Rapid determination of viral RNA sequences in mosquitoes collected in the field

A method for rapid determination of viral RNA sequences (RDV) was applied to homogenates of Aedes aegypti collected in Thailand in an area in which dengue fever (dengue hemorrhagic fever) is endemic, using the mosquito cell line C6/36. Nucleic acid sequences of dengue virus type 4 and cell fusing agent virus were detected. This RDV method has the potential to become a standard method for detection of both known and newly emerging, unknown mosquito-borne viruses.     

Effectiveness of 16S ribosomal DNA real-time PCR and sequencing for diagnosing bacterial keratitis

Graefe's Archive for Clinical and Experimental Ophthalmology - To evaluate the efficacy of real-time PCR for 16S ribosomal DNA (16S r-DNA) and sequencing for diagnosing microbial keratitis. We...     

Thromboembolism in lung cancer patients]

We reviewed 649 primary lung cancer patients with special reference to the occurrence of thromboembolism. Thirteen episodes of thromboembolism were detected in 12 (1.8%) of the 649. Eight of the 12 were men, and their mean age was 63. Adenocarcinoma was the predominant cell type. Most patients had an advanced stage of the disease, but in some in the cancer was at an early stage. In 5 cases, the finding of thromboembolisms led to diagnosis of the lung cancer (38.5%). Anticoagulant therapy was performed in 9 cases, of which 8 responded without serious complication. We emphasize the importance of anticoagulant therapy as a therapy indicated for thromboembolism in patients with lung cancer.     

A case of pleomorphic carcinoma of the pancreas showing sequential histological change by immunohistochemical study

Summary Pleomorphic carcinoma of the pancreas is a rare, histologically characterized pancreatic tumor with a rapid and fatal course. We report a case of a resected pleomorphic carcinoma located in the body of the pancreas in a 61-yr-old male. Histological analysis of the resected specimen revealed the coexistence of pleomorphic carcinoma with adenocarcinoma, but the recurrent tumors at autopsy 20 mo later were only of the adenocarcinomatous type. Cells in the adenocarcinomatous component showed a diffuse reactivity for CA19-9, CEA, and cytokeratin, and a focal reactivity for vimentin. In contrast, vimentin was diffusely expressed in pleomorphic lesion. Adenocarcinoma at autopsy expressed CA19-9, CEA, and cytokeratin, but not vimentin. These findings suggest that the recurrent adenocarcinoma may have developed as a consequence of sequential change in the nature of the tumor.     

Cumulative probability of prostate cancer detection in biopsy according to free/total PSA ratio in men with total PSA levels of 2.1–10.0 ng/ml at population screening

Purpose

The aim of this study was to investigate the cumulative probability of prostate cancer detection according to free/total prostate-specific antigen (PSA) ratio in men with PSA levels of 2.1–10.0 ng/ml and also likelihood of detecting clinically insignificant prostate cancer in population-based screening.

Methods

A total of 1,277 men aged between 55 and 69 years with total PSA (tPSA) levels of 2.1–10.0 ng/ml screened in population screening in Kanazawa city and underwent systematic transrectal ultrasonography-guided prostate biopsy between 2000 and 2011 were enrolled. The cumulative probability of prostate cancer detection in biopsy according to age, serum tPSA, and free-to-total PSA (f/t PSA) ratio was investigated. The clinicopathological features of screening-detected prostate cancer were also investigated.

Results

Of the 1,277 subjects in the study population, 320 (25.0 %) were diagnosed with prostate cancer during the observation period. The probabilities of prostate cancer detection at 3 years were 64.5, 41.2, 28.5, and 14.3 % for the men with f/t PSA ratio ≤0.08, 0.09–0.13, 0.14–0.22, and ≥0.23, respectively; the differences in probabilities of prostate cancer detection among men with different f/t PSA ratios were statistically significant. Among 320 patients, 84 (26.3 %) had favorable clinicopathological features that made them suitable for active surveillance. The f/t PSA ratio in unfavorable cancer patients was significantly lower that that in favorable cancer patients.

Conclusion

The present study demonstrated that the f/t PSA ratio was a strong predictor of future cancer detection and unfavorable cancerous features in prostate biopsy in men with total PSA levels of 2.1–10.0 ng/ml at population screening.     

C-terminal region of activation-induced cytidine deaminase (AID) is required for efficient class switch recombination and gene conversion

Activation-induced cytidine deaminase (AID) introduces single-strand breaks (SSBs) to initiate class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM). CSR is mediated by double-strand breaks (DSBs) at donor and acceptor switch (S) regions, followed by pairing of DSB ends in two S regions and their joining. Because AID mutations at its C-terminal region drastically impair CSR but retain its DNA cleavage and SHM activity, the C-terminal region of AID likely is required for the recombination step after the DNA cleavage. To test this hypothesis, we analyzed the recombination junctions generated by AID C-terminal mutants and found that 0- to 3-bp microhomology junctions are relatively less abundant, possibly reflecting the defects of the classical nonhomologous end joining (C-NHEJ). Consistently, the accumulation of C-NHEJ factors such as Ku80 and XRCC4 was decreased at the cleaved S region. In contrast, an SSB-binding protein, poly (ADP)-ribose polymerase1, was recruited more abundantly, suggesting a defect in conversion from SSB to DSB. In addition, recruitment of critical DNA synapse factors such as 53BP1, DNA PKcs, and UNG at the S region was reduced during CSR. Furthermore, the chromosome conformation capture assay revealed that DNA synapse formation is impaired drastically in the AID C-terminal mutants. Interestingly, these mutants showed relative reduction in GC compared with SHM in chicken DT40 cells. Collectively, our data indicate that the C-terminal region of AID is required for efficient generation of DSB in CSR and GC and thus for the subsequent pairing of cleaved DNA ends during recombination in CSR.Activation-induced cytidine deaminase (AID) is essential for three different genetic events: class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM), which contribute to Ig gene diversification (1–5). Although AID generates single-strand breaks (SSBs) in the Ig genes, subsequent repair steps for CSR and GC are similar to each other but are distinct from SHM in their mechanistic properties, i.e, in (i) generation of the double-strand breaks (DSBs), (ii) recombination, and (iii) the requirement for uracil-DNA-glycosylase (UNG) for the pairing of the DSB ends (6–10). Despite the similarities between GC and CSR, their repair mechanisms have distinct features: CSR recombination requires nonhomologous end joining (NHEJ), and GC depends on homologous recombination (HR). During CSR, DSB ends normally are joined by classical NHEJ (C-NHEJ), which requires specific repair proteins such as Ku80, XRCC4, or DNA ligase IV (11, 12). In the absence of C-NHEJ, a back-up end-joining pathway called “alternative end joining” (A-EJ), which is reported to be slower and also more error prone than C-NHEJ, joins the broken DSBs ends (13). On the other hand, HR, the most common form of homology-directed repair, requires long sequence homology between donor and acceptor DNA to complete the recombination step by recruiting a distinct set of repair proteins such as RAD54, RAD52, and RAD51 to the break sites (14, 15).Various studies on AID mutations in the N-terminal or C-terminal regions (4, 8, 9, 16–19) have shown that N-terminal AID mutants are compromised for CSR and are defective in SHM, indicating that the N-terminal region of AID is required for DNA cleavage (9, 16, 19). On the other hand, the C-terminal region of AID, which contains a nuclear-export signal and is responsible for AID’s shuttling activity between the nucleus and cytoplasm, is required for CSR-specific activity but not for DNA cleavage activity and SHM (8, 16). Among the series of AID C-terminal mutants examined, two mutants show characteristic features: P20, which has an insertion of 34 amino acids at residue 182 and normal nuclear-cytoplasmic shuttling activity, and JP8Bdel, which has a 16-amino acid truncation at residue 183, accumulates in the nucleus, and shows higher DNA break activity at the donor switch (S) region (16, 17). Although several reports suggest that the C-terminal region of AID is involved in protein stability (20, 21), C-terminal mutants of AID stabilized by fusing the hormone-binding domain of estrogen receptor (ER) also show similar CSR-defective phenotypes (8). Taken together, these data suggest that the DNA cleavage activity and CSR-specific activity depend on different regions of AID (8, 19). In addition, the C-terminal region of AID is essential for the interaction of AID with poly (A)⁺ RNA via a specific cofactor (22). Because CSR requires de novo protein synthesis, we proposed that after DNA cleavage the C-terminal region of AID may be involved in the regulation of the recombination step through generation of a new protein (8, 16, 22).DSBs induced by AID during CSR ultimately are joined by the efficient DNA repair pathway that requires C-NHEJ factors such as Ku70/80 (12, 23). However, in the absence of C-NHEJ, the A-EJ pathway that relies on microhomology can join the broken DNA ends, although this pathway is associated with chromosomal translocations (11, 24). Previously, we reported that JP8Bdel enhances aberrant c-myc/IgH translocations and that it fails to carry out the efficient recombination between donor and acceptor S regions in the IgH locus (8). Therefore, it is important to examine whether the AID C-terminal mutants affect the S–S joining in CSR.In the current work we examined whether the C-terminal region of AID is involved in DNA synapse formation and recombination during CSR in CH12F3-2 and spleen B cells. We also examined the effect of AID C-terminal mutations on GC in chicken DT40 cells, which depends on HR between pseudo V genes and the downstream IgVλ region. Using these CSR- and GC-monitoring systems, we demonstrate that efficient CSR and GC require the C-terminal region of AID for the formation of DSB from SSB and subsequent end synapse. Considering these findings together, we conclude that, in addition to DNA cleavage, AID has a unique function in the generation of DSBs, which is required for S–S synapse formation and joining in CSR and recombination in GC.     

Characterization of Multidrug-Resistant Group B Streptococci with Reduced Penicillin Susceptibility Forming Small Non-Beta-Hemolytic Colonies on Sheep Blood Agar Plates

We isolated and characterized three multidrug-resistant clinical isolates of group B streptococci with reduced penicillin susceptibility (PRGBS) that formed small non-beta-hemolytic colonies on sheep blood agar plates but grew well on chocolate agar plates. They can be overlooked in the bacterial identification step, leading to clinical misdiagnosis and treatment failure.