Metagenomic analysis reveals the prevalence and persistence of antibiotic- and heavy metal-resistance genes in wastewater treatment plant

The increased antibiotic resistance among microorganisms has resulted into growing interest for investigating the wastewater treatment plants (WWTPs) as they are reported to be the major source in the dissemination of antibiotic resistance genes (ARGs) and heavy metal resistance genes (HMRGs) in the environment. In this study, we investigated the prevalence and persistence of ARGs and HMRGs as well as bacterial diversity and mobile genetic elements (MGEs) in influent and effluent at the WWTP in Gwangju, South Korea, using high-throughput sequencing based metagenomic approach. A good number of broad-spectrum of resistance genes (both ARG and HMRG) were prevalent and likely persistent, although large portion of them were successfully removed at the wastewater treatment process. The relative abundance of ARGs and MGEs was higher in effluent as compared to that of influent. Our results suggest that the resistance genes with high abundance and bacteria harbouring ARGs and MGEs are likely to persist more through the treatment process. On analyzing the microbial community, the phylum Proteobacteria, especially potentially pathogenic species belonging to the genus Acinetobacter, dominated in WWTP. Overall, our study demonstrates that many ARGs and HMRGs may persist the treatment processes in WWTPs and their association to MGEs may contribute to the dissemination of resistance genes among microorganisms in the environment.     

Non-secretory solitary plasmacytoma of the spleen

A case of primary nonsecretory plasmacytoma of the spleen is reported. On laparotomy and splenectomy a 920 g spleen was removed, measuring 16×14×6 cm. The cut surface of the entire spleen showed that the tumour occupied most of the splenic tissue. A bone marrow aspirate and trephine, skeletal survey showed no signs of myeloma. Biopsy of the liver and regional lymph nodes was normal. Immunocytochemistry of the splenic tumour showed positivity for pan-B and plasma cell markers. After splenectomy the patient was treated with chemotherapy according to protocol VBCMP (M2).     

Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.     

Importance of sandy bottoms in coral reefs to the oscillation of daily rhythms in the tropical wrasse Halichoeres trimaculatus

Most wrasse species swim during the day and bury themselves in the sandy bottoms of shallow reefs at night. This study aimed to evaluate the importance of sandy bottoms to the day-active/night-inactive rhythmicity of the tropical wrasse Halichoeres trimaculatus. Actogram analysis revealed that fish were active during the photophase and inactive during the scotophase in aquariums with both sandy and bare bottoms. When fish were kept in aquariums with bare bottoms, rhythmicity was maintained under constant dark conditions (DD) but became obscured under constant light conditions (LL), suggesting that a day-active/night-inactive rhythmicity is regulated by the circadian system. Robust fluctuations in Period1 (wPer1) and Period2 (wPer2) expression were observed in the pectoral fin tissue under light–dark conditions (LD). Similar fluctuations in wPer1 expression persisted under DD. When fish were kept under LD conditions for 7 days and then DD for 20 days, the emergence of fish from the sandy bottom was delayed gradually. At the same time, the peak time of wPer1 expression under DD was retarded from 06:00 to 10:00. Although wPer2 expression was dampened under DD, it increased after exposing fish to light. These results suggest that wPer1 and wPer2 are differentially involved in the day-active/night-inactive rhythmicity, and that blocking light with a sandy bed at night and exposing fish to light during emergence in the morning play important roles in maintaining consistent activities in wrasse species.     

Growth Inhibitors of Lettuce Seedlings From Bacillus cereus EJ-121

The ethyl acetate extract of the Bacillus sp. EJ-121 culture broth exhibited growth inhibitory activity on a lettuce (Lactuca sativa L.) seedlings assay. Bacillus sp. EJ-121 was identified as Bacillus cereus by the morphological characteristic and nucleotide sequence of the 16S rDNA. The bioassay-guided fractionation of the ethyl acetate extract led to the isolation of two compounds. Their structures were deduced by spectroscopic methods and determined as sodium vanillate (1) and 2-aminobenzoic acid (2). Both compounds 1 and 2 inhibited more than 90% of root length at 50 ppm (0.26 and 0.36 mM, respectively) while they had a limited effect on shoot growth at the same concentration level. Roots and shoots of lettuce seedlings showed severe deterioration at 100 ppm. In order to study the fundamental structure–activity relationship, several structurally related benzoic acid derivatives were also assayed. The existence of a polar carboxyl moiety seemed to be responsible for the stronger activity.     

Epoxide Formation on the Aromatic B Ring of Flavanone by Biphenyl Dioxygenase of Pseudomonas pseudoalcaligenes KF707

Prokaryotic dioxygenase is known to catalyze aromatic compounds into their corresponding cis-dihydrodiols without the formation of an epoxide intermediate. Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 showed novel monooxygenase activity by converting 2(R)- and 2(S)-flavanone to their corresponding epoxides (2-(7-oxabicyclo[4.1.0]hepta-2,4-dien-2-yl)-2, 3-dihydro-4H-chromen-4-one), whereby the epoxide bond was formed between C2′ and C3′ on the B ring of the flavanone. The enzyme also converted 6-hydroxyflavanone and 7-hydroxyflavanone, which do not contain a hydroxyl group on the B-ring, to their corresponding epoxides. In a previous report (S.-Y. Kim, J. Jung, Y. Lim, J.-H. Ahn, S.-I. Kim, and H.-G. Hur, Antonie Leeuwenhoek 84:261-268, 2003), however, we found that the same enzyme showed dioxygenase activity toward flavone, resulting in the production of flavone cis-2′,3′-dihydrodiol. Extensive structural identification of the metabolites of flavanone by using high-pressure liquid chromatography, liquid chromatography/mass spectrometry, and nuclear magnetic resonance confirmed the presence of an epoxide functional group on the metabolites. Epoxide formation as the initial activation step of aromatic compounds by oxygenases has been reported to occur only by eukaryotic monooxygenases. To the best of our knowledge, biphenyl dioxygenase from P. pseudoalcaligenes KF707 is the first prokaryotic enzyme detected that can produce an epoxide derivative on the aromatic ring structure of flavanone.     

Hepatitis C virus (HCV) genotyping by annealing reverse transcription-PCR products with genotype-specific capture probes

The genotype of the hepatitis C virus (HCV) strain infecting a given patient is an important predictive factor for the clinical outcome of chronic liver disease and its response to anti-viral therapeutic agents. We herein sought to develop a new easy, sensitive and accurate HCV genotyping method using annealing genotype-specific capture probes (AGSCP) in an automation-friendly 96-well plate format. The validation of our new AGSCP was performed using the Standard HCV Genotype Panel. We then used both our AGSCP and the commercially available INNO-LiPA assay to analyze the HCV genotypes from 111 Korean patients. Discordant results were analyzed by direct sequencing. AGSCP successfully genotyped the standard panel. The genotypes of 111 patient samples were also obtained successfully by AGSCP and INNO-LiPA. We observed a high concordance rate (93 matched samples, 83.8%) between the two assays. Sequencing analysis of the 18 discordant results revealed that the AGSCP had correctly identified 12 samples, whereas the INNO-LiPA had correctly identified only 6. These results collectively indicate that AGSCP assay is a convenient and sensitive method for large-scale genotyping, and it may be a promising tool for the determination of HCV and other genotypes in clinical settings.     

The resolution of five racemic alpha-lactams by HPLC

The resolution of five racemic alpha-lactams (1a-d,g) using HPLC is reported. Five different Pirkle-type stationary phases were tested. The enantiomers of alpha-lactams containing the trityl group (1a-d) were separated (selectivity factors ranging from 1.08 to 1.20) using a mobile phase of hexane/2-propanol:98/2 and a stationary phase consisting of the 3,5-dinitroaniline derivative of (S)-valine with a urea linkage. Among the dialkyl-substituted alpha-lactams (1e-g), only 1,3-di-tert-butylaziridinone (1g) could be resolved, but only partially (selectivity factor = 1.07), with a mobile phase of hexane/1,2-dichloroethane:95/5 and the stationary phase consisting of the 3,5-dinitrobenzoic acid derivative of (R)-1-naphthylglycine.