BirthSeq, a new method to isolate and analyze dated cells in different vertebrates.

Embryonic development is a complex and dynamic process that unfolds over time and involves the production and diversification of increasing numbers of cells. The impact of developmental time on the formation of the central nervous system is well-documented, with evidence showing that time plays a critical role in establishing the identity of neuronal subtypes. However, the study of how time translates into genetic instructions driving cell fate is limited by the scarcity of suitable experimental tools. We introduce BirthSeq, a new method for isolating and analyzing cells based on their birth date. This innovative technique allows for in vivo labeling of cells, isolation via FACS, and analysis using high-throughput techniques. We tuned up BirthSeq in developmental organs across three vertebrate species (mouse, chick, and gecko), and fully made use of it for single-cell RNA sequencing and novel spatially resolved transcriptomic approaches in mouse and chick, respectively. Overall, BirthSeq provides a versatile tool for studying virtually any tissue in different vertebrate organism, helping to fill the necessity in developmental biology research by targeting cells and their temporal cues.

Expression of the cancer-associated DNA polymerase ε P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication.

Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few indel mutations. The mutations are equally distributed across different genomic regions, but in the immediate vicinity there is an asymmetry in AT frequency. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.

The transcription factor Fosl1 preserves Klotho expression and protects from acute kidney injury.

Increased expression of AP-1 transcription factor components has been reported in acute kidney injury (AKI). However, the role of specific components, such as Fosl1, in tubular cells or AKI is unknown. Upstream regulator analysis of murine nephrotoxic AKI transcriptomics identified AP-1 as highly upregulated. Among AP-1 canonical components, Fosl1 was found to be upregulated in two transcriptomics datasets from nephrotoxic murine AKI induced by folic acid or cisplatin and from proximal tubular cells exposed to TWEAK, a cytokine mediator of AKI. Fosl1 was minimally expressed in the kidneys of control uninjured mice. Increased Fosl1 protein was localized to proximal tubular cell nuclei in AKI. In human AKI, FOSL1 was found present in proximal tubular cells in kidney sections and in urine along with increased urinary FOSL1 mRNA. Selective Fosl1 deficiency in proximal tubular cells (Fosl1Δtub) increased the severity of murine cisplatin- or folate-induced AKI as characterized by lower kidney function, more severe kidney inflammation and Klotho downregulation. Indeed, elevated AP-1 activity was observed after cisplatin-induced AKI in Fosl1Δtub mice compared to wild-type mice. More severe Klotho downregulation preceded more severe kidney dysfunction. The Klotho promoter was enriched in Fosl1 binding sites and Fosl1 bound to the Klotho promoter in cisplatin-AKI. In cultured proximal tubular cells, Fosl1 targeting increased the proinflammatory response and downregulated Klotho. In vivo, recombinant Klotho administration protected Fosl1Δtub mice from cisplatin-AKI. Thus, increased proximal tubular Fosl1 expression during AKI is an adaptive response, preserves Klotho, and limits the severity of tubular cell injury and AKI.

Analysis of the Gene Therapies Authorized by the United States Food and Drug Administration and the European Medicines Agency.

BACKGROUND: Gene therapy, altering the genes inside human cells, has recently emerged as an alternative for preventing and treating disease. Concerns have been expressed about the clinical value and the high cost of gene therapies. OBJECTIVE: This study assessed the characteristics of the clinical trials, authorizations, and prices of gene therapies in the United States and the European Union. RESEARCH DESIGN: We collected regulatory information from the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) and manufacturer-listed prices from the United States, UK, and Germany. Descriptive statistics and t tests were conducted in the study. RESULTS: As of January 1, 2022, the FDA and EMA authorized 8 and 10 gene therapies, respectively. The FDA and EMA granted orphan designation to all gene therapies except talimogene laherparepvec. Pivotal clinical trials were nonrandomized, open level, uncontrolled, phase I-III, and included a limited number of patients. Study primary outcomes were mainly surrogate endpoints without demonstration of direct patient benefit. The price of gene therapies at market entry ranged from $200,064 to $2,125,000 million. CONCLUSIONS: Gene therapy is used to treat incurable diseases that affect only a small number of patients (orphan diseases). Based on this, they are approved by the EMA and FDA with insufficient clinical evidence to ensure safety and efficacy, in addition to the high cost.

Cost-effectiveness analysis of androgen deprivation therapy with relugolix for the treatment of advanced prostate cancer.

BACKGROUND: Relugolix treatment of advanced prostate cancer (APC), like other gonadotropin-releasing hormone-antagonists, results in rapid decrease in testosterone concentrations without the risk of flare, as seen in leuprolide. Despite this benefit over leuprolide, no economic evaluation assessment to ascertain the cost-effectiveness of relugolix has been conducted. Therefore, this study aims to assess the cost-effectiveness of androgen deprivation therapy (ADT) with 120 mg relugolix against 7.5 mg leuprolide for the treatment of APC. METHODS: A Markov model was used to assess and compare the costs of APC treatment from a health care payer's perspective and the effectiveness of ADT with relugolix and leuprolide at the 3 lines of APC treatment among modified intent-to-treat patients. Relative progression-free (PFS) and overall survival (OS) rates were estimated. Outcomes measured in the analyses included costs of the drugs and therapies, quality-adjusted life-years (QALYs), incremental cost-effectiveness ratios (ICERs), cost-effectiveness acceptability, and probability curves. RESULTS: The cost-effectiveness analysis showed the ICER for ADT with relugolix to be US $49,571.1 per QALY. At the ICER value, the sensitivity analysis indicated that ADT with leuprolide was dominant in 100% of the simulations. ADT acceptance with relugolix was 100% when a willingness-to-pay threshold was set at US $100,000/QALY. At 5-years, the relative PFS and OS rates for relugolix at the first line of therapy were 72.7% and 86.0%, respectively, compared to 61.0% and 85.90% for leuprolide. CONCLUSION: Though the influence of adverse events was not considered in the analysis, ADT with relugolix was not a cost-effective choice for APC management. While the analysis revealed a slight chance of sustaining testosterone suppression with relugolix, ADT with relugolix provided no significant survival advantages over ADT with leuprolide. Therefore, this analysis confirms no need for further assessment of APC interventions to make informed decisions beneficial to the APC patients, oncologists, and other stakeholders.

Revalorization of Microalgae Biomass for Synergistic Interaction and Sustainable Applications: Bioplastic Generation.

Microalgae and cyanobacteria are photosynthetic microorganisms' sources of renewable biomass that can be used for bioplastic production. These microorganisms have high growth rates, and contrary to other feedstocks, such as land crops, they do not require arable land. In addition, they can be used as feedstock for bioplastic production while not competing with food sources (e.g., corn, wheat, and soy protein). In this study, we review the macromolecules from microalgae and cyanobacteria that can serve for the production of bioplastics, including starch and glycogen, polyhydroxyalkanoates (PHAs), cellulose, polylactic acid (PLA), and triacylglycerols (TAGs). In addition, we focus on the cultivation of microalgae and cyanobacteria for wastewater treatment. This approach would allow reducing nutrient supply for biomass production while treating wastewater. Thus, the combination of wastewater treatment and the production of biomass that can serve as feedstock for bioplastic production is discussed. The comprehensive information provided in this communication would expand the scope of interdisciplinary and translational research.

Recent Advances in Prodigiosin as a Bioactive Compound in Nanocomposite Applications.

Bionanocomposites based on natural bioactive entities have gained importance due to their abundance; renewable and environmentally benign nature; and outstanding properties with applied perspective. Additionally, their formulation with biological molecules with antimicrobial, antioxidant, and anticancer activities has been produced nowadays. The present review details the state of the art and the importance of this pyrrolic compound produced by microorganisms, with interest towards Serratia marcescens, including production strategies at a laboratory level and scale-up to bioreactors. Promising results of its biological activity have been reported to date, and the advances and applications in bionanocomposites are the most recent strategy to potentiate and to obtain new carriers for the transport and controlled release of prodigiosin. Prodigiosin, a bioactive secondary metabolite, produced by Serratia marcescens, is an effective proapoptotic agent against bacterial and fungal strains as well as cancer cell lines. Furthermore, this molecule presents antioxidant activity, which makes it ideal for treating wounds and promoting the general improvement of the immune system. Likewise, some of the characteristics of prodigiosin, such as hydrophobicity, limit its use for medical and biotechnological applications; however, this can be overcome by using it as a component of a bionanocomposite. This review focuses on the chemistry and the structure of the bionanocomposites currently developed using biorenewable resources. Moreover, the work illuminates recent developments in pyrrole-based bionanocomposites, with special insight to its application in the medical area.

Spinal interleukin-1ß induces mechanical spinal hyperexcitability in rats: Interactions and redundancies with TNF and IL-6.

Both spinal tumor necrosis factor (TNF) and interleukin-6 (IL-6) contribute to the development of "mechanical" spinal hyperexcitability in inflammatory pain states. Recently, we found that spinal sensitization by TNF was significantly reduced by blockade of spinal IL-6 signaling suggesting that IL-6 signaling is involved in spinal TNF effects. Here, we explored whether spinal interleukin-1ß (IL-1ß), also implicated in inflammatory pain, induces "mechanical" spinal hyperexcitability, and whether spinal IL-1ß effects are related to TNF and IL-6 effects. We recorded the responses of spinal cord neurons to mechanical stimulation of the knee joint in vivo and used cellular approaches on microglial and astroglial cell lines to identify interactions of IL-1ß, TNF, and IL-6. Spinal application of IL-1ß in anesthetized rats modestly enhanced responses of spinal cord neurons to innocuous and noxious mechanical joint stimulation. This effect was blocked by minocycline indicating microglia involvement, and significantly attenuated by interfering with IL-6 signaling. In the BV2 microglial cell line, IL-1ß, like TNF, enhanced the release of soluble IL-6 receptor, necessary for spinal IL-6 actions. Different to TNF, IL-1ß caused SNB-19 astrocytes to release interleukin-11. The generation of "mechanical" spinal hyperexcitability by IL-1ß was more pronounced upon spinal TNF neutralization with etanercept, suggesting that concomitant TNF limits IL-1ß effects. In BV2 cells, TNF stimulated the release of IL-1Ra, an endogenous IL-1ß antagonist. Thus, spinal IL-1ß has the potential to induce spinal hyperexcitability sharing with TNF dependency on IL-6 signaling, but TNF also limited IL-1ß effects explaining the modest effect of IL-1ß.

Characteristics of available studies and dissemination of research using major clinical data sharing platforms.

BACKGROUND/AIMS: Over the past decade, numerous data sharing platforms have been launched, providing access to de-identified individual patient-level data and supporting documentation. We evaluated the characteristics of prominent clinical data sharing platforms, including types of studies listed as available for request, data requests received, and rates of dissemination of research findings from data requests. METHODS: We reviewed publicly available information listed on the websites of six prominent clinical data sharing platforms: Biological Specimen and Data Repository Information Coordinating Center, ClinicalStudyDataRequest.com, Project Data Sphere, Supporting Open Access to Researchers-Bristol Myers Squibb, Vivli, and the Yale Open Data Access Project. We recorded key platform characteristics, including listed studies and available supporting documentation, information on the number and status of data requests, and rates of dissemination of research findings from data requests (i.e. publications in a peer-reviewed journals, preprints, conference abstracts, or results reported on the platform's website). RESULTS: The number of clinical studies listed as available for request varied among five data sharing platforms: Biological Specimen and Data Repository Information Coordinating Center (n = 219), ClinicalStudyDataRequest.com (n = 2,897), Project Data Sphere (n = 154), Vivli (n = 5426), and the Yale Open Data Access Project (n = 395); Supporting Open Access to Researchers did not provide a list of Bristol Myers Squibb studies available for request. Individual patient-level data were nearly always reported as being available for request, as opposed to only Clinical Study Reports (Biological Specimen and Data Repository Information Coordinating Center = 211/219 (96.3%); ClinicalStudyDataRequest.com = 2884/2897 (99.6%); Project Data Sphere = 154/154 (100.0%); and the Yale Open Data Access Project = 355/395 (89.9%)); Vivli did not provide downloadable study metadata. Of 1201 data requests listed on ClinicalStudyDataRequest.com, Supporting Open Access to Researchers-Bristol Myers Squibb, Vivli, and the Yale Open Data Access Project platforms, 586 requests (48.8%) were approved (i.e. data access granted). The majority were for secondary analyses and/or developing/validating methods (ClinicalStudyDataRequest.com = 262/313 (83.7%); Supporting Open Access to Researchers-Bristol Myers Squibb = 22/30 (73.3%); Vivli = 63/84 (75.0%); the Yale Open Data Access Project = 111/159 (69.8%)); four were for re-analyses or corroborations of previous research findings (ClinicalStudyDataRequest.com = 3/313 (1.0%) and the Yale Open Data Access Project = 1/159 (0.6%)). Ninety-five (16.1%) approved data requests had results disseminated via peer-reviewed publications (ClinicalStudyDataRequest.com = 61/313 (19.5%); Supporting Open Access to Researchers-Bristol Myers Squibb = 3/30 (10.0%); Vivli = 4/84 (4.8%); the Yale Open Data Access Project = 27/159 (17.0%)). Forty-two (6.8%) additional requests reported results through preprints, conference abstracts, or on the platform's website (ClinicalStudyDataRequest.com = 12/313 (3.8%); Supporting Open Access to Researchers-Bristol Myers Squibb = 3/30 (10.0%); Vivli = 2/84 (2.4%); Yale Open Data Access Project = 25/159 (15.7%)). CONCLUSION: Across six prominent clinical data sharing platforms, information on studies and request metrics varied in availability and format. Most data requests focused on secondary analyses and approximately one-quarter of all approved requests publicly disseminated their results. To further promote the use of shared clinical data, platforms should increase transparency, consistently clarify the availability of the listed studies and supporting documentation, and ensure that research findings from data requests are disseminated.

Perioperative Prescribing Practices of Extended-Release Opioids in Noncancer Surgical Patients, 2015-2018.

BACKGROUND: Extended-release (ER) opioids are indicated for the management of persistent moderate to severe pain in patients requiring around-the-clock opioid analgesics for an extended period of time. Concerns have been raised regarding safety of ER opioids due to its potential for abuse and dependence. However, little is known about perioperative prescribing practices of ER opioids. This study assessed perioperative prescribing practices of ER opioids in noncancer surgical patients stratified by type of opioid exposure prior to admission and examined predictors of postoperative opioid administration in oral morphine equivalents (OME). METHODS: This was a retrospective cohort study using the University of California San Francisco Medical Center electronic health record data. This study included 25,396 adult noncancer patients undergoing elective surgery under general anesthesia in the period 2015-2018. The primary study outcome was predictors of postoperative administration of opioids in hospitalized surgical patients. Secondary outcomes included patients discontinued and initiated on ER opioids during their hospital stay. RESULTS: substance use disorder diagnosis and use of opioids, surgery type, and postoperative administration of nonopioid analgesics were associated with postoperative administration of opioids (P < .0001). The estimated adjusted mean (95% confidence interval [CI]) of postoperative administration of OME prior to admission in ER opioid users (170.08 mg; 147.08-196.67) was twice the amount for opioid-naïve patients (81.36 mg; 70.7-93.63; P < .0001). One in 5 prior to admission ER opioid users were weaned off ER opioids while hospitalized without adversely affecting their postoperative pain or hospital length of stay (LOS). Four of 5 patients who used ER opioids prior to admission also received ER opioids after surgery, whereas, 1 in 100 opioid-naïve patients received ER opioids during their hospital stay. CONCLUSIONS: We found significant variability in the perioperative prescribing practices of ER opioids in hospitalized noncancer surgical patients by use of opioids prior to admission and surgery type. Pain medicine practitioners and surgeons may play a significant role tackling the surgery-related risk of exposure to ER opioids and decreasing opioid-related complications.

Nucleosomal signatures impose nucleosome positioning in coding and noncoding sequences in the genome.

In the yeast genome, a large proportion of nucleosomes occupy well-defined and stable positions. While the contribution of chromatin remodelers and DNA binding proteins to maintain this organization is well established, the relevance of the DNA sequence to nucleosome positioning in the genome remains controversial. Through quantitative analysis of nucleosome positioning, we show that sequence changes distort the nucleosomal pattern at the level of individual nucleosomes in three species of Schizosaccharomyces and in Saccharomyces cerevisiae This effect is equally detected in transcribed and nontranscribed regions, suggesting the existence of sequence elements that contribute to positioning. To identify such elements, we incorporated information from nucleosomal signatures into artificial synthetic DNA molecules and found that they generated regular nucleosomal arrays indistinguishable from those of endogenous sequences. Strikingly, this information is species-specific and can be combined with coding information through the use of synonymous codons such that genes from one species can be engineered to adopt the nucleosomal organization of another. These findings open the possibility of designing coding and noncoding DNA molecules capable of directing their own nucleosomal organization.

A comparison of new drugs approved by the FDA, the EMA, and Swissmedic: an assessment of the international harmonization of drugs.

INTRODUCTION: This study compared the characteristics of new human drugs approved by the Food and Drug Administration (FDA), the European Medicine Agency (EMA), and Swissmedic (SMC) in the period 2007 to 2016. METHODS: The list of new drugs and therapeutic biologics approved by the FDA, the EMA, and SMC in the period 2007 to 2016 was collected from websites of those agencies. The study included regulatory information, approval date, and indication for each drug. Descriptive statistical t tests and x2-tests were performed for the analysis. RESULTS: From 2007 to 2016, 134 new drugs were approved by all three regulatory agencies. Overall, 66.4% of the drugs were first approved by the FDA, 30.6% by the EMA, and 3.0% by SMC. The difference in approval dates between SMC and the EMA, SMC and the FDA, and the FDA and the EMA were statistically significant. The indications approved by the FDA, the EMA, and SMC for the same drugs were similar in content for 23.1% drugs and different in 76.9% of the drugs. Significant differences in indications existed between the FDA and SMC and the FDA and the EMA, but not between the EMA and SMC. CONCLUSION: There were differences in the characteristics of new drugs approved by the EMA, the FDA, and SMC in the period 2007-2016. Overall, two thirds of the new drugs were first approved by the FDA. Differences in indications were found in three out of four new drugs approved by the three regulatory agencies. Despite international drug regulation harmonization efforts, significant differences in the characteristics of new drugs approved by different agencies persist.

Microcephaly epidemic related to the Zika virus and living conditions in Recife, Northeast Brazil.

BACKGROUND: Starting in August 2015, there was an increase in the number of cases of neonatal microcephaly in Northeast Brazil. These findings were identified as being an epidemic of microcephaly related to Zika virus (ZIKV) infection. The present study aims to analyse the spatial distribution of microcephaly cases in Recife (2015-2016), which is in Northeast Brazil, and its association with the living conditions in this city. METHODS: This was an ecological study that used data from reported cases of microcephaly from the State Health Department of Pernambuco (August 2015 to July 2016). The basic spatial unit of analysis was the 94 districts of Recife. The case definition of microcephaly was: neonates with a head circumference of less than the cut-off point of -2 standard deviations below the mean value from the established Fenton growth curve. As an indicator of the living conditions of the 94 districts, the percentage of heads of households with an income of less than twice the minimum wage was calculated. The districts were classified into four homogeneous strata using the K-means clustering algorithm. We plotted the locations of each microcephaly case over a layer of living conditions. RESULTS: During the study period, 347 microcephaly cases were reported, of which 142 (40.9%) fulfilled the definition of a microcephaly case. Stratification of the 94 districts resulted in the identification of four strata. The highest stratum in relation to the living conditions presented the lowest prevalence rate of microcephaly, and the overall difference between this rate and the rates of the other strata was statistically significant. The results of the Kruskal-Wallis test demonstrated that there was a strong association between a higher prevalence of microcephaly and poor living conditions. After the first 6 months of the study period, there were no microcephaly cases recorded within the population living in the richest socio-economic strata. CONCLUSION: This study showed that those residing in areas with precarious living conditions had a higher prevalence of microcephaly compared with populations with better living conditions.

Cost-effectiveness analysis of metformin+dipeptidyl peptidase-4 inhibitors compared to metformin+sulfonylureas for treatment of type 2 diabetes.

BACKGROUND: Patients with type 2 diabetes (T2D) typically use several drug treatments during their lifetime. There is a debate about the best second-line therapy after metformin monotherapy failure due to the increasing number of available antidiabetic drugs and the lack of comparative clinical trials of secondary treatment regimens. While prior research compared the cost-effectiveness of two alternative drugs, the literature assessing T2D treatment pathways is scarce. The purpose of this study was to evaluate the long-term cost-effectiveness of dipeptidyl peptidase-4 inhibitors (DPP-4i) compared to sulfonylureas (SU) as second-line therapy in combination with metformin in patients with T2D. METHODS: A Markov model was developed with four health states, 1 year cycle, and a 25-year time horizon. Clinical and cost data were collected from previous studies and other readily available secondary data sources. The incremental cost-effectiveness ratio (ICER) was estimated from the US third party payer perspective. Both, costs and outcomes, were discounted at a 3% annual discount rate. One way and probabilistic sensitivity analyses were performed to evaluate the impact of uncertainty on the base-case results. RESULTS: The discounted incremental cost of metformin+DPP-4i compared to metformin+SU was $11,849 and the incremental life-years gained were 0.61, resulting in an ICER of $19,420 per life-year gained for patients in the metformin+DPP-4i treatment pathway. The ICER estimated in the probabilistic sensitivity analysis was $19,980 per life-year gained. Sensitivity analyses showed that the results of the study were not sensitive to changes in the parameters used in base-case. CONCLUSIONS: The metformin+DPP-4i treatment pathway was cost-effective compared to metformin+SU as a long-term second-line therapy in the treatment of T2D from the US health care payer perspective. Study findings have the potential to provide clinicians and third party payers valuable evidence for the prescription and utilization of cost-effective second-line therapy after metformin monotherapy failure in the treatment of T2D.

Changes in the utilization of osteoporosis drugs after the 2010 FDA bisphosphonate drug safety communication.

INTRODUCTION: In October 2010, the US Food and Drug Administration (FDA) issued a safety communication regarding the risks of atypical fractures of the femur, with bisphosphonates drugs. This study evaluated the impact of the bisphosphonates FDA safety communication on the utilization of osteoporosis medications in Medicaid programs. METHODS: Osteoporosis drugs utilization data from the July 2006 to June 2014 were extracted from the national Summary Files from the Medicaid State Drug Utilization Data maintained by the Centers for Medicare & Medicaid Services (CMS). We performed an interrupted time series analyses to evaluate trends in utilization of osteoporosis drugs before and after the 2010 FDA safety commination. RESULTS: Time-series analyses of osteoporosis drug utilization in Medicaid program revealed a significant downward trend associated with the 2010 FDA bisphosphonates safety communication. Before the FDA safety communication was issued, the utilization rate was slightly decreased between 2006 and 2010. In the year following the FDA safety communication the bisphosphonate DDDs per 1000 beneficiaries of fell 22% yearly until the end of study period. CONCLUSIONS: The 2010 FDA bisphosphonates safety communication appeared to have influenced Osteoporosis utilization in Medicaid recipients. The 2010 FDA bisphosphonates safety communication was associated with a significant reduction in the utilization of bisphosphonates in the Medicaid program.

A conserved role of the RSC chromatin remodeler in the establishment of nucleosome-depleted regions.

The occupancy of nucleosomes governs access to the eukaryotic genomes and results from a combination of biophysical features and the effect of ATP-dependent remodelling complexes. Most promoter regions show a conserved pattern characterized by a nucleosome-depleted region (NDR) flanked by nucleosomal arrays. The conserved RSC remodeler was reported to be critical to establish NDR in vivo in budding yeast but other evidences suggested that this activity may not be conserved in fission yeast. By reanalysing and expanding previously published data, we propose that NDR formation requires, at least partially, RSC in both yeast species. We also discuss the most prominent biological role of RSC and the possibility that non-essential subunits do not define alternate versions of the complex.

Comparative analysis of methods for genome-wide nucleosome cartography.

Nucleosomes contribute to compacting the genome into the nucleus and regulate the physical access of regulatory proteins to DNA either directly or through the epigenetic modifications of the histone tails. Precise mapping of nucleosome positioning across the genome is, therefore, essential to understanding the genome regulation. In recent years, several experimental protocols have been developed for this purpose that include the enzymatic digestion, chemical cleavage or immunoprecipitation of chromatin followed by next-generation sequencing of the resulting DNA fragments. Here, we compare the performance and resolution of these methods from the initial biochemical steps through the alignment of the millions of short-sequence reads to a reference genome to the final computational analysis to generate genome-wide maps of nucleosome occupancy. Because of the lack of a unified protocol to process data sets obtained through the different approaches, we have developed a new computational tool (NUCwave), which facilitates their analysis, comparison and assessment and will enable researchers to choose the most suitable method for any particular purpose. NUCwave is freely available at http://nucleosome.usal.es/nucwave along with a step-by-step protocol for its use.

Replication dynamics in fission and budding yeasts through DNA polymerase tracking.

The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof-read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions.

Spinal extradural arteriovenous fistula with intradural drainage: three surgical cases with long-term follow-up.

Spinal extradural arteriovenous fistulas (SEDAVF) with intradural drainage are uncommon vascular lesions that cause venous congestive myelopathy. We present three SEDAVF with intradural drainage treated with surgical disconnection of the intradural drainage via a single level laminectomy, followed by transarterial embolization (TAE) with Onyx if spontaneous thrombosis of the extradural fistula did not occur spontaneously. All patients improved their neurological condition and no congestive myelopathy recurrence was noted after mean follow-up of 84 months. We believe that SEDAVF with intradural drainage constitute a specific entity that should be included in the classifications of spinal arteriovenous malformations.