The DNA–protein interaction modes of FEN-1 with gap substrates and their implication in preventing duplication mutations

Flap endonuclease-1 (FEN-1) is a structure-specific nuclease best known for its involvement in RNA primer removal and long-patch base excision repair. This enzyme is known to possess 5′-flap endo- (FEN) and 5′–3′ exo- (EXO) nuclease activities. Recently, FEN-1 has been reported to also possess a gap endonuclease (GEN) activity, which is possibly involved in apoptotic DNA fragmentation and the resolution of stalled DNA replication forks. In the current study, we compare the kinetics of these activities to shed light on the aspects of DNA structure and FEN-1 DNA-binding elements that affect substrate cleavage. By using DNA binding deficient mutants of FEN-1, we determine that the GEN activity is analogous to FEN activity in that the single-stranded DNA region of DNA substrates interacts with the clamp region of FEN-1. In addition, we show that the C-terminal extension of human FEN-1 likely interacts with the downstream duplex portion of all substrates. Taken together, a substrate-binding model that explains how FEN-1, which has a single active center, can have seemingly different activities is proposed. Furthermore, based on the evidence that GEN activity in complex with WRN protein cleaves hairpin and internal loop substrates, we suggest that the GEN activity may prevent repeat expansions and duplication mutations

Imaging and Pathological Features of Percutaneous Cryosurgery on Normal Lung Evaluated in a Porcine Model

Background and objective Lung cancer is one of the most commonly occurring malignancies and frequent causes of death in the world. Cryoablation is a safe and alternative treatment for unresectable lung cancer. Due to the lung being gas-containing organ and different from solid organs such as liver and pancreas, it is difficult to achieve the freezing range of beyond the tumor edge 1 cm safety border. The aim of this study is to examine the effect of different numbers of freeze cycles on the effectiveness of cryoablation on normal lung tissue and to create an operation guideline that gives the best effect. Methods Six healthy Tibetan miniature pigs were given a CT scan and histological investigation after percutaneous cryosurgery. Cryoablation was performed as 2 cycles of 10 min of active freezing in the left lung; each freeze followed by a 5 min thaw. In the right lung, we performed the same 2 cycles of 5 min of freezing followed by 5 min of thawing. However, for the right lung, we included a third cycle of consisting of 10 min of freezing followed by 5 min of thawing. Three cryoprobes were inserted into the left lung and three cryoprobes in the right lung per animal, one in the upper and two in the lower lobe, so as to be well away from each other. Comparison under the same experimental condition was necessary. During the experiment, observations were made regarding the imaging change of ice-ball. The lungs were removed postoperatively at 3 intervals: 4 h, 3 d of postoperation and 7 d of postoperation, respectively, to view microscopic and pathological change. Results The ice-ball grew gradually in relation to the increase in time, and the increase in number of cycles. The size of the cryolesion (hypothesis necrotic area) in specimens, over time, became larger in size than the size of the ice-ball during operation, regardless of whether 2 or 3 freeze-thaw cycles were performed. The area of necrosis was gradually increased over the course of time. The hypothesis necrotic area was equal to necrosis area 3 d after cryosurgery. Conclusion Percutaneous cryoablation of the lung can achieve complete ablation of target tissue. The freezing technique may be different depending on the individual circumstances of each tumor. In technology, 3 freeze-thaw cycles are recommended, and the range of cryoablation’s effective diameter may be not necessarily beyond the tumor edge at least 1 cm safe border during cryosurgery

Genomic Characterizations of Porcine Epidemic Diarrhea Viruses (PEDV) in Diarrheic Piglets and Clinically Healthy Adult Pigs from 2019 to 2022 in China

Porcine epidemic diarrhea virus (PEDV) is a major causative pathogen of diarrheic disease. In this study, the prevalence and evolution of PEDV was evaluated using intestinal samples collected from six provinces of China in 2019–2022. PEDV could not only be detected in diarrheic piglets but also in adult pigs without enteric diseases. The complete genomes of five temporal and geographical representative PEDV strains were determined. Genome-based phylogenetic analysis indicated that XJ1904-700 belongs to the G2-a subgroup, while the other strains are clustered within the S-INDEL subgroup. Recombination analyses supported that JSNJ2004-919 is an inter-subgroup recombinant from SD2014-like (G2-b), CHZ-2013-like (G2-b) and CV777-like (G1-b) isolates, while FJFZ2004-1017 is an intra-subgroup recombinant from XM1-2-like (S-INDEL) and LYG-2014-like (S-INDEL) isolates. Both JSNJ2004-919 and FJFZ2004-1017 were from adult pigs, providing evidence that adult pigs may also serve as the host of PEDV reservoirs for virus evolution. Overall, this study provides new insights into PEDV’s prevalence and evolution in both diseased piglets and clinically healthy adult pigs

Determining an Optimal Cutoff of Serum β-Human Chorionic Gonadotropin for Assisting the Diagnosis of Intracranial Germinomas.

BACKGROUND:Beta (β)-human chorionic gonadotropin (β-HCG) is used to confirm the diagnosis and plan treatment of intracranial germinomas. However, the cutoff values of serum β-HCG in diagnosis of intracranial germinomas reported in the literature are inconsistent. To establish an appropriate cutoff value of serum β-HCG for diagnosis of intracranial germinomas, we retrospectively reviewed the records of intracranial tumor patients who received serum β-HCG and α-fetoprotein (AFP) tests for diagnostic purposes at our hospital from 2005 to 2014. METHODS:A total of 93 intracranial germinomas and 289 intracranial non-germ cell tumors were included in this study. Receiver operating characteristic (ROC) analysis was used to evaluate the sensitivity and specificity of 3 cutoffs (0.1, 0.4, and 0.5 mIU/mL) for diagnosing intracranial germinomas. The serum β-HCG level of intracranial germinoma patients was further analyzed to investigate the effect of metastasis status and tumor location on serum β-HCG level. RESULTS:The area under the ROC curve was 0.81 (P < .001), suggesting β-HCG is an effective marker. Of the 3 cutoff values, 0.1 mIU/mL possessed a highest sensitivity (66.67%) and good specificity (91%). Although there was no β-HCG level difference between metastatic and non-metastatic intracranial germinoma patients, the diagnostic rate of metastatic neurohypophyseal germinomas was significantly higher than that of its non-metastatic counterpart (P < .05), implying that the location of the germinoma might need to be considered when β-HCG is used as a marker to predict metastasis. CONCLUSIONS:Determining an optimal cutoff of serum β-HCG is helpful for assisting the diagnosis of intracranial germinoma

Acquisition of a stable and transferable plasmid coharbouring hypervirulence and MDR genes with low fitness cost: Accelerating the dissemination of ST11-KL64 CR-HvKP

Objectives: This study aimed to delineate the ability of a plasmid, pS130–4, which harboured both hypervirulence and multidrug resistance genes, to disseminate within Klebsiella pneumoniae, as well as its potential formation mechanism. Methods: We employed whole-genome sequencing to decipher the genetic architecture of pS130–4. Its capability to conjugate and transfer was assessed through a series of experiments, including plasmid stability, competitive growth, and growth curve analysis. Its expression stability was further evaluated using drug sensitivity, larval survival, and biofilm formation tests. Results: pS130–4 contained four intact modules typical of self-transmissible plasmids. BLAST analysis revealed a sequence identity exceeding 90% with other plasmids from a variety of hosts, suggesting its broad prevalence. Our findings indicated the plasmid's formation resulted from IS26-mediated recombination, leading us to propose a model detailing the creation of this conjugative fusion plasmid housing both blaKPC-2 and hypervirulence genes. Our conjugation experiments established that pS130–4, when present in the clinical strain S130, was self-transmissible with an estimated efficiency between 10−5 and 10−4. Remarkably, pS130–4 showcased a 90% retention rate and did not impede the growth of host bacteria. Galleria mellonella larval infection assay demonstrated that S130 had pronounced toxicity when juxtaposed with high-virulence control strain NTUH-K2044 and low-toxicity control strain ATCC700603. Furthermore, pS130–4′s virulence remained intact postconjugation. Conclusion: A fusion plasmid, encompassing both hypervirulence and multidrug resistance genes, was viable within K. pneumoniae ST11-KL64 and incurred minimal fitness costs. These insights underscored the criticality of rigorous monitoring to pre-empt the escalation and distribution of this formidable super-plasmid