Reduced Geminin levels promote cellular senescence

Cellular senescence is a permanent out-of-cycle state regulated by molecular circuits acting during the G1 phase of the cell cycle. Cdt1 is a central regulator of DNA replication licensing acting during the G1 phase and it is negatively controlled by Geminin. Here, we characterize the cell cycle expression pattern of Cdt1 and Geminin during successive passages of primary fibroblasts and compare it to tumour-derived cell lines. Cdt1 and Geminin are strictly expressed in distinct subpopulations of young fibroblasts, similarly to cancer cells, with Geminin accumulating shortly after the onset of S phase. Cdt1 and Geminin are down-regulated when primary human and mouse fibroblasts undergo replicative or stress-induced senescence. RNAi-mediated Geminin knock-down in human cells enhances the appearance of phenotypic and molecular features of senescence. Mouse embryonic fibroblasts heterozygous for Geminin exhibit accelerated senescence compared to control fibroblasts. In contrast, ectopic expression of Geminin in mouse embryonic fibroblasts delays the appearance of the senescent phenotype. Taken together, our data suggest that changes in Geminin expression levels affect the establishment of senescence pathways.     

Ultrasensitive Amplification Refractory Mutation System Real-Time PCR (ARMS RT-PCR) Assay for Detection of Minority Hepatitis B Virus-Resistant Strains in the Era of Personalized Medicine

Resistance to antiviral treatment for chronic hepatitis B virus (HBV) has been associated with mutations in the HBV polymerase region. This study aimed at developing an ultrasensitive method for quantifying viral populations with all major HBV resistance-associated mutations, combining the amplification refractory mutation system real-time PCR (ARMS RT-PCR) with a molecular beacon using a LightCycler. The discriminatory ability of this method, the ARMS RT-PCR with molecular beacon assay, was 0.01 to 0.25% for the different HBV resistance-associated mutations, as determined by laboratory-synthesized wild-type (WT) and mutant (Mut) target sequences. The assay showed 100% sensitivity for the detection of mutant variants A181V, T184A, and N236T in samples from 41 chronically HBV-infected patients under antiviral therapy who had developed resistance-associated mutations detected by direct PCR Sanger sequencing. The ratio of mutant to wild-type viral populations (the Mut/WT ratio) was >1% in 38 (63.3%) of 60 samples from chronically HBV-infected nucleos(t)ide analogue-naive patients; combinations of mutations were also detected in half of these samples. The ARMS RT-PCR with molecular beacon assay achieved high sensitivity and discriminatory ability compared to the gold standard of direct PCR Sanger sequencing in identifying resistant viral populations in chronically HBV-infected patients receiving antiviral therapy. Apart from the dominant clones, other quasispecies were also quantified. In samples from chronically HBV-infected nucleos(t)ide analogue-naive patients, the assay proved to be a useful tool in detecting minor variant populations before the initiation of the treatment. These observations need further evaluation with prospective studies before they can be implemented in daily practice.     

Distal leg wear debris mass from a rotating hinged knee prosthesis

An 18-year-old woman presented with a gradually increasing distal leg mass 8 years after wide resection for an osteosarcoma and reconstruction of the proximal left tibia with a rotating hinged knee megaprosthesis. Open biopsy of the distal leg mass showed necrobiotic tissue, metallosis, fibroblasts, osteoblasts, histiocytes, and multinucleated giant cells. The patient underwent debridement of the distal leg mass, metallosis, and wear debris surrounding the tibial component, followed by revision of the destructed polyethylene-bearing components. At the latest follow-up, 4 years after the revision surgery, the patient is alive and tumor-free, asymptomatic, and has no clinical or imaging evidence of wear and metallosis.     

Targeted Virome Sequencing Enhances Unbiased Detection and Genome Assembly of Known and Emerging Viruses—The Example of SARS-CoV-2

Targeted virome enrichment and sequencing (VirCapSeq-VERT) utilizes a pool of oligos (baits) to enrich all known—up to 2015—vertebrate-infecting viruses, increasing their detection sensitivity. The hybridisation of the baits to the target sequences can be partial, thus enabling the detection and genomic reconstruction of novel pathogens with <40% genetic diversity compared to the strains used for the baits’ design. In this study, we deploy this method in multiplexed mixes of viral extracts, and we assess its performance in the unbiased detection of DNA and RNA viruses after cDNA synthesis. We further assess its efficiency in depleting various background genomic material. Finally, as a proof-of-concept, we explore the potential usage of the method for the characterization of unknown, emerging human viruses, such as SARS-CoV-2, which may not be included in the baits’ panel. We mixed positive samples of equimolar DNA/RNA viral extracts from SARS-CoV-2, coronavirus OC43, cytomegalovirus, influenza A virus H3N2, parvovirus B19, respiratory syncytial virus, adenovirus C and coxsackievirus A16. Targeted virome enrichment was performed on a dsDNA mix, followed by sequencing on the NextSeq500 (Illumina) and the portable MinION sequencer, to evaluate its usability as a point-of-care (PoC) application. Genome mapping assembly was performed using viral reference sequences. The untargeted libraries contained less than 1% of total reads mapped on most viral genomes, while RNA viruses remained undetected. In the targeted libraries, the percentage of viral-mapped reads were substantially increased, allowing full genome assembly in most cases. Targeted virome sequencing can enrich a broad range of viruses, potentially enabling the discovery of emerging viruses.     

An Alternative Macrophage Activation Pathway Regulator,CHIT1, May Provide a Serum and Synovial Fluid Biomarker of Periprosthetic Osteolysis

Background

Periprosthetic osteolysis (PPO) is a frequent indication for total hip replacement (THR) failure. Currently, PPO diagnosis occurs in advanced stages that often necessitate complex revisions due to bone loss. PPO biomarkers could facilitate earlier diagnosis. Alternative macrophage activation pathway regulators, chitotriosidase (CHIT1) and CC chemokine ligand 18 (CCL18), have increased periprosthetic expression in patients undergoing revision THR for osteolysis. We hypothesized that synovial fluid and serum levels of CHIT1 and CCL18 would be increased in patients undergoing revision THR for PPO versus controls without osteolysis.

Methods

In this prospective case-control study, 60 patients undergoing revision metal-on-polyethylene THR at Hospital for Special Surgery were screened preoperatively from January 2013 to December 2014. Twenty “osteolysis” patients who underwent revision for PPO (based on imaging and operative reports) and 10 “control” patients (with stable implants) who underwent revision for recurrent dislocation or a mechanical etiology were included. Among osteolysis and control patients, 11/20 and 4/10 were male; average age was 68 and 63 years, respectively; 9/20 and 3/10 had cemented femoral components; and average implant longevity was 15 and 5 years, respectively. Preoperative serum and intraoperative synovial fluid samples were collected. CHIT1 and CCL18 were quantified via enzyme-linked immunosorbent assay. Significance was assessed via nonparametric Mann-Whiney U test.

Results

CHIT1 was significantly increased in both synovial fluid (3727 versus 731 nanomoles [nM]) and serum (98 versus 39 nM) in the osteolysis versus control patients. CCL18 levels were also significantly increased in osteolysis versus control patients’ synovial fluid (425 versus 180 nM) but not their serum.

Conclusions

In this prospective case-control study, CHIT1 was identified as a novel synovial fluid and serum biomarker of PPO. CHIT1 expression is induced during macrophage activation in response to wear debris. CHIT1 monitoring may facilitate early diagnosis of THR PPO. Furthermore, CHIT1 may represent a novel therapeutic target for PPO.

     

Detection of specific antibodies to HCV-ARF/CORE+1 protein in patients treated with pegylated interferon plus ribavirin

Hepatitis C virus (HCV) infection is a major cause for chronic liver disease and hepatocellular carcinoma. The HCV-ARF/core+1 protein is an alternative product of HCV core-encoding sequence of unknown biological function. Highly purified HCV core and ARF/core+1 recombinant proteins from HCV genotype 1a and HCV-ARF/core+1 recombinant protein from HCV genotype 3a were expressed in Escherichia coli. Using an enzyme-linked immunosorbent assay, we assessed the prevalence of anti-ARF/core+1 antibodies in 90 chronic hepatitis C patients infected with HCV genotypes 1a/1b or 3a, treated with pegylated interferon (Peg-IFN-a-2a) plus ribavirin. Samples derived from 92 healthy blood donors were used as negative controls. All HCV-RNA-positive serum samples reacted with core 1a antigen, while 15 (37.5%) of 40 and 14 (28%) of 50 patients infected with HCV-1a/1b and HCV-3a, respectively, were found to have anti-ARF/core+1 antibodies into their serum before treatment initiation. These antibodies were persistently present during treatment follow-up and linked to elevated levels of HCV-RNA at baseline.     

Long-term effects of levosimendan infusion on inflammatory processes and sFas in patients with severe heart failure

BACKGROUND: The calcium sensitizer levosimendan improves myocardial contractility in patients with heart failure, although its effects on inflammation and apoptosis are unknown. AIM: To examine the effects of levosimendan on markers of inflammation and apoptosis, over a period of 30 d following a 24 h infusion, in patients with heart failure. METHODS: Thirty four patients with severe heart failure were randomised to receive a 24 h infusion of levosimendan or placebo, in a double-blind trial. Haemodynamic evaluation and blood sampling were performed at baseline, 24 h, 30 h, 48 h, 7 d and 30 d after the end of the infusion. RESULTS: Seven patients (1 levosimendan, 6 placebo), were excluded during follow-up. In the remaining 27 patients, levosimendan decreased serum IL-6 and sFAS, 24 h after the infusion (p<0.01 and p<0.05 vs baseline), an effect sustained for 7-30 d. Serum TNF-alpha and sTNF-R1 were decreased between 48 h (p<0.01 vs baseline for both) and 7 d (p<0.05 vs baseline for sTNF-R1) after infusion. Serum sTNF-R2 was decreased at 24 h (p<0.05 vs baseline) and remained lower than baseline for at least 7 d (p<0.05). CONCLUSIONS: These findings indicate that levosimendan decreases the expression of proinflammatory cytokines, TNF-alpha receptors and sFAS, immediately after infusion, an effect which persists for 7-30 d.