DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.     

Lysosomal leukocyte beta-D-glucuronidase during enzyme replacement therapy in Fabry disease

OBJECTIVE: Fabry disease results from a deficiency in the activity of alpha-d-galactosidase A and subsequent accumulation of neutral glycosphingolipids in lysosomes. This study investigated whether lysosomal enzymes can indicate biochemical changes in the lysosomal apparatus induced by enzyme replacement therapy (ERT). DESIGN AND METHODS: Eight patients were monitored by clinical and biochemical tests and several lysosomal glycohydrolases were measured in plasma and leucocytes. RESULTS: Before starting ERT, beta-d-glucuronidase in leukocytes was markedly increased. After 1 month of therapy, enzyme levels dropped in all patients. In the patients who regularly followed the therapy, the enzyme levels remained stable for the next 20 months. In one patient who interrupted therapy for 2 months, the enzyme levels rose again. CONCLUSIONS: Lysosomal enzymes can be useful for monitoring biochemical changes in patients with Fabry disease receiving ERT. Though these findings refer to only a small number of patients, the correlation between beta-d-glucuronidase levels and ERT is interesting and might serve as a basis for further studies to define the potential of this enzyme in monitoring the effects of ERT in lysosomal storage disorders.     

Exome sequences and multi‐environment field trials elucidate the genetic basis of adaptation in barley

Broadening the genetic base of crops is crucial for developing varieties to respond to global agricultural challenges such as climate change. Here, we analysed a diverse panel of 371 domesticated lines of the model crop barley to explore the genetics of crop adaptation. We first collected exome sequence data and phenotypes of key life history traits from contrasting multi‐environment common garden trials. Then we applied refined statistical methods, including some based on exomic haplotype states, for genotype‐by‐environment (G×E) modelling. Sub‐populations defined from exomic profiles were coincident with barley's biology, geography and history, and explained a high proportion of trial phenotypic variance. Clear G×E interactions indicated adaptation profiles that varied for landraces and cultivars. Exploration of circadian clock‐related genes, associated with the environmentally adaptive days to heading trait (crucial for the crop's spread from the Fertile Crescent), illustrated complexities in G×E effect directions, and the importance of latitudinally based genic context in the expression of large‐effect alleles. Our analysis supports a gene‐level scientific understanding of crop adaption and leads to practical opportunities for crop improvement, allowing the prioritisation of genomic regions and particular sets of lines for breeding efforts seeking to cope with climate change and other stresses.     

Why we should take care of the competing risk bias in survival analysis: A phase II trial on the toxicity profile of radiotherapy for prostate cancer

AimThe aim of the present study is to evaluate and quantify the bias of competing risks in an Italian oncologic cohort comparing results from different statistical analysis methods.BackgroundCompeting risks are very common in randomized clinical trials and observational studies, in particular oncology and radiotherapy ones, and their inappropriate management causes results distortions widely present in clinical scientific articles.Materials and methodsThis is a single-institution phase II trial including 41 patients affected by prostate cancer and undergoing radiotherapy (IMRT-SIB) at the University Hospital of Udine.Different outcomes were considered: late toxicities, relapse, death.Death in the absence of relapse or late toxicity was considered as a competing event.ResultsThe Kaplan Meier method, compared to cumulative incidence function method, overestimated the probability of the event of interest (toxicity and biochemical relapse) and of the competing event (death without toxicity/relapse) by 9.36%. The log-rank test, compared to Gray's test, overestimated the probability of the event of interest by 5.26%.The Hazard Ratio's and cause specific hazard's Cox regression are not directly comparable to subdistribution hazard's Fine and Gray's modified Cox regression; nonetheless, the FG model, the best choice for prognostic studies with competing risks, found significant associations not emerging with Cox regression.ConclusionsThis study confirms that using inappropriate statistical methods produces a 10% overestimation in results, as described in the literature, and highlights the importance of taking into account the competing risks bias.     

Long-Term Structural and Functional Myocardial Adaptations in Healthy Living Kidney Donors: A Pilot Study

Background and Aims

Compensatory renal hypertrophy following unilateral nephrectomy (UNX) occurs in the remaining kidney. However, the long-term cardiac adaptive process to UNX remains poorly defined in humans. Our goal was to characterize myocardial structure and function in living kidney donors (LKDs), approximately 12 years after UNX.

Methods and Results

Cardiac function and structure in 15 Italian LKDs, at least 5 years after UNX (median time from donation = 8.4 years) was investigated and compared to those of age and sex matched U.S. citizens healthy controls (n = 15). Standard and speckle tracking echocardiography (STE) was performed in both LKDs and controls. Plasma angiotensin II, aldosterone, atrial natriuretic peptide (ANP), N terminus pro B-type natriuretic peptide (NT-proBNP), cyclic guanylyl monophosphate (cGMP), and amino-terminal peptide of procollagen III (PIIINP) were also collected. Median follow-up was 11.9 years. In LKDs, LV geometry and function by STE were similar to controls, wall thickness and volumes were within normal limits also by CMR. In LKDs, CMR was negative for myocardial fibrosis, but apical rotation and LV torsion obtained by STE were impaired as compared to controls (21.4 ± 7.8 vs 32.7 ± 8.9 degrees, p = 0.04). Serum creatinine and PIIINP levels were increased [1.1 (0.9–1.3) mg/dL, and 5.8 (5.4–7.6)] μg/L, respectively), while urinary cGMP was reduced [270 (250–355) vs 581 (437–698) pmol/mL] in LKDs. No LKD developed cardiovascular or renal events during follow-up.

Conclusions

Long-term kidney donors have no apparent structural myocardial abnormalities as assessed by contrast enhanced CMR. However, myocardial deformation of the apical segments, as well as apical rotation, and LV torsion are reduced. The concomitant increase in circulating PIIINP level is suggestive of fibrosis. Further studies, focused on US and EU patients are warranted to evaluate whether these early functional modifications will progress to a more compromised cardiac function and structure at a later time.     

Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.