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Loss of biodiversity is a serious concern, and amphibians are particularly threatened. Most small salamanders in Japan are endangered. Distributional information is fundamental to the conservation of these rare species; however, small salamanders are generally difficult to locate or catch. Environmental DNA analysis is an effective survey method for monitoring such rare species. The conventional polymerase chain reaction (PCR) method, which combines PCR amplification with subsequent electrophoresis, and the real-time PCR method, which uses fluorescent material, are commonly used for this purpose. In this study, a comparison of these two detection methods was conducted using a rare salamander species, Hynobius boulengeri, as a model case. We compared three points: (i) detection sensitivity, (ii) influence of environmental factors related to detection, and (iii) time and financial costs of the two methods. To perform this comparison, we developed a real-time PCR detection assay, conducted field surveys, and compared the time and financial costs of conventional and real-time PCR methods. The comparison showed no statistical difference in the detection sensitivity from field samples, and the effects of environmental factors tended to be similar. In addition, the financial cost was lower for the conventional PCR method while the time cost was lower for the real-time PCR method. Therefore, selecting eDNA detection methods based on objectives, time, and financial costs will promote efficient monitoring and contribute to the conservation of rare species., (© 2023. The Author(s), under exclusive licence to The Japan Society for Analytical Chemistry.)

Background: Traumatic tension gastrothorax is a rare and potentially fatal condition occurring in patients with congenital or acquired diaphragmatic defects. Traumatic tension gastrothorax leads to acute and severe respiratory distress. Delayed tension gastrothorax that develops late during injury can be more severe., Case Presentation: An 84-year-old woman was brought to our facility with cardiac arrest and returned to spontaneous circulation after 2 min of cardiopulmonary resuscitation. Computed tomography showed diaphragmatic injury and tension gastrothorax due to trauma because of a fall episode few days earlier. Emergency thoracotomy and laparotomy was performed, because nasogastric tube insertion failed. There was a partially necrotic stomach in the chest cavity. The stomach was retracted from the thoracic cavity into the abdominal cavity and placed in its proper position. There was a 5 cm tear of the diaphragm. The tear was sutured and closed and then the necrotic area of the stomach was resected. Although the surgery relieved the intrathoracic compression, it resulted in re-expansion pulmonary edema immediately after surgery and hypoxemia. The patient was unable to overcome the hypoxemic state and eventually died., Conclusions: Delayed tension gastrothorax can lead not only to obstructive shock due to intrathoracic compression but also to more severe organ ischemia and re-expansion pulmonary edema due to insufflation., (© 2022. The Author(s).)

This study was a prospective, multicentre, cohort study on 685 patients who had undergone oncologic surgery. The patients were divided into two groups according to the presence or absence of postoperative pneumonia. The two groups were compared with respect to their background, index operation, food eaten, oral condition, contents of oral care and dental treatment, laboratory data, and bacterial flora. All postoperative pneumonias occurred in six cases within four days postoperatively. The multivariable logistic regression analysis showed that preoperative serum C-reactive protein was the strongest predictor of postoperative pneumonia. In addition, decreased postoperative Candida albicans colonies was an effective predictor of postoperative pneumonia. For patients with predictors of postoperative pneumonia, perioperative strategies for its prevention should be considered in addition to professional oral health care. This study was approved by the National Hospital Organization's Central Ethics Review Board and was also approved by the directors of the participating institutions.

Ubiquitin and its polymeric forms are conjugated to intracellular proteins to regulate diverse intracellular processes. Intriguingly, polyubiquitin has also been identified as a component of pathological protein aggregates associated with Alzheimer's disease and other neurodegenerative disorders. We recently found that polyubiquitin can form amyloid-like fibrils, and that these fibrillar aggregates can be degraded by macroautophagy. Although the structural properties appear to function in recognition of the fibrils, no structural information on polyubiquitin fibrils has been reported so far. Here, we identify the core of M1-linked diubiquitin fibrils from hydrogen-deuterium exchange experiments using solution nuclear magnetic resonance (NMR) spectroscopy. Intriguingly, intrinsically flexible regions became highly solvent-protected in the fibril structure. These results indicate that polyubiquitin fibrils are formed by inter-molecular interactions between relatively flexible structural components, including the loops and edges of secondary structure elements., Competing Interests: The authors declare no conflict of interest.

Background: The purpose of this retrospective study was to accumulate data regarding the quality of postoperative neurosensory function after inferior alveolar nerve (IAN) transposition for dental implant placement., Methods: The study included seven consecutive patients who underwent IAN transposition surgery for the insertion of a dental implant into the atrophic posterior mandible. Of these, six patients (seven sides) were available for long-term assessment of postoperative IAN function. Neurosensory disturbance of the IAN was assessed objectively using the modified SW perception test reported by Semmes and Weinstein. In addition, the quality of nerve paralysis was assessed according to the criteria reported by Highet., Results: The median follow-up time was 49 months (range 12-105 months). No implant loss was observed during the follow-up. All patients experienced numbness immediately and the days after surgery. Complete recovery of neural function was observed on two sides; weak hypoesthesia was observed on two sides, moderate hypoesthesia on two sides, and severe hypoesthesia on one side. However, only one patient expressed concern about IAN function., Conclusions: IAN transposition is a useful method for placing implants in the atrophic posterior mandible. However, the procedure is complicated, with the possibility of some degree of neurosensory disturbance, although in most of our cases, it resolved within a clinically acceptable period.

A novel series of pyridyl carboxamide-based CCR5 inhibitors was designed, synthesized, and demonstrated to be highly potent against HIV-1 infection in both HOS and PBL assays. Attempts to evaluate this series of compounds in a rat PK model revealed its instability in rat plasma. A hypothesis for this liability was proposed, and strategies to overcome this issue were pursued, leading to discovery of highly potent 40 and 41, which featured dramatically improved rat PK profiles., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Based on the original spirodiketopiperazine design framework, further optimization of an orally available CCR5 antagonist was undertaken. Structural hybridization of the hydroxylated analog 4 derived from one of the oxidative metabolites and the new orally available non-hydroxylated benzoic acid analog 5 resulted in another potent orally available CCR5 antagonist 6a as a clinical candidate. Full details of a structure-activity relationship (SAR) study and ADME properties are presented., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Following the discovery that hydroxylated derivative 3 (Fig. 1) was one of the oxidative metabolites of the original lead 1, it was found that hydroxylated compound 4 possesses higher in vitro anti-HIV potency than the corresponding non-hydroxylated compound 2. Structural hybridation of 4 with the orally available analog 5 resulted in another orally-available spirodiketopiperazine CCR5 antagonist 6a that possesses more favorable pharmaceutical profile for use as a drug candidate., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objectives: Fatty acid metabolism is controlled not only by the acyl-coenzyme A (CoA) synthetases but by some enzymes in the β-oxidation cycle. Medium-chain and long-chain acyl-CoA esters are key metabolites in fatty acid metabolism. We have developed an enzymatic assay method for determining chain shortening of the acyl-CoAs via β-oxidation from palmitic and octanoic acids in liver mitochondria. We have evaluated the assay method for detecting whether drugs influence the activation or the β-oxidation of fatty acids., Methods: Liver mitochondria were used for investigating the effect of drugs on fatty acid metabolism. The drugs selected were salicylic acid, diclofenac, valproic acid and paracetamol. Each acyl-CoA formed was analysed by liquid chromatography-tandem mass spectrometry., Key Findings: After less than 5 min of incubation, the levels of acyl-CoAs reflected the acyl-CoA synthetase activity, whereas after 60-min incubation they reflected the activity of some enzymes in the β-oxidation cycle. Salicylic acid, diclofenac and valproic acid inhibited the medium-chain acyl-CoA synthetases, whereas valproic acid only exhibited a weak inhibitory activity toward the β-oxidation of the medium-chain fatty acids. In the case of long-chain fatty acid metabolism, salicylic acid and diclofenac inhibited both the activation and β-oxidation, whereas valproic acid was a weak inhibitor for only the β-oxidation activity. Paracetamol showed hardly any influence on the metabolism of medium-chain and long-chain fatty acids., Conclusions: These findings suggest that salicylic acid, diclofenac, valproic acid and paracetamol exert a different influence on fatty acid metabolism depending on the length of the acyl chain. This assay allows sensitive and selective analysis for predicting the pathways by which drugs exert a greater influence over fatty acid metabolism., (© 2010 The Authors. JPP © 2010 Royal Pharmaceutical Society of Great Britain.)

The purpose of this study was to analyze the impact of different surgical margin conditions on local recurrence of oral squamous cell carcinoma (SCC) in 148 consecutive patients who underwent planned radical resection of oral SCC. The patients were classified into four categories according to the status of the surgical margin: clear (no SCC within 5mm, n=103), close (SCC within 5mm, n=21), dysplasia (dysplastic epithelium at margin, n=13), and involved (SCC at margin, n=11). Cox's proportional hazard model showed that the status of the surgical margin had a significant impact on local recurrence (p<0.003); hazard ratio was 3.79 (95%CI: 1.17-12.28) with a close and 7.89 (2.38-26.17) with an involved margin. The presence of mucosal dysplasia at the surgical margin was also a significant predictor of local recurrence (hazard ratio: 5.29, 95%CI: 1.31-21.29). Local recurrence was observed only with severe dysplasia, while no recurrence with mild and moderate dysplasia. In the patients with a clear and closed surgical margin, local recurrence was related with T4 tumor and an advanced mode of tumor invasion. The results of this study suggested that the presence of tumor cells at or close to the surgical margin increased the risk of local recurrence. The presence of dysplastic epithelium (especially severe dysplasia) at the mucosal surgical margin has a significant impact on local control. It was also suggested that not simply the width of the free margin but also clinical and histological risk factors should be included in deciding the necessity for adjuvant radiotherapy., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Using the previously reported novel spirodiketopiperazine scaffold, the design and synthesis of orally available CCR5 antagonists was undertaken. Compounds possessing a carboxylic acid function in the appropriate position showed improved oral exposure (AUC) relative to the initial chemical leads without reduction in the antagonist activity. The optimized compound 40 was found to show potent anti-HIV activity. Full details of structure-activity relationship (SAR) study are presented., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Spirodiketopiperazine-based CCR5 antagonists, showing improved pharmacokinetic profiles without reduction in antagonist activity, were designed and synthesized. We also demonstrate the anti-HIV activity of a representative compound 12, as measured in a p24 assay., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Oral lichen planus (OLP) is a refractory mucosal disease. Its pathogenesis is thought to involve immunologic and genetic alterations. To gain a better understanding of the genetic risk factors, the authors evaluated associations between 14 functional gene polymorphisms and OLP. 32 Japanese patients with OLP and 99 unrelated healthy Japanese controls were genotyped for 14 single nucleotide polymorphisms (SNPs) of genes that regulate host immune responses. Genotyping was performed with a modified version of the serial invasive signal amplification reaction. A trend towards over-representation of tumor necrosis factor receptor 2 (TNFR2) +587 G allele was found in the patients compared with the controls (allele frequency: P=0.049). The other 13 SNPs were unassociated with OLP. These results suggest that TNFR2 +587 gene polymorphism may be associated with susceptibility to OLP.

Background: Matrix metalloproteinase (MMP) is known to be involved in the initial and progressive stages of cancer development, and in the aggressive phenotypes of cancer. This study examines the association of single nucleotide polymorphisms in promoter regions of MMP-1 and MMP-3 with susceptibility to oral squamous cell carcinoma (OSCC)., Methods: We compared 170 Japanese OSCC cases and 164 healthy controls for genotypes of MMP-1 and MMP-3., Results: The frequency of the MMP-1 2G allele was higher and that of the 1G homozygote was lower in the OSCC cases (p = 0.034). A multivariate logistic regression analysis revealed that subjects who were 45 years old or older had a significantly increased (2.47-fold) risk of OSCC (95%CI 1.47-4.14, p = 0.0006), and those carrying the MMP-1 2G allele had a 2.30-fold risk (95%CI 1.15-4.58, p = 0.018), indicating independent involvement of these factors in OSCC. One of the key discoveries of this research is the apparent reduction of the MMP-1 1G/1G and 1G/2G genotype distributions among the early onset OSCC cases under the ages of 45 years. It should be noted that the tongue was the primary site in 86.2% of these early onset cases. This could suggest the specific carcinogenic mechanisms, i.e. specific carcinogenic stimulations and/or genetic factors in the tongue., Conclusion: Since the 2G allele is a majority of the MMP-1 genotype in the general population, it seems to act as a genetic pre-condition in OSCC development. However this report suggests a crucial impact of the MMP-1 2G allele in the early onset OSCC.

Hydroxylated derivatives were designed and synthesized based on the information of oxidative metabolites. Compounds derived from beta-substituted (2R,3R)-2-amino-3-hydroxypropionic acid showed improved inhibitory activities against the binding of MIP-1alpha to human CCR5, compared with the non-hydroxylated derivatives and the other isomers.

We quantified telomerase activity (TA) in patients with oral and maxillofacial malignant and nonmalignant lesions, and compared it with their clinical status and grade of malignancy. Fifty-two malignant and 52 nonmalignant lesions were analyzed. All malignant lesions were pathologically diagnosed as oral squamous cell carcinoma (OSCC). Normal gingival tissue served as a control. These specimens were obtained by biopsy or surgical resection, and stored at -80 degrees C until use. TA was quantified by a fluorescence-based TRAP method. TA levels ranged from 0.00 to 95.24 (average 33.24)U/microgP in 52 malignant lesions, and from 0.00 to 79.35 (average 11.91)U/microgP in 52 nonmalignant lesions (P < 0.0001). TA was detected in 96.2% of malignant and 65.4% of nonmalignant lesions. There was no relationship between TA levels and clinical stages or YK classification. However, under WHO classification, there were significant differences (P < 0.05) between Grades I and III or II + III. Among nonmalignant lesions, epithelial dysplasia showed a significantly higher TA level than that of oral lichen planus (P < 0.05) and other benign lesions (P < 0.0001). Oral lichen planus also significantly differed from other benign lesions (P < 0.05). These results suggest that TA is related to the histological grade of malignancy, and is also useful as a prognostic predictor for precancerous lesions and conditions.

Two cases of recurrent gastric cancer are described. Recurrence at anastomosis was detected following total gastrectomy in both patients. They declined surgery but wanted medical treatment. Therefore, 100 mg/kg of TS-1 was administered with informed consent. Symptoms related to stenosis were relieved after the first course, and complete responses were confirmed by endoscopic and pathological studies after completion of 4 courses in both patients. Palliative surgery remains a major option of treatments for patients with recurrence at anastomosis after total gastrectomy. TS-1, however, should be considered a treatment for such cases with satisfactory QOL, especially for outpatients who do not want surgery.

Bestatin, an immunomodulator useful in cancer treatment with the structure: N-[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, has been synthesized from N-acyl-alpha-aminoacetophenone. This novel method using readily available reagents and mild conditions can be applied to a large scale production of this useful agent. The overall yield of bestatin was 10.5% from N-acetyl-alpha-aminoacetophenone and 7.8% from N-benzoyl-alpha-aminoacetophenone.

The series was collected in 1973-1977 from 15 hospitals in Gunma Prefecture where all pregnant women were Rh-typed and blood samples were checked for antibodies by this laboratory. The total number of Rho(D) negative pregnant women was 435 and of these, 20 had developed antibodies. Three of these 20 patients had received blood transfusions. Six of the antibody containing patients were first immunized during the period of observation. Three of these 6 produced the antibody in the course of a first pregnancy and 2 in second pregnancy and 1 in third. The antibody titers increased after 20th gestational week, especially after 30th through 40th week in most of the patients. Three out of 9 infants, whose mother's antibody titers increased no more than 256 folds by papain treated red cells received phototherapy. In the cases of antibody titers more than 256 folds, 9 out of 11 babies received exchange blood transfusion. The mature infants were all alive healthily. There was no Rho(D) negative woman who repeated intrauterine fetal death by hemolytic disease of the fetuses.

Stereoisomers and analogues of bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, were synthesized and tested for aminopeptidase B and leucine aminopeptidase inhibiting activity. Among the eight stereoisomers, the 2S stereoisomers exhibited strong activity. In a series of compounds in which the L-leucine residue of bestatin was substituted with other amino acids, only the one containing isoleucine showed more activity than bestatin. Norleucine, norvaline, methionine, valine, serine, glutamine, phenylalanine, glutamic acid, proline, and lysine analogues gave, in that order, decreasing activity. Alkyl and phenyl sub stitution for the benzyl group of bestatin decreased the activity markedly. p-Methyl-, p-chloro-, and p-nitrobestatins showed greater activity than bestatin.

A lambda clone carrying a rat DNA fragment of 11.9 kb was isolated from a rat gene library with total rat tRNA as a probe. Nucleotide sequence analysis revealed that the DNA fragment contained six tRNA genes, three for tRNAPro and three for tRNALys. Of the six genes all but one tRNAPro gene have the same polarity. Each tRNA gene is separated by a DNA region of 0.1 to 3.6 kb. The 5'-flanking regions of the six rat genes in the cluster do not have any significant sequence homology, but in the 3'-flanking region, each gene has a short T cluster, which is supposed to be a transcription termination signal.

Stable spergualin analogues were synthesized by substitutions of the alpha-hydroxyglycine residue of spergualin with various alpha- or omega-amino acids. The antitumor activity of these analogues against L1210 and their immunosuppressive effects on delayed-type hypersensitivity and antibody formation was then examined. Analogues substituted with glycine and L-serine showed significant biological activity but were less potent than 15-deoxyspergualin. Among the analogues synthesized so far, 10-[N-4-(4-guanidinophenyl)butyryl-L-seryl]-1,5,10-triazadecane has possessed the strongest antitumor and immunosuppressive activities.

Eight tripeptides containing L-Phe-L-Tyr were synthesized, and their effects on the excitability of a giant neurone (tonically autoactive neurone, TAN), identified in the suboesophageal ganglion of an African giant snail (Achiatina fulica Férussac), were examined. Of these tripeptides, the following three showed a marked inhibitory effect: L-Glu-L-Phe-L-Tyr (most effective; critical concentration: 3 X 10(-6)--10(-5) M), Gly-L-Phe-L-Tyr and sarcosine-L-Phe-L-Tyr. The TAN inhibition caused by L-Glu-L-Phe-L-Tyr was not due to an increased membrane permeability to chloride ions.

p-Hydroxyubenimex, (2S,3R)-3-amino-2-hydroxy-4-p-hydroxyphenylbutyryl-L-leucine, was synthesized starting from D-tyrosine. The structure and stereochemistry of the synthesized product were confirmed by comparison with p-hydroxyubenimex that was chemically transformed from ubenimex, an aminopeptidase inhibitor of microbial origin. Compared to ubenimex, p-hydroxyubenimex is more active against aminopeptidase B but less active against leucine aminopeptidase. By using the synthetic p-hydroxyubenimex as a reference sample, one of the metabolites of ubenimex was identified as p-hydroxyubenimex. The (2R,3R)-stereoisomer of p-hydroxyubenimex was also prepared. However, its activity against aminopeptidases was much weaker.

The linear DNA killer plasmids (pGKL1 and pGKL2) isolated from a Kluyveromyces lactis killer strain are also maintained and expressed its killer character in Saccharomyces cerevisiae. After these killer plasmid DNAs isolated from S. cerevisiae were treated with alkali, four terminal fragments from each plasmid DNAs were cloned separately. Using these and other cloned DNA fragments, the terminal nucleotide sequences of pGKL2 and the complete nucleotide sequence of pGKL1 were determined. The inverted terminal repetitions of 202 bp and 182 bp were found in pGKL1 and pGKL2, respectively. The pGKL1 sequence showed an extremely high A + T content of 73.2% and it contained five large open reading frames. The largest of these open reading frame was suggested to code for a membrane-bound precursor of glycoprotein subunit of the killer toxin.