinical survey on patients with taste disorders in Japan: A comparative study.

OBJECTIVE: To investigate changes in the clinical state of taste disorders between 1990, 2003, and 2019 using the same methodology as that in previous studies. MATERIALS AND METHODS: In June 2019, we mailed a questionnaire to 1100 otolaryngologists belonging to the Japan Society of Stomato-pharyngology and investigated three question categories: "Institution", "Number of patients for 3 months", and "Treatment". In addition, we analyzed some results by the class of institution. RESULTS: The rate of patients who complained of taste disorders in the 2019 survey (220/100,000 persons/year) was twice that of the 1990 survey (110/100,000 persons/year), and slightly higher than that of the 2003 survey (192/100,000 persons/year). The rate of female patients was higher than that of male patients in all age groups. The number of patients was correlated with age up to 70 years of age in both genders. The rates of performing taste tests to assess taste function in the 2019 survey were significantly decreased compared with a 2003 survey (electrogustometry: p<0.001, filter paper disk method: p<0.05 in university). The rate of examination of the serum zinc in the 2019 survey was increased compared with the 1990 survey (p<0.001). Zinc oral therapy was used for the treatment of taste disorders in 239/299 (79.9%) patients/institutes for 3 months. In addition, 213 institutions (69.6%) answered that zinc oral therapy was efficacious for taste disorders. CONCLUSION: The patients who complained of taste disorder have increased. The zinc administration is an appropriate clinical treatment for taste disorders in Japan. To enhance treatment for taste disorders, simpler methods for assessing taste function need to be developed, and the pathological mechanisms of taste disorders other than zinc deficiency need to be clarified.

Validity of dietary isothiocyanate intake estimates from a food frequency questionnaire using 24 h urinary isothiocyanate excretion as an objective biomarker: the JPHC-NEXT protocol area.

BACKGROUND/OBJECTIVES: Isothiocyanate (ITC) is formed via the hydrolysis of glucosinolates by myrosinase, found in cruciferous vegetables. Although myrosinase is inactivated by the cooking process, no studies have incorporated the effect of cooking into the estimation of dietary ITC intake or evaluated the validity. We evaluated the validity of dietary ITC intake estimated from a food frequency questionnaire (FFQ), and urinary ITC levels using 24 h urine samples or a WFR (weighed food record), and evaluated the reproducibility of dietary ITC in two FFQs administered at an interval of 1-year. SUBJECTS/METHODS: The JPHC-NEXT Protocol Area included a total of 255 middle-aged participants across Japan. We calculated dietary ITC intake from WFR and two FFQs by assuming that cooked cruciferous vegetables contain zero ITC. Urinary ITC excretion was measured at two points during summer and winter. The validity and reproducibility of dietary ITC intake estimated by FFQ were assessed using Spearman's correlation coefficients. RESULTS: Although we observed a moderate correlation between dietary ITC intake derived from a 12-day WFR and urinary ITC excretion, notwithstanding the cooking process, the correlation between dietary ITC intake estimated by FFQ and mean urinary ITC excretion was low. However, the correlation was improved when we compared urinary ITC excretion and a 3-day WFR or FFQ collected during winter. Our FFQ showed good reproducibility. CONCLUSION: Although seasonality is a critical factor, dietary ITC intake estimated using an FFQ showed moderate validity and reproducibility and can be used in future epidemiological studies.

Structural Changes during the Photorepair and Binding Processes of Xenopus (6-4) Photolyase with (6-4) Photoproducts in Single- and Double-Stranded DNA.

Photolyases (PHRs) repair ultraviolet (UV)-induced DNA photoproducts into normal bases. In this study, we measured the conformational changes upon photoactivation and photorepair processes of a PHR and its specific substrates, (6-4)PHR and a pyrimidine(6-4)pyrimidone photoproduct ((6-4)PP), by light-induced difference Fourier transform infrared (FT-IR) spectroscopy. The single-stranded DNA with (6-4)PP (ss(6-4)PP) was used as a substrate and the resultant FT-IR spectra were compared with the previous results on double-stranded DNA with (6-4)PP (ds(6-4)PP). In the excess amount of substrate to the enzyme, different ss(6-4)PP photorepair FT-IR signals were obtained in an illumination time-dependent manner. As reported for ds(6-4)PP, the early stages of the photoreaction involve the changes in the ss(6-4)PP only, while the late stages of the reaction involve the ss(6-4)PP repair-associated changes and dissociation from (6-4)PHR. From these spectra, difference spectra originating from the binding/dissociation spectrum were extracted. The signals of the C═O stretches of (6-4)PP and repaired thymines in the single- and double-stranded DNA were tentatively assigned. The C═O stretches of (6-4)PP were observed at frequencies that reflect single- and double-stranded DNA environments in aqueous solution, reflecting the different hydrogen-bonding environments. The conformational changes of PHR upon binding of ss(6-4)PP and ds(6-4)PP were similar, suggesting that the conformational change is limited to the (6-4)PP binding pocket region. We interpreted that ds(6-4)PP may be bound together without any special mechanism for flipping out.