Circulating tumor DNA and radiological tumor volume identify patients at risk for relapse with resected, early-stage non-small-cell lung cancer.

BACKGROUND: Predicting relapse and overall survival (OS) in early-stage non-small-cell lung cancer (NSCLC) patients remains challenging. Therefore, we hypothesized that detection of circulating tumor DNA (ctDNA) can identify patients with increased risk of relapse and that integrating radiological tumor volume measurement along with ctDNA detectability improves prediction of outcome. PATIENTS AND METHODS: We analyzed 366 serial plasma samples from 85 patients who underwent surgical resections and assessed ctDNA using a next-generation sequencing liquid biopsy assay, and measured tumor volume using a computed tomography-based three-dimensional annotation. RESULTS: Our results showed that patients with detectable ctDNA at baseline or after treatment and patients who did not clear ctDNA after treatment had a significantly worse clinical outcome. Integrating radiological analysis allowed the stratification in risk groups prognostic of clinical outcome as confirmed in an independent cohort of 32 patients. CONCLUSIONS: Our findings suggest ctDNA and radiological monitoring could be valuable tools for guiding follow-up care and treatment decisions for early-stage NSCLC patients.

Assessing the clinical utility of genetic profiling in fracture risk prediction: a decision curve analysis.

Using decision curve analysis on 2188 women and 1324 men, we found that an osteogenomic profile constructed from 62 genetic variants improved the clinical net benefit of fracture risk prediction over and above that of clinical risk factors and BMD. INTRODUCTION: Genetic profiling is a promising tool for assessing fracture risk. This study sought to use the decision curve analysis (DCA), a novel approach to determine the impact of genetic profiling on fracture risk prediction. METHODS: The study involved 2188 women and 1324 men, aged 60 years and above, who were followed for up to 23 years. Bone mineral density (BMD) and clinical risk factors were obtained at baseline. The incidence of fracture and mortality were recorded. A weighted individual genetic risk score (GRS) was constructed from 62 BMD-associated genetic variants. Four models were considered: CRF (clinical risk factors); CRF + GRS; Garvan model (GFRC) including CRF and femoral neck BMD; and GFRC + GRS. The DCA was used to evaluate the clinical net benefit of predictive models at a range of clinically reasonable risk thresholds. RESULTS: In both women and men, the full model GFRC + GRS achieved the highest net benefits. For 10-year risk threshold > 18% for women and > 15% for men, the GRS provided net benefit above those of the CRF models. At 20% risk threshold, adding the GRS could help to avoid 1 additional treatment per 81 women or 1 per 24 men compared with the Garvan model. At lower risk thresholds, there was no significant difference between the four models. CONCLUSIONS: The addition of genetic profiling into the clinical risk factors can improve the net clinical benefit at higher risk thresholds of fracture. Although the contribution of genetic profiling was modest in the presence of BMD + CRF, it appeared to be able to replace BMD for fracture prediction.

Molecular identification and characterisation of a novel chicken leukocyte immunoglobulin-like receptor A5.

1. Leukocyte immunoglobulin-like receptor A5 (LILRA5) is a key molecule that regulates the immune system. However, the LILRA5 gene has not been characterised in avian species, including chickens. The present study aimed to identify and functionally characterise LILRA5 identified from two genetically disparate chicken lines, viz., Marek's disease (MD)-resistant (R) line 6.3 and MD-susceptible (S) line 7.2. 2. Multiple sequence alignment and phylogenetic analyses confirmed that the identity and similarity homologies of amino acids of LILRA5 in chicken lines 6.3 and 7.2 ranged between 93% and 93.7%, whereas those between chicken and mammals ranged between 20.9% and 43.7% and 21.1% to 43.9%, respectively. The newly cloned LILRA5 from chicken lines 6.3 and 7.2 revealed high conservation and a close relationship with other known mammalian LILRA5 proteins. 3. The results indicated that LILRA5 from chicken lines 6.3 and 7.2 was associated with phosphorylation of Src kinases and protein tyrosine phosphatase non-receptor type 11 (SHP2), which play a regulatory role in immune functions. Moreover, the results demonstrated that LILRA5 in these lines was associated with the activation of major histocompatibility complex (MHC) class I and ß2-microglobulin and induced the expression of the transporter associated with antigen processing. In addition, LILRA5 in both chicken lines activated and induced Janus kinase (JAK)-signal transducer and the activator of transcription (STAT), nuclear factor kappa B (NF-κB), phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) and the extracellular signal-regulated kinase (ERK)1/2 signalling pathways; toll-like receptors; and Th1-, Th2-, and Th17- cytokines. 4. The data suggested that LILRA5 has innate immune receptors essential for macrophage immune response and provide novel insights into the regulation of immunity and immunopathology.

The level and pattern of physical activity among fifth-grade students in Ho Chi Minh City, Vietnam.

OBJECTIVES: Despite the benefits of physical activity (PA), a significant proportion of children do not meet physical activity guidelines (PAGs). However, most studies were among secondary-school-aged youth and relied on PA self-report. In addition, information regarding children's PA behaviors during specific segments of day/week is not usually collected. This study, therefore, investigated the level and pattern of PA among fifth-grade students in Ho Chi Minh City (HCMC), Vietnam. STUDY DESIGN: A complex cross-sectional survey was conducted on a representative sample of 619 fifth-grade students in eight public schools in urban areas of HCMC in 2016. METHODS: Demographic/anthropometric characteristics were measured using standard protocols. PA was measured using pedometers. After-school activities were measured using the Previous Day Physical Activity Recall questionnaire. Survey procedures with sampling weights were used for analyses. RESULTS: Approximately 18% of children met the PAG; 52.7% were overweight (OW)/obese (OB). On average, students recorded about 8800 steps/day. Boys were more active than girls at school and on weekdays. Students were more active at school on physical education (PE) days vs non-PE days and weekdays vs weekends. OW/OB students were more active at school on PE days. After-school PAs differed between boys and girls, whereas sedentary activities were popular among both the genders. CONCLUSIONS: Most fifth-grade students had insufficient PA levels. Patterns of PA are different at various times during the day and week. The finding emphasized an urgent need for interventions to improve children's PA and obesity in this area.

Integrating cytokines and angiogenic factors and tumour bulk with selected clinical criteria improves determination of prognosis in advanced renal cell carcinoma.

BACKGROUND: In two clinical trials of the vascular endothelial growth factor (VEGF) receptor inhibitor pazopanib in advanced renal cell carcinoma (mRCC), we found interleukin-6 as predictive of pazopanib benefit. We evaluated the prognostic significance of candidate cytokines and angiogenic factors (CAFs) identified in that work relative to accepted clinical parameters. METHODS: Seven preselected plasma CAFs (interleukin-6, interleukin-8, osteopontin, VEGF, hepatocyte growth factor, tissue inhibitor of metalloproteinases (TIMP-1), and E-selectin) were measured using multiplex ELISA in plasma collected pretreatment from 343 mRCC patients participating in the phase 3 registration trial of pazopanib vs placebo (NCT00334282). Tumour burden (per sum of longest diameters (SLD)) and 10 other clinical factors were also analysed for association with overall survival (OS; based on initial treatment assignment). RESULTS: Osteopontin, interleukin-6, and TIMP-1 were independently associated with OS in multivariable analysis. A model combining the three CAFs and five clinical variables (including SLD) had higher prognostic accuracy than the International Metastatic Renal Cell Carcinoma Database Consortium criteria (concordance-index 0.75 vs 0.67, respectively), and distinguished two groups of patients within the original intermediate risk category. CONCLUSIONS: A prognostic model incorporating osteopontin, interleukin-6, TIMP-1, tumour burden, and selected clinical criteria increased prognostic accuracy for OS determination in mRCC patients.

Protection of Penaeus monodon against white spot syndrome by continuous oral administration of a low concentration of Bacillus subtilis spores expressing the VP28 antigen.

In this study, Bacillus subtilis spores expressing a chimeric protein, CotB-VP28, were used as a probiotic vaccine to protect black tiger shrimps (Penaeus monodon) against white spot syndrome virus (WSSV) infection. Oral administration of pellets coated with CotB-VP28 spores (at ≥1 × 109  CFU per g pellet) to shrimps induced immune-relating phenoloxydase activity (PO) in shrimps after 14 days of feeding (prior challenge) and at day 3 post challenge (1·26 and 1·70 fold increase respectively). A 75% protection rate was obtained by continuous feeding of the spore-coated pellets at ≥1 × 109  CFU per g for 14 days prior to WSSV challenge and during all the postchallenge period. Even when the amount of CotB-VP28 spores in feed pellets was reduced down to ≥5 × 107  CFU per g and ≥1 × 106  CFU per g, relatively high protection rates of 70 and 67·5%, respectively, were still obtained. By contrast, feeding pellets without spores (untreated group) and with naked spores (PY79 group) at ≥1 × 109  CFU per g could not protect shrimps against WSSV. These data suggest that supplementation of CotB-VP28 spores at low dose of ≥1 × 106  CFU per g could be effective as a prophylactic treatment of WSS for black tiger shrimps. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the protective efficacy of Bacillus subtilis CotB-VP28 spores on black tiger shrimps (Penaeus monodon) against white spot syndrome virus infection. Oral administration of pellets coated with CotB-VP28 spores (≥1 × 109  CFU per g) conferred 75% protection after white spot syndrome virus challenge. Even after reducing CotB-VP28 spores in feed pellets to ≥1 × 106  CFU per g, 67·5% protections was still obtained. These data indicate that supplementation of CotB-VP28 spores at a low dose of ≥1 × 106  CFU per g could be effective in prophylaxis against white spot syndrome in black tiger shrimps.

Phase II trial of everolimus and erlotinib in patients with platinum-resistant recurrent and/or metastatic head and neck squamous cell carcinoma.

BACKGROUND: Enhanced phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is one of the key adaptive changes accounting for epidermal growth factor receptor (EGFR) inhibitor-resistant growth in head and neck squamous cell carcinoma (HNSCC). We designed a phase II clinical trial of EGFR tyrosine kinase inhibitor (TKI), erlotinib, in association with the mTOR inhibitor, everolimus, based on the hypothesis that the downstream effects of Akt through inhibition of mTOR may enhance the effectiveness of the EGFR-TKI in patients with recurrent/metastatic HNSCC. PATIENTS AND METHODS: Patients with histologically or cytologically confirmed platinum-resistant HNSCC received everolimus 5 mg and erlotinib 150 mg daily orally until disease progression, intolerable toxicity, investigator or patient decision. Cytokines and angiogenic factors profile, limited mutation analysis and p16 immunohistochemistry status were included in the biomarker analysis. RESULTS: Of the 35 assessable patients, 3 (8%) achieved partial response at 4 weeks, 1 confirmed at 12 weeks; overall response rate at 12 weeks was 2.8%. Twenty-seven (77%) patients achieved disease stabilization at 4 weeks, 11 (31%) confirmed at 12 weeks. Twelve-week progression-free survival (PFS) was 49%, median PFS 11.9 weeks and median overall survival (OS) 10.25 months. High neutrophil gelatinase lipocalin (P = 0.01) and vascular endothelial growth factor (VEGF) (P = 0.04) plasma levels were significantly associated with worse OS. CONCLUSIONS: The combination of erlotinib and everolimus did not show significant benefit in unselected patients with platinum-resistant metastatic HNSCC despite a manageable toxicity profile. Markers of tumor invasion and hypoxia identify a group of patients with particularly poor prognosis. CLINICAL TRIAL NUMBER: NCT00942734.

Monitoring bypassing agent therapy - a prospective crossover study comparing thromboelastometry and thrombin generation assay.

The aim of this study was to evaluate the capability of thromboelastometry (ROTEM) and thrombin generation assay (TGA) to monitor the treatment response of bypassing agent (BPA) therapy and to study whether one method is superior to another. In a prospective crossover study haemophilia A patients with high titre inhibitors were included to receive a dose of 75 U kg(-1) activated prothrombin complex concentrates (aPCC) intravenously. Blood sampling was performed at baseline, 15, 30 min, 1, 2, 3 and 4 h post-infusion for TGA and ROTEM analysis. After a washout period of 14 days the subjects received recombinant FVIIa (rFVIIa) at a dose of 90 µg kg(-1) and similar blood sampling was performed. Healthy subjects were used as controls. Six haemophilia A patients with inhibitors were included. We found that TGA parameters endogenous thrombin potential (ETP) and peak thrombin increased 2-3 folds from baseline 15-30 min after infusion. ROTEM parameters MaxVel and maximum clot firmness increased to a level comparable to that of healthy controls. An individual difference in response was observed for different parameters among participants. ETP and peak thrombin were almost two-fold greater following aPCC infusion compared to rFVIIa, whereas ROTEM parameters showed no difference in response between the two products. The study showed that ROTEM and TGA have a great potential to evaluate the effect of BPA in haemophilia patients with inhibitors. TGA seemed to be more sensitive than ROTEM in reflecting the difference in treatment response between aPCC and rFVIIa. Additional prospective clinical studies are needed to clarify which assay and what parameters are clinically predictive.

Use of thromboelastography and thrombin generation assay to predict clinical phenotype in patients with severe FVII deficiency.

Bleeding tendency is weakly correlated with the activity of factor VII (FVII) in the plasma of patients with FVII deficiency. A laboratory method for predicting bleeding risk in patients with this coagulation disorder is lacking. We investigated whether global coagulation assays, specifically thromboelastography (TEG) and thrombin generation assay (TGA), could be used to predict bleeding risk. We also sought to identify factors that may explain the differences in bleeding phenotype observed among individuals with severe FVII deficiency. The study comprised 12 patients with severe FVII deficiency (FVII activity <1%). Eleven patients were homozygous for the Gln100Arg mutation and one patient was compound heterozygous. Clinically, 10 patients had increased haemorrhagic diathesis, whereas two patients were asymptomatic. Blood sampling was performed at baseline for TEG and TGA analyses. The platelet aggregation assay was performed and the plasma level of anticoagulation inhibitors and thrombophilic risk factors assessed. No difference in the TEG and TGA results was observed in all FVII-deficient individuals. The level of free tissue factor pathway inhibitor was within the normal range and similar in symptomatic and asymptomatic subjects. None of the participants had the FV Leiden mutation, prothrombin gene mutation, or abnormal anticoagulant inhibitor levels. Asymptomatic subjects showed normal platelet aggregation. These data suggested that TEG and TGA were not suitable methods for predicting the clinical phenotype in FVII-deficient subjects.

Tranexamic acid as adjunct therapy to bypassing agents in haemophilia A patients with inhibitors.

Haemophilia patients with inhibitors require bypassing agents (BPA) like activated prothrombin complex concentrate (aPCC) and recombinant activated factor VII (rFVIIa) to control bleeds. Adjunct tranexamic acid (TXA) may improve haemostasis. The objective of this study was to investigate safety and haemostatic effect of TXA given in combination with BPA. Healthy volunteers (N = 5) and haemophilia inhibitor patients (N = 6) were enrolled in a prospective case crossover design. Controls were treated with TXA 20 mg kg(-1) orally (O.R.) Patients were treated with aPCC 75 IU kg(-1) intravenous (I.V.) on day 1 followed by TXA 20 mg kg(-1) O.R. combined with aPCC 75 IU kg(-1) I.V. on day 2. A 14-day washout occurred before crossover to rFVIIa 90 µg kg(-1) I.V. ±TXA. Safety evaluation and blood sampling processes were performed at baseline, 15, 30, 60, 120, 180 and 240 min post treatment. Primary outcome was maximum clot firmness (MCF) evaluated by whole blood thromboelastometry using a TF + tissue plasminogen activator-based assay. Healthy controls showed a 20-fold increase in MCF following TXA. Adjunct TXA to aPCC or rFVIIa induced a significant increase in MCF (P < 0.0001) reaching levels indistinguishable from healthy controls treated with TXA (P > 0.05). Infusion of aPCC or rFVIIa alone induced only 3-10 fold increase in MCF from baseline, with a decline in MCF starting after 60-120 min. TXA did not increase the endogenous thrombin potential. No clinical or laboratory signs of thromboembolic events, disseminated intravascular coagulation, or hypercoagulability were observed. Combination of aPCC or rFVIIa with TXA normalizes clot stability in haemophilia patients with inhibitor as compared to healthy controls. No clinical or laboratory adverse events were observed.

Enhancing classical swine fever virus identification: the advantages of Field-LAMP testing.

Classical swine fever virus (CSFV) identification has witnessed significant advancements with the development of rapid reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays. However, conventional RT-LAMP assays for CSFV diagnosis are hindered by a laborious RNA extraction step. Moreover, the need for thermal incubators and expensive micropipettes has limited their application in field settings. Addressing these challenges, our study presents a groundbreaking solution-an electro-free and point-of-care (POC) tool known as the field-LAMP assay-for the rapid clinical detection of CSFV. By eliminating the RNA extraction requirement, advancing the colorimetric read-out and lyophilized reaction reagents, our field-LAMP assay streamlines the diagnostic process, saving valuable time and effort. This novel approach also overcomes the dependency on electric-dependent thermal incubators and expensive micropipettes, making it practical and accessible for use in the field. The successful development of the field-LAMP assay marks a significant milestone in CSFV detection. This electro-free and POC tool offers several advantages, including its ability to deliver rapid results without compromising accuracy, facilitating prompt response and containment measures.

A cytokine and angiogenic factor (CAF) analysis in plasma for selection of sorafenib therapy in patients with metastatic renal cell carcinoma.

BACKGROUND: We investigated cytokines and angiogenic factors (CAFs) in patients with metastatic renal cell carcinoma (mRCC) treated in a randomized phase II clinical trial of sorafenib versus sorafenib+ interferon-α (IFN-α) that yielded no differences in progression-free survival (PFS). We aimed to link the CAF profile to PFS and select candidate predictive and prognostic markers for further study. METHODS: The concentrations of 52 plasma CAFs were measured pretreatment (n = 69), day 28, and day 56 using multiplex bead arrays and enzyme-linked immunosorbent assay. We investigated the association between baseline levels of CAFs with PFS and posttreatment changes. RESULTS: Unsupervised CAF clustering analysis revealed two distinct mRCC patient groups with elevated proangiogenic or proinflammatory mediators. A six-marker baseline CAF signature [osteopontin, vascular endothelial growth factor (VEGF), carbonic anhydrase 9, collagen IV, VEGF receptor-2, and tumor necrosis factor-related apoptosis-inducing ligand] correlated with PFS benefit (hazard ratio 0.20 versus 2.25, signature negative versus positive, respectively; P = 0.0002). While changes in angiogenic factors were frequently attenuated by the sorafenib+ IFN combination, most key immunomodulatory mediators increased. CONCLUSIONS: Using CAF profiling, we identified two mRCC patient groups, a candidate plasma signature for predicting PFS benefit, and distinct marker changes occurring with each treatment. This platform may provide valuable insights into renal cell carcinoma biology and the molecular consequences of targeted therapies.

Combined treatment with APCC (FEIBA®) and tranexamic acid in patients with haemophilia A with inhibitors and in patients with acquired haemophilia A--a two-centre experience.

The management of bleeding in haemophilia patients with inhibitors can be challenging when using monotherapy with either activated prothrombin complex concentrate (APCC) or recombinant activated FVII (rFVIIa) fail. The antifibrinolytic agent tranexamic acid (TXA) increases clot stability and is used concomitantly with coagulation factor replacement to improve haemostasis in haemophilia patients without inhibitors in many countries in Europe. Combined treatment with TXA and rFVIIa is not contraindicated in haemophilia patients with inhibitors. However, the combined approach of TXA and APCC has not been investigated due to safety concerns of increased risk of thrombosis or disseminated intravasal coagulation (DIC). The aim of this study is to report our experience of concomitant use of APCC and TXA in haemophilia A patients with inhibitor and in patients with acquired haemophilia A with respect to safety and efficacy. Seven (n = 6) haemophilia A patients with inhibitors and one (n = 1) with acquired haemophilia A from Oslo (Norway) and Stockholm (Sweden) were included in the study. The APCC was given at doses consistent to the manufacturers' recommendation. TXA was administered concomitantly either 10 mg kg(-1) every 6-8 h intravenously or 20 mg kg(-1) every 6-8 h orally. Haemostatic response was assessed by thromboelastography (TEG) and thrombin generation assay (TGA) in three of the patients. A total number of three bleeding episodes and two minor and six major surgical procedures were performed under the coverage with APCC and TXA. Haemostatic outcome was rated excellent or good in 10 of 11 (91%) treatment episodes. One episode was rated with poor effect. No episodes of arterial, venous thrombosis or DIC occurred during or after the treatment. Data from TEG and TGA analysis showed no signs of hypercoagulability following the combined treatment. This report demonstrates that, in a limited number of patients, combined treatment with APCC and TXA seemed to be safe, tolerated and relatively effective in management of bleeding episodes and in preventing haemorrhage during surgery in haemophilia patients with inhibitors and in a patient with acquired haemophilia A. Further studies should be performed to confirm these data.

Association between asymptomatic hyperuricemia and knee osteoarthritis in older outpatients.

OBJECTIVE: To investigate the association between asymptomatic hyperuricemia and knee osteoarthritis in older outpatients in Vietnam. PATIENTS AND METHODS: This cross-sectional study included 257 older outpatients (195 in the knee osteoarthritis group and 62 in the non-knee osteoarthritis group) aged ≥60 years (mean age 73.31 ± 7.96 years) attending rheumatologic and geriatric clinics from November 2020 to May 2021. Data were collected for both groups, including demographics, symptoms and signs of knee osteoarthritis, serum uric acid levels, and knee radiographs. The association between asymptomatic hyperuricemia and knee osteoarthritis was assessed using logistic regression. RESULTS: The mean serum uric acid level among patients with knee osteoarthritis was higher than that among patients without knee osteoarthritis (6.3 ± 1.74 mg/dl vs. 5.71 ± 1.45 mg/dl, p = 0.017). Hyperuricemia was more common among older outpatients with knee osteoarthritis than among those without knee osteoarthritis (39% vs. 19%, p = 0.005). After adjusting for age, sex, body mass index (BMI), and other comorbidities, the association between asymptomatic hyperuricemia and knee osteoarthritis remained significant (odds ratio [OR] 2.61, 95% confidence interval [CI] 1.22-5.60, p = 0.013). Subgroup analyses were performed according to sex and BMI groups. Significant associations between asymptomatic hyperuricemia and knee osteoarthritis were observed among women (p = 0.017) and among individuals who were underweight-normal-weight according to BMI (p = 0.009). CONCLUSIONS: Asymptomatic hyperuricemia is a common comorbidity among older outpatients with knee osteoarthritis. An independent association was identified between asymptomatic hyperuricemia and knee osteoarthritis among older Vietnamese outpatients, although sex and BMI may be confounding factors that impact this association.

rFVIIa administered by continuous infusion during surgery in patients with severe congenital FVII deficiency.

The use of recombinant FVIIa (rFVIIa) to control bleed in individuals with FVII deficiency has been proven to be effective. The main problems associated with its use are that it requires frequent bolus injections to counteract its short half-life and high cost. Our study aimed to evaluate whether any advantage could be gained by providing rFVIIa by continuous infusion during surgery with regard to haemostatic efficacy, safety and cost. The prospective study included 10 patients with severe FVII deficiency, who underwent 25 surgical procedures (13 major and 12 minor procedures) and were treated with rFVIIa administered by continuous infusion. Tranexamic acid was given concomitantly every 8 h. Prothrombin time, FVII:C assay and thrombin generation assay were used to monitor the treatment. The mean total dose given was 10 mg during a major surgery and 4.4 mg during a minor surgery for a mean treatment duration of 7.5 and 4.0 days respectively. This corresponds to a reduction of 70-90% in drug usage and medication cost compared with bolus injections. Except for one major perioperative bleeding, excellent haemostasis was achieved in all procedures. One patient developed a transient inhibitory activity. None of these events affected the postoperative course or prolonged the hospital stay. Our study demonstrated that continuous infusion of rFVIIa during surgery is safe, effective and highly cost effective.

The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3' exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae.

Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5'-->3' Exo1 and the 3'-->5' DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and highly sensitive mutation detection systems that are based on long and short homonucleotide runs. Genetic interactions were examined among DNA polymerase epsilon (pol2-4) and delta (pol3-01) mutants defective in 3'-->5' proofreading exonuclease, mutants defective in the 5'-->3' exonuclease Exo1, and mismatch repair mutants (msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01 mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they participate directly in postreplication mismatch repair as well as in other DNA metabolic processes.

Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.

Homonucleotide runs in coding sequences are hot spots for frameshift mutations and potential sources of genetic changes leading to cancer in humans having a mismatch repair defect. We examined frameshift mutations in homonucleotide runs of deoxyadenosines ranging from 4 to 14 bases at the same position in the LYS2 gene of the yeast Saccharomyces cerevisiae. In the msh2 mismatch repair mutant, runs of 9 to 14 deoxyadenosines are 1,700-fold to 51,000-fold, respectively, more mutable for single-nucleotide deletions than are runs of 4 deoxyadenosines. These frameshift mutations can account for up to 99% of all forward mutations inactivating the 4-kb LYS2 gene. Based on results with single and double mutations of the POL2 and MSH2 genes, both DNA polymerase epsilon proofreading and mismatch repair are efficient for short runs while only the mismatch repair system prevents frameshift mutations in runs of > or = 8 nucleotides. Therefore, coding sequences containing long homonucleotide runs are likely to be at risk for mutational inactivation in cells lacking mismatch repair capability.

Destabilization of yeast micro- and minisatellite DNA sequences by mutations affecting a nuclease involved in Okazaki fragment processing (rad27) and DNA polymerase delta (pol3-t).

We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase delta) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1- to 8-bp repeat units). Both the rad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the "proofreading" exonuclease domain of DNA polymerase delta) mutations were synthetically lethal.

Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes.

Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication.

A novel role in DNA metabolism for the binding of Fen1/Rad27 to PCNA and implications for genetic risk.

Fen1/Rad27 nuclease activity, which is important in DNA metabolism, is stimulated by proliferating cell nuclear antigen (PCNA) in vitro. The in vivo role of the PCNA interaction was investigated in the yeast Rad27. A nuclease-defective rad27 mutation had a dominant-negative effect that was suppressed by a mutation in the PCNA binding site, thereby demonstrating the importance of the Rad27-PCNA interaction. The PCNA-binding defect alone had little effect on mutation, recombination, and the methyl methanesulfonate (MMS) response in repair-competent cells, but it greatly amplified the MMS sensitivity of a rad51 mutant. Furthermore, the PCNA binding mutation resulted in lethality when combined with a homozygous or even a heterozygous pol3-01 mutation in the 3'-->5' exonuclease domain of DNA polymerase delta. These results suggest that phenotypically mild polymorphisms in DNA metabolic proteins can have dramatic consequences when combined.