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Transposase genes are ubiquitous in all domains of life and provide a rich reservoir for the evolution of novel protein functions. Here we report deep evolutionary links between bacterial IS110-family transposases, which catalyse RNA-guided DNA recombination using bridge RNAs, and archaeal/eukaryotic Nop5-family proteins, which promote RNA-guided RNA 2'-O-methylation using C/D-box snoRNAs. On the basis of conservation of protein sequence, domain architecture, three-dimensional structure and non-coding RNA features, alongside phylogenetic analyses, we propose that programmable RNA modification emerged through the exaptation of components derived from IS110-like transposons. These findings underscore how recurrent domestication events of transposable elements have driven the evolution of RNA-guided mechanisms., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature Limited.)

Double-strand breaks (DSBs) in DNA are challenging to repair. Cells employ at least three DSB-repair mechanisms, with a preference for non-homologous end joining (NHEJ) over homologous recombination (HR) and microhomology-mediated end joining (MMEJ). While most eukaryotic DNA is transcribed into RNA, providing complementary genetic information, much remains unknown about the direct impact of RNA on DSB-repair outcomes and its role in DSB-repair via end joining. Here, we show that both sense and antisense-transcript RNAs impact DSB repair in a sequence-specific manner in wild-type human and yeast cells. Depending on its sequence complementarity with the broken DNA ends, a transcript RNA can promote repair of a DSB or a double-strand gap in its DNA gene via NHEJ or MMEJ, independently from DNA synthesis. The results demonstrate a role of transcript RNA in directing the way DSBs are repaired in DNA, suggesting that RNA may directly modulate genome stability and evolution., (© 2024. The Author(s).)

TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life. IS605-family TnpB homologs function as programmable RNA-guided homing endonucleases in bacteria, driving transposon maintenance through DNA double-strand break-stimulated homologous recombination. In this work, we uncovered molecular mechanisms of the transposition life cycle of IS607-family elements that, notably, also encode group I introns. We identified specific features for a candidate "IStron" from Clostridium botulinum that allow the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts evolved an ability to balance competing and mutually exclusive activities that promote selfish transposon spread while limiting adverse fitness costs on the host. Collectively, this work highlights molecular innovation in the multifunctional utility of transposon-encoded noncoding RNAs.

Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination 1-4 . These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12 (refs. 5,6 ). We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Cas adaptive immunity. Here, using phylogenetics, structural predictions, comparative genomics and functional assays, we uncover multiple independent genesis events of programmable transcription factors, which we name TnpB-like nuclease-dead repressors (TldRs). These proteins use naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPR interference technologies invented by humans 7 . Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility 8 , phage susceptibility 9 , and host immunity 10 . Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of transposon-encoded genes, and reveals the evolutionary trajectory of diverse RNA-guided transcription factors., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Transposase genes are ubiquitous in all domains of life and provide a rich reservoir for the evolution of novel protein functions. Here we report deep evolutionary links between bacterial IS110 transposases, which catalyze RNA-guided DNA recombination using bridge RNAs, and archaeal/eukaryotic Nop5-family proteins, which promote RNA-guided RNA 2'-O-methylation using C/D-box snoRNAs. Based on conservation in the protein primary sequence, domain architecture, and three-dimensional structure, as well as common architectural features of the non-coding RNA components, we propose that programmable RNA modification emerged via exaptation of components derived from IS110-like transposons. Alongside recent studies highlighting the origins of CRISPR-Cas9 and Cas12 in IS605-family transposons, these findings underscore how recurrent domestication events of transposable elements gave rise to complex RNA-guided biological mechanisms., Competing Interests: COMPETING INTERESTS The authors declare no competing interests.

Ribonucleoside monophosphates (rNMPs) are abundantly found within genomic DNA of cells. The embedded rNMPs alter DNA properties and impact genome stability. Mutations in ribonuclease (RNase) H2, a key enzyme for rNMP removal, are associated with the Aicardi-Goutières syndrome (AGS), a severe neurological disorder. Here, we engineered orthologs of the human RNASEH2A-G37S and RNASEH2C-R69W AGS mutations in yeast Saccharomyces cerevisiae : rnh201 -G42S and rnh203 -K46W. Using the ribose-seq technique and the Ribose-Map bioinformatics toolkit, we unveiled rNMP abundance, composition, hotspots, and sequence context in these AGS-ortholog mutants. We found a high rNMP presence in the nuclear genome of rnh201 -G42S-mutant cells, and an elevated rCMP content in both mutants, reflecting preferential cleavage of RNase H2 at rGMP. We discovered unique rNMP patterns in each mutant, showing differential activity of the AGS mutants on the leading or lagging replication strands. This study guides future research on rNMP characteristics in human genomes with AGS mutations., Competing Interests: We have a patent related to this study: Storici, F., Hesselberth, J.R., and Koh, K. D. Methods to detect Ribonucleotides in deoxyribonucleic acids. GTRC-6522, 2013; U.S. 10,787,703 B1 Sep. 29, 2020. https://uspto.report/patent/grant/10,787,703., (© 2024 The Authors.)

Transposon-encoded tnpB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination 1-4 . This widespread gene family was repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12 5,6 . We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Cas. Here, using phylogenetics, structural predictions, comparative genomics, and functional assays, we uncover multiple instances of programmable transcription factors that we name TnpB-like nuclease-dead repressors (TldR). These proteins employ naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPRi technologies invented by humans 7 . Focusing on a TldR clade found broadly in Enterobacteriaceae , we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility 8 , phage susceptibility 9 , and host immunity 10 . Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of genes encoded by transposable elements, and reveals that RNA-guided transcription factors emerged long before the development of dCas9-based editors.

TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life, presumably due to the critical roles they play in transposon proliferation. IS605family TnpB homologs function in bacteria as programmable homing endonucleases by exploiting transposon-encoded guide RNAs to cleave vacant genomic sites, thereby driving transposon maintenance through DSB-stimulated homologous recombination. Whether this pathway is conserved in other genetic contexts, and in association with other transposases, is unknown. Here we uncover molecular mechanisms of transposition and RNA-guided DNA cleavage by IS607-family elements that, remarkably, also encode catalytic, self-splicing group I introns. After reconstituting and systematically investigating each of these biochemical activities for a candidate 'IStron' derived from Clostridium botulinum , we discovered sequence and structural features of the transposon-encoded RNA that satisfy molecular requirements of a group I intron and TnpB guide RNA, while still retaining the ability to be faithfully mobilized at the DNA level by the TnpA transposase. Strikingly, intron splicing was strongly repressed not only by TnpB, but also by the secondary structure of ωRNA alone, allowing the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts have evolved a sensitive equilibrium to balance competing and mutually exclusive activities that promote transposon maintenance while limiting adverse fitness costs on the host. Collectively, this work explains how diverse enzymatic activities emerged during the selfish spread of IS607-family elements and highlights molecular innovation in the multi-functional utility of transposon-encoded noncoding RNAs., Competing Interests: Competing interests: Columbia University has filed a patent application related to this work. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences, a scientific advisor to CrisprBits and Prime Medicine, and an equity holder in Dahlia Biosciences and CrisprBits.

Ribonucleoside monophosphates (rNMPs) are abundantly found within genomic DNA of cells. The embedded rNMPs alter DNA properties and impact genome stability. Mutations in ribonuclease (RNase) H2, a key enzyme for rNMP removal, are associated with the Aicardi-Goutières syndrome (AGS), a severe neurological disorder. Here, we engineered two AGS-ortholog mutations in Saccharomyces cerevisiae : rnh201 -G42S and rnh203 -K46W. Using the ribose-seq technique and the Ribose-Map bioinformatics toolkit, we unveiled rNMP abundance, composition, hotspots, and sequence context in these yeast AGS-ortholog mutants. We found higher rNMP incorporation in the nuclear genome of rnh201 -G42S than in wild-type and rnh203- K46W-mutant cells, and an elevated rCMP content in both mutants. Moreover, we uncovered unique rNMP patterns in each mutant, highlighting a differential activity of the AGS mutants towards rNMPs embedded on the leading or on the lagging strand of DNA replication. This study guides future research on rNMP characteristics in human genomic samples carrying AGS mutations., Competing Interests: DECLARATION OF INTERESTS We have a patent related to this study: Storici, F., Hesselberth, J.R., and Koh, K. D. Methods to detect Ribonucleotides in deoxyribonucleic acids. GTRC-6522, 2013; U.S. 10,787,703 B1 Sep. 29, 2020. https://uspto.report/patent/grant/10,787,703

Insertion sequences are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance 1 . IS200- and IS605-family transposons undergo 'peel-and-paste' transposition catalysed by a TnpA transposase 2 , but they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively 3,4 . Recent studies have demonstrated that TnpB and IscB function as RNA-guided DNA endonucleases 5,6 , but the broader biological role of this activity has remained enigmatic. Here we show that TnpB and IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related insertion sequences from Geobacillus stearothermophilus that encode several TnpB and IscB orthologues, and showed that a single TnpA transposase was broadly active for transposon mobilization. The donor joints formed upon religation of transposon-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB and IscB nucleases, and co-expression of TnpB and TnpA led to substantially greater transposon retention relative to conditions in which TnpA was expressed alone. Notably, TnpA and TnpB also stimulated recombination frequencies, surpassing rates observed with TnpB alone. Collectively, this study reveals that RNA-guided DNA cleavage arose as a primal biochemical activity to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defence., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS 200 /IS 605 elements undergo 'peel-and-paste' transposition catalyzed by a TnpA transposase, but intriguingly, they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively. Recent studies demonstrated that TnpB-family enzymes function as RNA-guided DNA endonucleases, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB/IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related IS elements from Geobacillus stearothermophilus that encode diverse TnpB/IscB orthologs, and showed that a single TnpA transposase was active for transposon excision. The donor joints formed upon religation of IS-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB/IscB nucleases, and co-expression of TnpB together with TnpA led to significantly greater transposon retention, relative to conditions in which TnpA was expressed alone. Remarkably, TnpA and TnpB/IscB recognize the same AT-rich transposon-adjacent motif (TAM) during transposon excision and RNA-guided DNA cleavage, respectively, revealing a striking convergence in the evolution of DNA sequence specificity between collaborating transposase and nuclease proteins. Collectively, our study reveals that RNA-guided DNA cleavage is a primal biochemical activity that arose to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defense.

DNA double-stranded breaks (DSBs) are dangerous lesions threatening genomic stability. Fidelity of DSB repair is best achieved by recombination with a homologous template sequence. In yeast, transcript RNA was shown to template DSB repair of DNA. However, molecular pathways of RNA-driven repair processes remain obscure. Utilizing assays of RNA-DNA recombination with and without an induced DSB in yeast DNA, we characterize three forms of RNA-mediated genomic modifications: RNA- and cDNA-templated DSB repair (R-TDR and c-TDR) using an RNA transcript or a DNA copy of the RNA transcript for DSB repair, respectively, and a new mechanism of RNA-templated DNA modification (R-TDM) induced by spontaneous or mutagen-induced breaks. While c-TDR requires reverse transcriptase, translesion DNA polymerase ζ (Pol ζ) plays a major role in R-TDR, and it is essential for R-TDM. This study characterizes mechanisms of RNA-DNA recombination, uncovering a role of Pol ζ in transferring genetic information from transcript RNA to DNA., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Double strand-breaks (DSBs) of genomic DNA caused by ionizing radiation or mutagenic chemicals are a common source of mutation, recombination, chromosomal aberration, and cell death. Linker histones are DNA packaging proteins with established roles in chromatin compaction, gene transcription, and in homologous recombination (HR)-mediated DNA repair. Using a machine-learning model for functional prioritization of eukaryotic post-translational modifications (PTMs) in combination with genetic and biochemical experiments with the yeast linker histone, Hho1, we discovered that site-specific phosphorylation sites regulate HR and HR-mediated DSB repair. Five total sites were investigated (T10, S65, S141, S173, and S174), ranging from high to low function potential as determined by the model. Of these, we confirmed S173/174 are phosphorylated in yeast by mass spectrometry and found no evidence of phosphorylation at the other sites. Phospho-nullifying mutations at these two sites results in a significant decrease in HR-mediated DSB repair templated either with oligonucleotides or a homologous chromosome, while phospho-mimicing mutations have no effect. S65, corresponding to a mammalian phosphosite that is conserved in yeast, exhibited similar effects. None of the mutations affected base- or nucleotide-excision repair, nor did they disrupt non-homologous end joining or RNA-mediated repair of DSBs when sequence heterology between the break and repair template strands was low. More extensive analysis of the S174 phospho-null mutant revealed that its repression of HR and DSB repair is proportional to the degree of sequence heterology between DSB ends and the HR repair template. Taken together, these data demonstrate the utility of machine learning for the discovery of functional PTM hotspots, reveal linker histone phosphorylation sites necessary for HR and HR-mediated DSB repair, and provide insight into the context-dependent control of DNA integrity by the yeast linker histone Hho1., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.

Purpose: To evaluate the feasibility of robot-assisted retinal vein cannulation for retinal vein occlusion., Methods: Prospective experimental study performed in in vivo porcine eyes. A standard three port pars plana vitrectomy was followed by laser-induced branch retinal vein occlusion. Consequently, a retinal vein cannulation with the help of a surgical robot and a microneedle was performed. Complete success was defined as a stable intravenous position of the needle tip confirmed by blood washout for at least 3 min. Secondary outcomes were the occurrence of intra-operative complications and technical failures., Results: Cannulation was successful in 15 of 18 eyes with a complete success rate (duration of infusion of more than 3 min) of 73% after exclusion of two eyes from analysis due to failure in establishing a blood clot. There were no technical failures regarding the robotic device. The intravessel injections of ocriplasmin in two of two eyes led to a clot dissolution. In a subset of five eyes, a second cannulation attempt at the border of the optic disc resulted in a stable intravessel position and infusion during 362 (±138) seconds., Conclusion: Robot-assisted retinal vein cannulation with prolonged infusion time is technically feasible. Human experiments are required to analyse the clinical benefit of this new therapy., (© 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Purpose: To develop a methodology for cannulating porcine retinal venules using a robotic assistive arm after inducing a retinal vein occlusion using the photosensitizer rose bengal., Methodology: Retinal vein occlusions proximal to the first vascular branch point were induced following intravenous injection of rose bengal by exposure to 532nm laser light delivered by slit-lamp or endolaser probe. Retinal veins were cannulated by positioning a glass catheter tip using a robotically controlled micromanipulator above venules with an outer diameter of 80μm or more and performing a preset piercing maneuver, controlled robotically. The ability of a balanced salt (BSS) solution to remove an occlusion by repeat distention of the retinal vein was also assessed., Results: Cannulation using the preset piercing program was successful in 9 of 9 eyes. Piercing using the micromanipulator under manual control was successful in only 24 of 52 attempts, with several attempts leading to double piercing. The best location for cannulation was directly proximal to the occlusion. Infusion of BSS did not result in the resolution of the occlusion., Conclusion: Cannulation of venules using a robotic microassistive arm can be achieved with consistency, provided the piercing is robotically driven. The model appears robust enough to allow testing of therapeutic strategies aimed at eliminating a retinal vein thrombus and its evolution over time., Competing Interests: MDdS received grant support from TG and is a patent holder (without royalty) of ocriplasmin. He is also a founding member and chief medical officer of Preceyes NV.

Cells are continuously exposed to both endogenous and exogenous sources of genomic stress. To maintain chromosome stability, a variety of mechanisms have evolved to cope with the multitude of genetic abnormalities that can arise over the life of a cell. Still, failures to repair these lesions are the driving force of cancers and other degenerative disorders. DNA double-strand breaks (DSBs) are among the most toxic genetic lesions, inhibiting cell ability to replicate, and are sites of mutations and chromosomal rearrangements. DSB repair is known to proceed via two major mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR reliance on the exchange of genetic information between two identical or nearly identical DNA molecules offers increased accuracy. While the preferred substrate for HR in mitotic cells is the sister chromatid, this is limited to the S and G2 phases of the cell cycle. However, abundant amounts of homologous genetic substrate may exist throughout the cell cycle in the form of RNA. Considered an uncommon occurrence, the direct transfer of information from RNA to DNA is thought to be limited to special circumstances. Studies have shown that RNA molecules reverse transcribed into cDNA can be incorporated into DNA at DSB sites via a non-templated mechanism by NHEJ or a templated mechanism by HR. In addition, synthetic RNA molecules can directly template the repair of DSBs in yeast and human cells via an HR mechanism. New work suggests that even endogenous transcript RNA can serve as a homologous template to repair a DSB in chromosomal DNA. In this perspective, we will review and discuss the recent advancements in DSB repair by RNA via non-templated and templated mechanisms. We will provide current findings, models and future challenges investigating RNA and its role in DSB repair., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the process of RNA-driven DNA repair and recombination, and provide further data in support of our model of DSB repair by transcript RNA in cis. We show that a DSB is precisely repaired predominately by transcript RNA and not by residual cDNA in conditions in which formation of cDNA by reverse transcription is inhibited. Additionally, we demonstrate that defects in ribonuclease (RNase) H stimulate precise DSB repair by homologous RNA or cDNA sequence, and not by homologous DNA sequence carried on a plasmid. These results highlight an antagonistic role of RNase H in RNA-DNA recombination. Ultimately, we discuss several questions that should be addressed to better understand mechanisms and implications of RNA-templated DNA repair and recombination.

Relaxing the standard lung donor criteria may significantly increase the reported 15% organ yield but post-transplant recipient outcome should be carefully monitored. Charts from all consecutive deceased organ donors within our hospital network were reviewed over a 2-year period. Reasons for lung refusals and number of lungs transplanted were analysed. Hospital outcome including early recipient survival was compared between standard- and extended criteria donors. Out of 283 referrals, 164 (58%) qualified as donor of any organ. The majority (65.9%) of these effective donors were declined for lung donation because of chest X-ray abnormalities (20%), age >70 years (13%), poor oxygenation (10%), or aspiration (9%). Out of 56 (34.1%) accepted lung donors, 50 transplants were performed at our center, 23 from standard criteria donors versus 27 from extended criteria donors. There were no significant differences in hospital outcome and in early survival between lung recipients from both donor groups. Lung acceptance rate (34.1%) in our donor network is 10-20% higher than reported figures. The number of lung transplants in our center doubled by accepting extended criteria donors. This policy did not negatively influence our results after lung transplantation.

Background: The use of non-heart-beating donors (NHBD) to overcome organ shortage is moving into the clinic. In 2007, 5 of 51 lung transplantations (LTx) in our center were performed with lungs from controlled NHBD., Methods: Our aim was to describe these 5 NHBD LTx recipients and compare early outcome (

The determination of age on the basis of aspartic acid (Asp) racemization in teeth is one of the most reliable and accurate methods to date. In this paper, the usefulness of HPLC coupled with fluorescence detection for determination of Asp racemization was evaluated. A modified sample preparation is proposed for better stability of o-phthaldialdehyde-N-acetyl-L-cysteine derivatives of D/L-Asp (due to the instability below pH 7). To ensure the accuracy of the method, the validation parameters' specificity, precision, linearity, and LOD were determined. Three dentin samples of premolar teeth, extracted from living individuals (bucco-lingual longitudinal sections of 1 mm thickness), were analyzed and quantitative results are discussed.

The estimation of chronological age in cadavers, human remains and in living human beings by various methods is discussed. These methods, which are based on the age dependent non-enzymatic changes of l-form amino acids to d-form amino acids, mainly aspartic acid, are among the most reliable and accurate methods to date. Most of these methods use gas chromatography (GC). In this review, results of aspartic acid racemization in dentin at different targets are discussed. In addition, pre-considerations and guidelines are given for the selection of dentin from teeth. A pilot project was run to evaluate the efficiency of high performance liquid chromatography (HPLC) coupled with fluorescence detection. New buffer conditions were found to obtain stable derivatives of aspartic acid enantiomers for the estimation of racemization.

Impaired vascular refilling and declining plasma osmolality contribute to the incidence of complications during hemodialysis therapy. Interventions such as ultrafiltration and dialysate sodium profiling can be utilized to make dialysis treatments more effective and comfortable for patients. Selecting appropriate profiles requires careful clinical assessment and an understanding of the principles supporting these interventions. This paper describes fluid distribution and factors influencing vascular refilling during hemodialysis. The principles of ultrafiltration and dialysate sodium profiling are discussed and validated. Guidelines for clinical assessment and selection of profiles are presented.

Objective: Although earlier research has suggested that baseline prealbumin level is an independent predictor of outcome among dialysis patients, the prognostic importance of serial prealbumin levels is less clear. The present study had 3 objectives: first, to determine if prealbumin (a marker of visceral protein stores with a relatively short half-life) predicts subsequent albumin levels taken at least 1 month later; second, to examine the association between serial prealbumin levels and clinical outcome; and third, to examine the association between changes in prealbumin level and outcome., Design: The prognostic value of serial prealbumin levels was examined by linear regression analysis and Cox hazard models in an observational cohort study using a repeated measures design and time-dependent covariates., Setting: Patients were followed by a tertiary care center, receiving hemodialysis (HD; at either an in-center dialysis unit or one of several satellite units operated by the hospital) or home peritoneal dialysis (PD)., Patients: A retrospective cohort was identified consisting of 268 incident and prevalent chronic HD and PD patients receiving dialysis from June 1998 to September 1999., Main Outcome: The study examined the association between serial prealbumin measurements and future laboratory and clinical outcomes (albumin, hospitalization, and death)., Results: Serial prealbumin values were independent predictors of future albumin levels among HD patients (P =.04), but not PD patients. Independent predictors of hospitalization included diabetes for PD patients (P =.0012) and advanced age for HD patients (P =.0008). Advanced age and diabetes were independent predictors of death for both HD (P =.0001 and P =.0368) and PD patients (P =.0014 and P =.0164). Serial prealbumin values, measured as time-dependent covariates, did not predict hospitalization or death. Further analyses examined the prognostic value of changes in prealbumin and albumin values as time-dependent covariates. The final multivariate analysis identified low baseline albumin level as an independent predictor of hospitalization among HD patients (P =.0282), whereas low baseline prealbumin was an independent predictor of death for HD patients (P =.0001). Interestingly, negative changes in serial prealbumin measurements were also independent predictors of death among HD patients (P =.0025)., Conclusion: Serial prealbumin measurements predict subsequent albumin values among HD patients. As well, low baseline prealbumin level is an independent predictor of adverse outcome among HD patients. Although repeated prealbumin measurements in and of themselves were of no added prognostic value, falling prealbumin values identified by repeated measurements were additional independent predictors of death. These results support the clinical utility of regular prealbumin monitoring among HD patients., (Copyright 2001 by the National Kidney Foundation, Inc.)

The objective of this study was to determine if patient complications and nursing interventions during hemodialysis could be reduced using gradient ultrafiltration and gradient sodium dialysate. Twenty outpatients who had been on hemodialysis for at least 3 months, and using gradient sodium dialysate for at least 1 month, participated. Patients received either ultrafiltration at a constant hourly rate or gradient ultrafiltration, in which the ultrafiltration rate was set higher initially, then decreased step-wise mid-dialysis. Patients received each protocol for 3 months, using a randomized cross-over design. Both protocols used gradient sodium dialysate (150 mEq/L x 3 hrs, 140 mEq/L x 1 hr). There were significantly fewer complications and interventions using gradient ultrafiltration, as compared to constant ultrafiltration. No differences were found in interdialytic weight gain, intradialytic weight loss, or orthostatic blood pressure. These results indicate that gradient ultrafiltration combined with gradient sodium dialysate enhances patient well-being and reduces nursing interventions during hemodialysis.

Thrombus formation within hemodialysis catheters contributes to inadequate dialysis and adverse patient outcomes. A thrombolytic agent may be required to restore patency and improve blood flow. This study evaluates the efficacy of instilling low dose (1 mg/ml) t-PA in catheter lumens to restore patency in malfunctioning catheters. t-PA was utilized to treat suspected catheter thrombus over a four-month period. Seventeen patients with 21 catheters (12 temporary, 9 permanent) received 40 doses of t-PA. Catheter function was restored in 39 of 40 cases (97.5%). Significant improvement in blood flow was confirmed by paired t-test (p < 0.001). Sustained improvement in blood flow was confirmed by ANOVA (p < 0.001). The mean primary patency of all catheters was 29.7 days (SD = 27.0 days). No adverse patient effects were noted. These results demonstrate that t-PA can safely and effectively restore blood flow and extend patency in hemodialysis catheters.

As renal function declines, changes in mineral metabolism occur including phosphate retention, calcitriol deficiency, and the development of secondary hyperparathyroidism. Renal bone disease related to disordered mineral metabolism may result in increased patient morbidity and mortality. Uncontrolled parathyroid hormone (PTH) secretion will result in osteitis fibrosa, a high turnover bone disease. The use of calcium and aluminum-based phosphate binders and vitamin D sterols may contribute to the development of low turnover bone diseases such as osteomalacia and aplastic bone disease. Prevention and control of renal bone disease is an important goal for the interdisciplinary health care team. This paper discusses disordered mineral metabolism and its relationship to renal bone disease. Case studies illustrate the development and treatment of renal bone disease related to secondary hyperparathyroidism and aluminum intoxication.

Thrombus formation is a common cause of hemodialysis catheter malfunction. When thrombus or fibrin sheath restrict the flow of blood through one or both lumens, the catheter may need to be replaced. A less invasive, potentially lower cost option may be the instillation of low dose urokinase to degrade fibrin and restore catheter function. This study examines the efficacy of urokinase in improving blood flow and maintaining catheter patency. In a one-year period, urokinase was utilized in 25 dual lumen hemodialysis catheters (20 temporary, five permanent) in 22 patients. Blood flow and arterial and venous pressures were monitored before and after instillation. Urokinase administration successfully restored function in 20 catheters (80%). Paired t-tests demonstrated a significant improvement in blood flow and arterial pressure (p < 0.01) following urokinase. Catheter patency was extended for a mean of 18.0 days (range 0-90 days). The cost effectiveness of urokinase was evaluated in terms of direct costs, such as the cost of urokinase or materials to replace catheters, and indirect costs such as nursing and physician time and delays in dialysis scheduling. The results of this study suggest that judicious use of urokinase is a cost-effective, non-invasive method of restoring blood flow and extending patency in hemodialysis catheters.

Patient autonomy, sense of control, and well-being are thought to be enhanced by self-care hemodialysis as a therapy for end-stage renal disease. Dialysis in a satellite setting reduces travel time and can diminish therapy intrusiveness. Health-related quality of life (HRQOL), in terms of functional status and well-being, was measured in a group of patients trained for self-care, and then measured again after these patients were transferred to a satellite unit. Comparison was made with an age- and comorbidity-matched cohort of full-care patients. Patients trained for self-care tended to score higher than the full-care patients in the psychosocial domains of HRQOL, such as role function, social function, and emotional well-being, before and after transfer to the satellite unit. Physiological measurements did not differ significantly between groups at any time during the study, indicating that differences in HRQOL were not attributable to differences in metabolic stability. We conclude that patients trained for self-care hemodialysis experience better subjective quality of life than their full-care counterparts. This study highlights both the usefulness of measuring HRQOL as an outcome of hemodialysis therapy and the potential benefits of therapies such as self-care and satellite dialysis.

Objectives: To determine the feasibility and practicality of measuring general health status (GHS) in an outpatient peritoneal dialysis population. To determine whether GSH correlated intuitively with biochemical, socio-demographic and co-morbidity measurements., Design: The Medical Outcomes Study 20-item short form was administered on a voluntary basis in the outpatient setting. Demographic and current biochemical data were extracted from the medical record. The effects of the socio-demographic, biochemical and physiologic variables on the six subscales of GHS generated by the questionnaire were estimated using multivariate linear regression analysis resulting in the development of six separate models., Setting: Peritoneal dialysis program of a University Hospital., Patients: Sixty stable patients on home peritoneal dialysis completed the GHS questionnaire during regularly scheduled outpatient visits. Ages ranged from 13 to 81 years. The study group included 14 diabetics (23%)., Results: Administering the questionnaire caused no logistical difficulties in the outpatient setting. Regression models for predicting GHS were both significant and intuitively correct. The presence of co-morbidities such as diabetes mellitus (p = 0.002; Social Subscale) and peripheral vascular disease (p = 0.016: General Health Subscale) had the most significant negative impact on GHS. An increased length of time on dialysis was associated with a higher GHS (p = 0.002; Physical Subscale)., Conclusion: General Health Status questionnaires can be readily administered to peritoneal dialysis patients in the outpatient setting. They have face validity as a measurement of wellness and functioning. The longitudinal use of such instruments in conjunction with clinical and laboratory findings may identify both medical and non-medical factors impacting on our peritoneal dialysis population.

Assessment of biochemical responses to therapy is routine in the management of patients with end stage renal disease (ESRD). Assessment of health-related quality of life (HRQOL), however, is less common. Previous research indicates that HRQOL is a meaningful indicator that should be integrated into clinical practice. HRQOL is longitudinally evaluated in in-centre hemodialysis patients using the RAND 36-item Health Survey 1.0. Caregivers incorporate scores from this instrument into their assessment of patient functioning and well-being. HRQOL scores can be utilized to evaluate responses to changes in therapy, and to direct clinical decision-making, adding an important dimension to holistic, quality care for ESRD patients.

The diversity and complexity of nephrology is increasing and specialized knowledge and skills are required to practise this subspeciality. As a result, specialization and certification by the Canadian Nurses' Association (CNA) in nephrology nursing has become a reality. Specialization benefits patients, nurses and employers by ensuring high professional standards, providing an increased sense of professional achievement and innovative ways of providing quality care. Maintaining these high standards requires continuing education and recertification. A number of educational opportunities meet these needs. Recognition of speciality services would motivate more nurses to commit to nephrology certification. Specialization demands a greater commitment from the health care system, but provides significant dividends to both the health care system and the patient.

To test the feasibility of using general health status as a practical dialysis outcome measure, in a longitudinal pilot study, two well-validated instruments were administered to patients from our dialysis unit or clinic. The instruments were administered three times, at eight-week intervals for seven months to 41 hemodialysis (HD) patients. Forty-five transplant (Tx) patients were surveyed once as a validating control. Sociodemographic, imaging and biochemical data were tested as outcome predictors. Unexpected hospitalizations and adverse intercurrent events were used as additional outcome measures. Preliminary analysis shows HD patients scoring low, while Tx patients scored higher (healthier). These preliminary results suggest that assessment of general health status is a valid and practical outcome measure.

Noncompliance is a nursing diagnosis frequently encountered by nephrology nurses in their practice. Patient assessment, as part of the nursing process, gives us insight into the numerous factors contributing to noncompliance. An assessment approach has been developed to assist nurses in acquiring the knowledge, empathy and understanding to plan effective care for the noncompliant patient.