[Inhibitory effect of small-molecule compound AM679 targeting elongation-factor binding protein 2 on hepatitis B virus in vitro].

Objective: To explore the antiviral activity of the small-molecule compound AM679 in hepatitis B virus (HBV) replication and infection cell models. Methods: The positive regulatory effect of AM679 on EFTUD2 expression was validated by qPCR and Western blotting. HepAD38 and HepG2-NTCP cells were treated with AM679 (0.5, 1, and 2 nmol/L). Negative control, positive control, and AM679 combined with the entecavir group were set up. HBV DNA intra-and extracellularly, as well as the expression levels of intracellular HBV total RNAs and 3.5kb-RNA changes, were detected with qPCR. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels were measured in the cell supernatant by an enzyme-linked immunosorbent assay (ELISA). The t-test method was used for the statistical analysis of the mean difference between groups. Results: EFTUD2 mRNA and protein expression levels were significantly increased in HepAD38 and HepG2-NTCP cells following AM679 treatment, with a statistically significant difference (P < 0.001). Intra-and extracellular indicators such as HBV DNA, HBV RNAs, HBV 3.5kb-RNA, HBsAg, and HBeAg were decreased to varying degrees in both cell models, and the decrease in these indicators was more pronounced with the increase in AM679 concentration and prolonged treatment duration, while the combined use of AM679 and entecavir had a more significant antiviral effect. The HBV DNA inhibition rates in the supernatant of HepAD38 cells with the use of 2 nmol/L AM679 were 21% and 48% on days three and nine, respectively. The AM679 combined with the ETV treatment group had the most significant inhibitory effect (62%), with a P < 0.01. More active HBV replication was observed after silencing EFTUD2, while the antiviral activity of AM679 was significantly weakened. Conclusion: AM679 exerts anti-HBV activity in vitro by targeting the regulation of EFTUD2 expression.

Mobilization of recalcitrant phosphorous and enhancement of pepper P uptake and yield by a new biocontrol and bioremediation bacterium Burkholderia cepacia CQ18.

AIMS: Phosphorus (P) is a finite resource and inoculation of phosphorus-mobilizing bacteria (PMB) is a promising approach for the enhancement of soil P availability and plant P uptake. This drives scientists to search for the microbes effective in mobilizing legacy P in soils. METHODS AND RESULTS: The current incubation and greenhouse pot experiments were conducted to investigate P mobilization and pepper P uptake as affected by a new biocontrol and bioremediation bacterium Burkholderia cepacia CQ18. This bacterium converted Ca3 (PO4 )2 , FePO4 , AlPO4 , and lecithin into soluble inorganic P in the culture solutions and increased available P (including water-soluble P and Olsen P) in the soil. There were positive correlations between the soluble inorganic phosphorus and the exudates (protons, organic acids (oxalate and gluconate), siderophores and phosphatases) in culture solutions. Pepper plant biomass, fruit yield and P uptake changed in the sequence: chemical fertilizers plus bacterial inoculant >only chemical fertilizers >only bacterial inoculant >blank control. CONCLUSIONS: Taking into account the wide spectrums of P mobilization and simultaneous production of acid, neutral and alkaline phosphatases at a given pH, B.cepacia CQ18 may be a potential PMB used in soils with wide pH ranges. The mechanisms employed by this bacterium in the solubilization of recalcitrant inorganic P could be the efflux of protons, organic acids (oxalate and gluconate) and siderophores. Phosphatases could be of utmost importance in the mineralization of the organic P. The production of siderophores and phosphatases by of B.cepacia CQ18 could thus be crucial for not only the antagonism against plant pathogens but also the mobilization of soil sparingly available P. SIGNIFICANCE AND IMPACT OF THE STUDY: Burkholderia cepacia CQ18 could be potentially developed into a biofertilizer.

Clinical outcomes and predictive relapse factors of IgG4-related disease following treatment: a long-term cohort study.

OBJECTIVES: The aim of this study was to investigate the predictive factors for relapse of IgG4-related disease (IgG4-RD) and observe the long-term clinical outcomes in patients with IgG4-RD. METHODS: We included in the present analysis 122 patients who were newly diagnosed with IgG4-RD, treated with glucocorticoid (GC) monotherapy or GC and immunosuppressant combination therapy, and followed for at least 3 years. Clinical relapse, response and side effects were recorded. RESULTS: The cumulative relapse rates of patients in this study were 10.66%, 22.95% and 27.87% at 12, 24 and 36 months, respectively. Complete drug withdrawal was an independent risk factor for disease relapse. Higher serum IgG4 concentrations, involvement of more organs, higher IgG4 RI scores and elevation of eosinophils at baseline were closely associated with disease relapse. Re-elevation of serum IgG4 concentrations and low GC maintenance dosage during the follow-up period were significantly associated with clinical relapse. The GC dosage should be more than 6.25 mg day-1 as monotherapy during the maintenance stage; moreover, combining with immunosuppressants can reduce the GC dosage. Adding GC or immunosuppressants for patients with re-elevation of serum IgG4 levels could prevent later disease relapse. No serious complications were noted during long-term follow-up. CONCLUSIONS: The combination of GC with immunosuppressants was more effective than GC monotherapy during the steroid tapering and maintenance stages. Higher serum IgG4 levels, involvement of more organs, higher IgG4 RI scores, history of allergy, eosinophil elevation at baseline, re-elevation of serum IgG4 levels and lower GC maintenance dosage at follow-up might be predictive of relapse.

[Analysis of single-nucleotide polymorphism of Sonic hedgehog signaling pathway in non-syndromic cleft lip and/or palate in the Chinese population].

OBJECTIVE: To study the relationship between Sonic hedgehog (Shh) associated single-nucleotide polymorphism (SNP) and non-syndromic cleft lip and/or palate (NSCL/P), and to explore the risk factors of cleft lip and/or palate. Many studies suggest that the pathogenesis of NSCL/P could be related to genes that control early development, in which the Shh signaling pathway plays an important role. METHODS: Peripheral blood was collected from 197 individuals (100 patients with NSCL/P and 97 healthy controls). Haploview software was used for haplotype analysis and Tag SNP were selected, based on the population data of Han Chinese in Beijing of the international human genome haplotype mapping project. A total of 27 SNP were selected for the 4 candidate genes of SHH, PTCH1, SMO and GLI2 in the Shh signaling pathway. The genotypes of 27 SNP were detected and analyzed by Sequenom mass spectrometry. The data were analyzed by chi-squared test and an unconditional Logistic regression model. RESULTS: The selected SNP basically covered the potential functional SNP of the target genes, and its minimum allele frequency (MAF) was >0.05: GLI2 73.5%, PTCH1 91.0%, SMO 100.0%, and SHH 75.0%. It was found that the genotype frequency of SNP (rs12674259) located in SMO gene and SNP (rs2066836) located in PTCH1 gene were significantly different between the NSCL/P group and the control group. Linkage disequilibrium was also found on 3 chromosomes (chromosomes 2, 7 and 9) where the 4 candidate genes were located. However, in the analysis of linkage imbalance haplotype, there was no significant difference between the disease group and the control group. CONCLUSION: In China, NSCL/P is the most common congenital disease in orofacial region. However, as it is a multigenic disease and could be affected by multiple factors, such as the external environment, the etiology of NSCL/P has not been clearly defined. This study indicates that Shh signaling pathway is involved in the occurrence of NSCL/P, and some special SNP of key genes in this pathway are related to cleft lip and/or palate, which provides a new direction for the etiology research of NSCL/P and may provide help for the early screening and risk prediction of NSCL/P.

Interaction of thymic stromal lymphopoietin, IL-33, and their receptors in epithelial cells in eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: Thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 system contribute to the initiation and development of Th2 responses. This study aimed to explore the involvement of TSLP, IL-25, IL-33, and their receptors in type 2 T-helper (Th) responses in chronic rhinosinusitis with nasal polyps (CRSwNPs) and their cross-regulation in human nasal epithelial cells (HNECs). METHODS: Immunohistochemistry, quantitative RT-PCR, ELISA, Bio-Plex assay, and flow cytometry were used to detect the expression of TSLP/common γ-like TSLP receptor (TSLPR)/IL-7 receptor α (IL-7Rα), IL-25/IL-17B receptor (IL-17RB), and IL-33/membrane-bound ST2 (ST2L)/soluble ST2 (sST2) in sinonasal mucosa and HNECs. HNECs cultured at an air-liquid interface were used to explore the expression in regulation of these cytokine systems. RESULTS: Compared with controls and noneosinophilic CRSwNP, the expression of TSLP/TSLPR/IL-7Rα and ST2L/sST2 was significantly increased in eosinophilic CRSwNP, predominantly in epithelial cells. In contrast, the expression of IL-33 and IL-25/IL-17RB was enhanced in epithelial cells in both eosinophilic and noneosinophilic CRSwNP compared to controls. The expression of TSLP, TSLPR, and ST2L was positively correlated with symptom and computer tomography scan scores in eosinophilic CRSwNP and with Th2 cytokine expression in sinonasal mucosa. The expression of ST2L was correlated with TSLP and its receptor expression. TSLP could induce ST2L expression that promoted IL-33-induced TSLP expression in HNECs. In addition, TSLP/TSLPR/IL-7Rα and ST2L could be induced by Th2 cytokines, while IL-25/IL-17RB and IL-33 could be upregulated by Th1/Th17 cytokines, in HNECs. CONCLUSIONS: The positive feedback loop between TSLP, IL-33 and their receptors, and Th2 cytokines may facilitate Th2-skewed inflammation in eosinophilic CRSwNP.

Rapid genomic DNA changes in allotetraploid fish hybrids.

Rapid genomic change has been demonstrated in several allopolyploid plant systems; however, few studies focused on animals. We addressed this issue using an allotetraploid lineage (4nAT) of freshwater fish originally derived from the interspecific hybridization of red crucian carp (Carassius auratus red var., ♀, 2n=100) × common carp (Cyprinus carpio L., ♂, 2n=100). We constructed a bacterial artificial chromosome (BAC) library from allotetraploid hybrids in the 20th generation (F20) and sequenced 14 BAC clones representing a total of 592.126 kb, identified 11 functional genes and estimated the guanine-cytosine content (37.10%) and the proportion of repetitive elements (17.46%). The analysis of intron evolution using nine orthologous genes across a number of selected fish species detected a gain of 39 introns and a loss of 30 introns in the 4nAT lineage. A comparative study based on seven functional genes among 4nAT, diploid F1 hybrids (2nF1) (first generation of hybrids) and their original parents revealed that both hybrid types (2nF1 and 4nAT) not only inherited genomic DNA from their parents, but also demonstrated rapid genomic DNA changes (homoeologous recombination, parental DNA fragments loss and formation of novel genes). However, 4nAT presented more genomic variations compared with their parents than 2nF1. Interestingly, novel gene fragments were found for the iqca1 gene in both hybrid types. This study provided a preliminary genomic characterization of allotetraploid F20 hybrids and revealed evolutionary and functional genomic significance of allopolyploid animals.

[Implant restoration and stomatognathic system rehabilitation].

Stomatognathic system rehabilitation (SSR) is an important component of dental implant therapy, involving multiple disciplines and factors. This article focuses on the importance of clinical issues, such as mandibular position, vertical distance, occlusion and temporomandibular joint in SSR, in order to provide reference for dentists in clinical diagnosis and treatment.

[Clinical analysis of nodular fasciitis in the head and neck].

Objective: To analyze the clinical features, diagnosis, treatment and prognosis of nodular fasciitis (NF) in the head and neck. Methods: Seven cases of primary NF in the head and neck admitted to Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from 1990 to 2022 were retrospectively analyzed, including 3 males and 4 females, aged from 2 to 67 years, and the location, course of disease, clinical manifestations, imaging findings, and treatment results of the disease were summarized. Results: Of the 7 patients, the primary sites were respectively nasal vestibule, paranasal sinus, nasal dorsum, sella turcica and clivus, neck, subglottis, and tonsil. Three cases presented with rapidly growing subcutaneous painless masses within 2 months, 1 case presented with hemoptysis, hoarseness and asthma for 28 days, 1 case presented with blood in the nasal discharge for 2 months, 1 case presented with headache for 1 month, and 1 case was found to have tonsillar neoplasms on physical examination for 3 days. CT was performed in 5 patients and the lesions showed soft tissue density shadows, and MRI was also performed in 2 of them, which showed T1 isointensity or T2 slightly long abnormal signal shadows. All patients underwent simple surgical resection of the mass. The patients were followed up for 13 months to 10 years, and none had recurrence. Conclusion: Primary NF in the head and neck is rare, with atypical clinical symptoms and imaging findings. Surgery is the mainstay of treatment for NF with good results.

Stabilization of source-separated urine by biological nitrification process: treatment performance and nitrite accumulation.

A laboratory study on nitrification of high-strength source-separated urine was conducted by means of sequencing batch reactors (SBR) and membrane bioreactors (MBR). The highest influent ammonia concentration for SBR and MBR reached more than 2,400 and 1,000 mg N/L, while the maximum pH was about 9 and 8.9, respectively. The ammonia oxidizing efficiency in both SBRs and MBRs was around 50%, which was restrained mainly by the deficiency of alkalinity in bulks. Meanwhile, the nitrite accumulation did also dominate in these two systems, and the major factor to inhibit the nitrite oxidization was thought to be the high free ammonia and free nitrous acid content in bulks. Hence, an ammonia nitrite solution was achieved with concentration ratio of 1:1; after that ammonia oxidation was restrained owing to the deficiency of alkalinity in urine. The temperature and influent ammonia content have no great influence on the nitrification process in both kinds of bioreactors. The nitrification can be progressed under a solids retention time (SRT) longer than 30 d; however, termination of ammonia oxidization was observed as the SRT fell below 20 d. The nitrifier biomass showed an excellent settleability, such that the suspended solids (SS) in effluent was of a low average, about 60 mg/L. This study on the stabilization of human urine will be useful to understand the process of urine separation from source.

[Mechanism of pepsin promoting lingual tonsil hypertrophy by stimulating macrophage].

Objective: To investigate the possible pathophysiological mechanism of laryngopharyngeal reflux (LPR) in the development of lingual tonsil hypertrophy (LTH). Methods: The lingual tonsil tissues were collected from 73 patients [48 males and 25 females, aged from 24 to 76 (52.86±12.04) years] who underwent surgery for laryngopharyngeal diseases at the Department of Otolaryngology and Head and Neck Surgery, Southern Hospital of Southern Medical University from October 2019 to December 2020, and the lingual tonsil grade (LTG), reflux symptom index (RSI) and reflux finding score (RFS) were assessed. The expression of pepsin in LTH was detected by immunohistochemistry. The coexpression of pepsin and macrophages were detected by immunohistofluorescence. In vitro, cytological experiments and pathway assays were performed on macrophages stimulated by pepsin. Pathway alterations of macrophages in pepsin-positive high-grade LTH were detected by double-fluorescence immunohistochemistry. Data were analyzed by SPSS 20.0 software. Results: There were 44 clinically significant LPRD patients with LTG 3 and 4, and the pepsin positive rate was 88.6% (39/44). While, the pepsin positive rate of LTG 1 and 2 was 48.3% (14/29). LTG was significantly positively correlated with RFS/RSI positive rate(χ2=23.01/19.62, P<0.001/0.001; r=0.54/0.51, P<0.001/0.001) and pepsin tissue staining intensity (H=21.58, P<0.001; r=0.53, P<0.001), respectively. Pepsin and macrophages were clearly colocalized in high grade LTH. In vitro, pepsin promoted macrophage proliferation (P<0.05) and production of IL-6/IL-8 (P<0.05). Pepsin significantly up-regulated the p38/JNK MAPK pathway in macrophages (P<0.05). Pepsin up-regulated the expression of IL-6 and IL-8 of macrophages by activating the p38 MAPK pathway (P<0.05), and up-regulated the expression of IL-8 by activating the JNK pathway (P<0.05). The p38/JNK MAPK pathways were highly expressed in macrophages of pepsin-positive LTH (P<0.05). Conclusions: LPR is an important pathogenic factor in LTH. Macrophages may mediate pepsin-induced inflammation and the pathogenesis of LTH.

[Scan time and accuracy of full-arch scans with intraoral scanners: a comparative study on conditions of the intraoral head-simulator and the hand-held model].

Objective: To comparatively evaluate the scan time and the accuracy of maxillary full-arch scans using four intraoral scanners (IOS) on conditions of the intraoral head-simulator and the hand-held model, and to evaluate the influence of different scanning conditions on digital scan. Methods: A upper dental arch model with melamine-formaldehyde resin teeth and silica gel gingiva that could be fixed on a head simulator was scanned with an optical scanner (ATOS Core) in order to obtain the standard tessellation language dataset as reference. Intraoral scans were performed on the model fixed on the head simulator by three researchers with four IOS [A: TRIOS 3; B: CS 3600; C: CEREC Omnicam; D: iTero]. For each scanner and each researcher, six scans were performed, to obtain the datasets as the head simulator group. And another six scans with each of the four intraoral scanners were performed by each researcher on the hand-held model to obtain the STL datasets as the hand-held group. The scan time were recorded for each scan. In the Geomagic Wrap software, the digital models were trimmed with only the teeth information retained and supreimposed by best fit alignment function and compared to obtain the root mean square (RMS) values of the discrepancies by three-dimensional compare function. The test datasets of each group were compared with the reference dataset for trueness. The six test scanning datasets with the same scanner of the same researcher were cross compared for precision. Mann Whitney U test was used to statistically analyze the difference values of the scan time, trueness and precision of the same intraoral scanner between head simulator group and hand-held group. Results: Compared to the hand-held group, the scan time of A [142(82) s] and D [119(52) s], which two IOS both with handle, were longer in head simulator group [A: 98(28) s; D: 85(22) s] (P<0.01). However there were no significant differences between the two groups for scan time of IOS B and C (P>0.05). For full-arch scan accuracy (trueness and precision), there were no significant differences between the two groups of IOS A and B (P>0.05), while the trueness of C (P<0.05) and the precision of D (P<0.01) were better in head simulator group [C: 112(38) µm; D: 43(13) µm] compared to hand-held group [C: 135(47) µm; D: 53(18) µm]. However, there were no significant differences for the precision of C (P>0.05) and the trueness of D (P>0.05). Conclusions: The scan time and the accuracy of full-arch digital scans with different IOS may be effected by the scan conditions. For in vitro study of intraoral scanning, head-simulator can simulate the intraoral environment of the real patient to some extent. Meanwhile, the position of the dentist and the patient, and also the limited intraoral space during intraoral scanning are also simulated.

[Effect of digital intraoral full-arch scan strategies on scan time and accuracy on conditions of intraoral head-simulator].

Objective: To comparatively evaluate the accuracy and the scan time of three full-arch scan strategies on the head-simulator, to explore a full-arch scan strategy with better clinical operability and high accuracy. Methods: A cross-controlled study design was used. A model with melamine-formaldehyde resin teeth and silica gel gingiva of an upper dental arch which can be fixed on a head simulator was scanned with an optical scanner (ATOS Core) in order to obtain the standard tessellation language (STL) dataset as reference. Intraoral scans were performed on the model fixed on the head simulator with four intraoral scanners (IOS) [A (TRIOS 3), B (CS 3600), C (CEREC Omnicam), D (iTero)]. The STL datasets were obtained from each of the four different IOS systems by using three scan strategies (scan strategies 1, 2 and 3 were composed of 10, 5 and 7 paths respectively) all by one attending doctor with 3 years of intraoral scanning experience. For each scanner and each scan strategy, nine scans were acquired. And the scan time was recorded for each scan. Following the scan strategy, the scan path was completed to obtain a full-arch digital model, and the scan time was recorded as full-arch scan time. Complementary scans were performed to fill the missing image, and this scan time was recorded as complementary scan time. The total scan time was obtained by adding full-arch scan time and complementary scan time. Through the Geomagic Wrap software, the three-dimensional (3D) models were overlaid by best fit alignment function and compared to obtain the root mean square values of the discrepancies by 3D compare function. The intraoral scanning datasets were compared with the reference for trueness. The nine intraoral scanning datasets were cross compared with same scan strategy and same intraoral scanner for precision. Results: There were no significant differences among the three scan strategies for trueness (P>0.05), while the differences among the three scan strategies for precision were affected by difference IOSs (P<0.05), and only scan strategy 3 showed the highest precision with all the four IOS. The full-arch scan time of scan strategies 1, 2 and 3 were (130±24), (72±17) and (90±19) s respectively (P<0.05). For complementary scan time, scan strategy 2 [(50±24) s] took longer time than scan strategy 1 [(26±18) s] and scan strategy [(25±21) s] (P<0.05), while no significant differences between the latter two (P>0.05). For total scan time, scan strategy 1 [(156±31) s] took longer time than scan strategy 2 [(122±30) s ] and scan strategy 3 [(115±29) s ] (P<0.05), while no significant differences between the latter two (P>0.05). Conclusions: Full-arch scanning on the head-simulator with scan strategy 3 which can obtain scanning datasets with high accuracy, was more convenient to operate and took shorter scan time, and is generally suitable for intraoral scanners commonly used in clinic.

Unique histological features of the left atrial posterior wall.

The left atrial posterior wall (LAPW) plays a critical role in atrial fibrillation, but the underlying mechanism remains unclear. In the present study, we sought to characterize the histological features of the LAPW. Different atrial regions were dissected from hearts of normal Sprague-Dawley rats and humans. Haematoxylin/eosin and van Gieson staining were used to analyse atrial cardiomyocyte arrangement and collagen distribution, respectively. Intercellular junctions were evaluated by transmission electron microscopy. In contrast with other atrial regions, the LAPW exhibited more disorganized cardiomyocytes, larger intercellular spaces and variable myocardial fibre arrangement. The proportion of collagen was significantly higher in the LAPW than in other atrial regions. Interestingly, desmosomes were sparse along with intercellular gaps in the LAPW. In summary, distinct disarrangement of cardiomyocytes and an abundance of collagen exist in the LAPW. The sparsity of desmosomes in the LAPW may be related to the heterogeneous distribution and separation of atrial myocytes.

Constituents of Craspedolobium schochii.

Nine phenolic compounds, including a new one, were isolated from 70% acetone extract of Craspedolobium schochii. The new compound was identified as 3-(3,4-dimethoxy-2-hydroxyphenyl)-7-hydroxy-coumarin (1) on the basis of spectroscopic evidence.

Constituents from Lonicera japonica.

Five caffeoylquinic acids and esters (1-5), including a new compound, 3,5-dicaffeoylquinic acid butyl ester (5), were isolated from the flowers and buds of Lonicera japonica and their structures were determined by NMR spectral analysis.

Analysis of mitochondrial respiratory-related genes reveals nuclear and mitochondrial genome cooperation in allotetraploid hybrid.

An allotetraploid hybrid lineage derived from the distant hybridization of red crucian carp (Carassius auratus red var., ♀, 2n =100) × common carp (Cyprinus carpio L., ♂, 2n =100) was investigated for its mitochondrial and nuclear genome inheritance patterns. Based on liver transcriptomic data for this hybrid, red crucian carp, and common carp, we identified 94, 136, and 86 contigs corresponding to 41, 46, and 37 mitochondrial respiratory chain nuclear genes, respectively. Mitochondrial respiratory chain nuclear gene sequences from red crucian carp and common carp were both detected in the allotetraploid hybrid, indicating that both parental nuclear genomes were participated in the synthesis of mitochondrial respiratory protein complexes in the hybrid. For mitochondrial respiratory related genes, high sequence similarity (>90%) and a low nucleotide divergence rate (<0.2) between red crucian carp and common carp could be a critical factor allowing cooperation of the three genomes (red crucian carp mitochondrial genome, red crucian and common carp nuclear genomes) in the allotetraploid hybrid lineage. Interestingly, gene duplication events were identified in the allotetraploid hybrid, red crucian and common carp, as confirmed by analysis of orthologous gene trees for these fish. Our findings provide valuable information with which to study cooperation between the nuclear and mitochondrial genomes of other hybrids, and will provide basic genetic information of relevance to mitochondrial-related diseases in humans and animals.

Minifish shows high genetic variation in mtDNA size.

The genus Paedocypris is a newly described taxon of minifish species that are characterized by extensive chromosome evolution and one of the smallest known vertebrate nuclear genomes. Paedocypris features a tiny adult size, a short generation time, low fecundity and fragmented tropical habitats, which are factors that favor rapid speciation. Most recently, we have revealed that P. progenetica (Pp), the type species of the genus Paedocypris, has an unusual mtDNA bearing - within its D-loop - a tandem array of a 34-bp repeat sequence called the minifish repeat, which shows compromised replication efficiency in vitro. Here we report that Pp exhibits high genetic variation in mtDNA size. The efficiency of D-loop amplification was found to depend upon primers. Interestingly, Pp individuals of one and the same population differed drastically in mtDNA size resulting from varying copy numbers of the minifish repeat. We conclude that minifish has a high mutation rate and perhaps represents a rapidly evolving taxon of vertebrates.

Minifish mtDNA has abundance of repeat sequences and inefficient replication in vitro.

Paedocypris is a newly described minifish genus endemic to Southeast Asia. Besides a tiny adult size of ~8 mm in length, minifish feature fragmentary habitats of acidic peat blackwater swamps, an unusual reproduction mode, truncated development and one of the smallest known genomes. A complete sequence is absent for the minifish mitochondrial DNA (mtDNA). Here we report the complete mtDNA sequence and its unusual feature in the minifish P. progenetica (Pp). We show that the Pp mtDNA is a circular molecule of 17,382 bp in length and has the same number of similarly oriented genes as in other vertebrates. Specifically, it comprises 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 D-loop. Surprisingly, the D-loop is elusive for amplification by standard PCR conditions. The D-loop possesses a 28-bp dinucleotide TA repeat and more intriguingly, up to 25 copies of a 34-bp tandem repeat sequence. These tandem repeats predict the formation of paired regions. Hence, besides a generally conserved mtDNA with other vertebrates, the Pp mtDNA features an unusual D-loop and compromised DNA replication in vitro.

Constituents from Limonia crenulata.

A new indole alkaloid, crenulatine (1), along with twenty known compounds, was isolated from the stems of Limonia Crenulata. Their structures were identified by spectral means. Those compounds include four alkaloids, four coumarins, two flavanones, three tetranortriterpenoids, one triterpenoid, three steroids, two lignans and two aromatic compounds.