Novel 3D printable PEEK-HA-Mg2SiO4 composite material for spine implants: biocompatibility and imaging compatibility assessments.

PURPOSE: To develop a novel 3D printable polyether ether ketone (PEEK)-hydroxyapatite (HA)-magnesium orthosilicate (Mg2SiO4) composite material with enhanced properties for potential use in tumour, osteoporosis and other spinal conditions. We aim to evaluate biocompatibility and imaging compatibility of the material. METHODS: Materials were prepared in three different compositions, namely composite A: 75 weight % PEEK, 20 weight % HA, 5 weight % Mg2SiO4; composite B: 70 weight% PEEK, 25 weight % HA, 5 weight % Mg2SiO4; and composite C: 65 weight % PEEK, 30 weight % HA, 5 weight % Mg2SiO4. The materials were processed to obtain 3D printable filament. Biomechanical properties were analysed as per ASTM standards and biocompatibility of the novel material was evaluated using indirect and direct cell cytotoxicity tests. Cell viability of the novel material was compared to PEEK and PEEK-HA materials. The novel material was used to 3D print a standard spine cage. Furthermore, the CT and MR imaging compatibility of the novel material cage vs PEEK and PEEK-HA cages were evaluated using a phantom setup. RESULTS: Composite A resulted in optimal material processing to obtain a 3D printable filament, while composite B and C resulted in non-optimal processing. Composite A enhanced cell viability up to ~ 20% compared to PEEK and PEEK-HA materials. Composite A cage generated minimal/no artefacts on CT and MR imaging and the images were comparable to that of PEEK and PEEK-HA cages. CONCLUSION: Composite A demonstrated superior bioactivity vs PEEK and PEEK-HA materials and comparable imaging compatibility vs PEEK and PEEK-HA. Therefore, our material displays an excellent potential to manufacture spine implants with enhanced mechanical and bioactive property.

Design and 3D printing of novel titanium spine rods with lower flexural modulus and stiffness profile with optimised imaging compatibility.

PURPOSE: To manufacture and test 3D printed novel design titanium spine rods with lower flexural modulus and stiffness compared to standard solid titanium rods for use in metastatic spine tumour surgery (MSTS) and osteoporosis. METHODS: Novel design titanium spine rods were designed and 3D printed. Three-point bending test was performed to assess mechanical performance of rods, while a French bender was used to assess intraoperative rod contourability. Furthermore, 3D printed spine rods were tested for CT & MR imaging compatibility using phantom setup. RESULTS: Different spine rod designs generated includes shell, voronoi, gyroid, diamond, weaire-phelan, kelvin, and star. Tests showed 3D printed rods had lower flexural modulus with reduction ranging from 2 to 25% versus standard rod. Shell rods exhibited highest reduction in flexural modulus of 25% (~ 77.4 GPa) and star rod exhibited lowest reduction in flexural modulus of 2% (100.8GPa). 3D printed rod showed reduction in stiffness ranging from 40 to 59%. Shell rod displayed highest reduction in stiffness of 59% (179.9 N/mm) and gyroid had least reduction in stiffness of 40% (~ 259.2 N/mm). Rod bending test showed that except gyroid, other rod designs demonstrated lesser bending difficulty versus standard rod. All 3D printed rods demonstrated improved CT/MR imaging compatibility with reduced artefacts versus standard rod. CONCLUSION: By utilising novel design approach, we successfully generated a spine rod design portfolio with lower flexural modulus/stiffness profile and better CT/MR imaging compatibility for potential use in MSTS/other conditions such as osteoporosis. Thus, exploration of new rod designs in surgical application could enhance treatment outcome and improve quality of life for patients.

Thermo-Responsive Hydrogel-Based Soft Valves with Annular Actuation Calibration and Circumferential Gripping.

Valves are largely useful for treatment assistance devices, e.g., supporting fluid circulation movement in the human body. However, the valves presently used in biomedical applications still use materials that are rigid, non-compliant, and hard to integrate with human tissues. Here, we propose biologically-inspired, stimuli-responsive valves and evaluate N-Isopropylacrylamide hydrogels-based valve (NPHV) and PAAm-alginate hydrogels-based valve (PAHV) performances with different chemical syntheses for optimizing better valve action. Once heated at 40 ∘C, the NPHV outperforms the PAHV in annular actuation (NPHV: 1.93 mm displacement in 4 min; PAHV: 0.8 mm displacement in 30 min). In contrast, the PAHV exhibits a flow rate change of up to 20%, and a payload of 100% when the object is at 100 ∘C. The PAHV demonstrated a completely soft, stretchable circular gripper with a high load-to-weight ratio for diversified applications. These valves are fabricated with a simple one-pot method that, once further optimized, can offer transdisciplinary applications.

Evolution of materials for implants in metastatic spine disease till date - Have we found an ideal material?

"Metastatic Spine Disease" (MSD) often requires surgical intervention and instrumentation with spinal implants. Ti6Al4V is widely used in metastatic spine tumor surgery (MSTS) and is the current implant material of choice due to improved biocompatibility, mechanical properties, and compatibility with imaging modalities compared to stainless steel. However, it is still not the ideal implant material due to the following issues. Ti6Al4V implants cause stress-shielding as their Young's modulus (110 gigapascal [GPa]) is higher than cortical bone (17-21 GPa). Ti6Al4V also generates artifacts on CT and MRI, which interfere with the process of postoperative radiotherapy (RT), including treatment planning and delivery. Similarly, charged particle therapy is hindered in the presence of Ti6Al4V. In addition, artifacts on CT and MRI may result in delayed recognition of tumor recurrence and postoperative complications. In comparison, polyether-ether-ketone (PEEK) is a promising alternative. PEEK has a low Young's modulus (3.6 GPa), which results in optimal load-sharing and produces minimal artifacts on imaging with less hinderance on postoperative RT. However, PEEK is bioinert and unable to provide sufficient stability in the immediate postoperative period. This issue may possibly be mitigated by combining PEEK with other materials to form composites or through surface modification, although further research is required in these areas. With the increasing incidence of MSD, it is an opportune time for the development of spinal implants that possess all the ideal material properties for use in MSTS. Our review will explore whether there is a current ideal implant material, available alternatives and whether these require further investigation.

Readmission-Free Survival Analysis in Metastatic Spine Tumour Surgical Patients: A Novel Concept.

BACKGROUND: Outcomes commonly used to ascertain success of metastatic spine tumour surgery (MSTS) are 30-day complications/mortality and overall/disease-free survival. We believe a new, effective outcome indicator after MSTS would be the absence of unplanned hospital readmission (UHR) after index discharge. We introduce the concept of readmission-free survival (ReAFS), defined as 'the time duration between hospital discharge after index operation and first UHR or death'. The aim of this study is to identify factors influencing ReAFS in MSTS patients. PATIENTS AND METHODS: We retrospectively analysed 266 consecutive patients who underwent MSTS between 2005 and 2016. Demographics, oncological characteristics, procedural, preoperative and postoperative details were collected. ReAFS of patients within 2 years or until death was reviewed. Perioperative factors predictive of reduced ReAFS were evaluated using multivariate regression analysis. RESULTS: Of 266 patients, 230 met criteria for analysis. A total of 201 had UHR, whilst 1 in 8 (29/230) had no UHR. Multivariate analysis revealed that haemoglobin ≥ 12 g/dL, ECOG score of ≤ 2, primary prostate, breast and haematological cancers, comorbidities ≤ 3, absence of preoperative radiotherapy and shorter postoperative length of stay significantly prolonged the time to first UHR. CONCLUSIONS: Readmission-free survival is a novel concept in MSTS, which relies on patients' general condition, appropriateness of interventional procedures and underlying disease burden. Additionally, it may indicate the successful combination of a multi-disciplinary treatment approach. This information will allow oncologists and surgeons to identify patients who may benefit from increased surveillance following discharge to increase ReAFS. We envisage that ReAFS is a concept that can be extended to other surgical oncological fields.

Can Polyether Ether Ketone Dethrone Titanium as the Choice Implant Material for Metastatic Spine Tumor Surgery?

Instrumentation during metastatic spine tumor surgery (MSTS) provides stability to the spinal column in patients with pathologic fracture or iatrogenic instability produced while undergoing extensive decompression. Titanium is the current implant material of choice in MSTS. However, it hinders radiotherapy planning and generates artifacts, with magnetic resonance imaging and computed tomography scans used for postoperative evaluation of tumor recurrence and/or complications. The high modulus of elasticity of titanium (110 GPa) results in stress shielding, which may lead to construct failure at the bone-implant interface. Polyether ether ketone (PEEK), a thermoplastic polymer, is an emerging alternative to titanium for use in MSTS. The modulus of elasticity of PEEK (3.6 GPa) is close to that of cortical bone (17-21 GPa), resulting in minimal stress shielding. Its radiolucent and nonmetallic properties cause minimal interference with magnetic resonance imaging and computed tomography scans. PEEK also causes low-dose perturbation for radiotherapy planning. However, PEEK has reduced bioactivity with bone and lacks sufficient rigidity to be used as rods in MSTS. The reduced bioactivity of PEEK may be addressed by 1) surface modification (introducing porosity or bioactive coating with hydroxyapatite [HA] or titanium) and 2) forming composites with HA/titanium. The mechanical properties of PEEK may be improved by forming composites with HA or carbon fiber. Despite these modifications, all PEEK and PEEK-based implants are difficult to handle and contour intraoperatively. Our review provides a comprehensive overview of PEEK and modified PEEK implants, with a description of their properties and limitations, potentially serving as a basis for their future development and use in MSTS.

The "Spinal Metastasis Invasiveness Index": A Novel Scoring System to Assess Surgical Invasiveness.

STUDY DESIGN: Retrospective review. OBJECTIVE: The aim of this study was to develop a surgical invasiveness index for metastatic spine tumor surgery (MSTS) that can serve as a standardized tool in predicting intraoperative blood loss and surgical duration; for the purpose of ascertaining resource requirements and aiding in patient education. SUMMARY OF BACKGROUND DATA: Magnitude of surgery is important in the metastatic spine disease (MSD) population since these patients have a continuing postoperative oncological process; a consideration that must be taken into account to maintain or improve quality of life. Surgical invasiveness indices have been established for general spine surgery, adult deformity, and cervical deformity, but not yet for spinal metastasis. METHODS: Demographic, oncological, and procedural data were collected from consecutive patients that underwent MSTS. Binary logistic regression, using median values for surgical duration and intraoperative estimated blood loss (EBL), was used to determine statistical significance of variables to be included in the "spinal metastasis invasiveness index" (SMII). The corresponding weightage of each of these variables was agreed upon by experienced spine surgeons. Multivariable regression analysis was used to predict operative time and EBL while controlling for demographical, procedural, and oncological characteristics. RESULTS: Two hundred and sixty-one MSD patients were included with a mean age of 59.7-years and near equal sex distribution. The SMII strongly predicted extended surgical duration (R2 = 0.28, P < 0.001) and high intraoperative blood loss (R2 = 0.18, P < 0.001). When compared to a previously established surgical invasiveness index, the SMII accounted for more variability in the outcomes. For every unit increase in score, there was a 42-mL increase in mean blood loss (P < 0.001) and 5-minute increase in mean operative time (P < 0.001). CONCLUSION: Long surgical duration and high blood loss were strongly predicted by the newly developed SMII. The use of the SMII may aid in preoperative risk assessment with the goal of improving patient outcomes and quality of life.Level of Evidence: 4.

Asymptomatic Construct Failure after Metastatic Spine Tumor Surgery: A New Entity or a Continuum with Symptomatic Failure?

STUDY DESIGN: Retrospective cohort study. PURPOSE: To study the incidence, onset, underlying mechanism, clinical course, and factors leading to asymptomatic construct failure (AsCF) after metastatic spinal tumor surgery (MSTS). OVERVIEW OF LITERATURE: The reported incidence rates for implant and/or construct failure after MSTS are low (1.9%-16%) and based on clinical presentations and revisions required for symptomatic failures (SFs). AsCF after MSTS has not been reported. METHODS: We conducted a retrospective analysis of 288 patients (246 for final analysis) who underwent MSTS between 2005-2015. Data collected were demographics and peri/postoperative clinical and radiological features. Early and late radiological AsCF were defined as presentation before and after 3 months, respectively. We analyzed patients with AsCF for risk factors and survival duration by performing competing risk regression analyses where AsCF was the event of interest, with SF and death as competing events. RESULTS: We observed AsCF in 41/246 patients (16.7%). The mean time to onset of AsCF after MSTS was 2 months (range, 1-9 months). Median survival of patients with AsCF was 20 and 41 months for early and late failures, respectively. Early AsCF accounted for 80.5% of cases, while late AsCF accounted for 19.5%. The commonest radiologically detectable AsCF mechanism was angular deformity (increase in kyphus) in 29 patients. Increasing age (p<0.02) and primary breast (13/41, 31.7%) (p<0.01) tumors were associated with higher AsCF rates. There was a non-significant trend towards AsCF in patients with a spinal instability neoplastic score ≥7, instrumentation across junctional regions, and construct lengths of 6-9 levels. None of the patients with AsCF underwent revision surgery. CONCLUSIONS: AsCF after MSTS is a distinct entity. Most patients with early AsCF did not require intervention. Patients who survived and maintained ambulation for longer periods had late failure. Increasing age and tumors with a better prognosis have a higher likelihood of developing AsCF. AsCF is not necessarily an indication for aggressive/urgent intervention.

Asymptomatic Construct Failure after Metastatic Spine Tumor Surgery: A New Entity or a Continuum with Symptomatic Failure?

Methods@#We conducted a retrospective analysis of 288 patients (246 for final analysis) who underwent MSTS between 2005–2015. Data collected were demographics and peri/postoperative clinical and radiological features. Early and late radiological AsCF were defined as presentation before and after 3 months, respectively. We analyzed patients with AsCF for risk factors and survival duration by performing competing risk regression analyses where AsCF was the event of interest, with SF and death as competing events. @*Results@#We observed AsCF in 41/246 patients (16.7%). The mean time to onset of AsCF after MSTS was 2 months (range, 1–9 months). Median survival of patients with AsCF was 20 and 41 months for early and late failures, respectively. Early AsCF accounted for 80.5% of cases, while late AsCF accounted for 19.5%. The commonest radiologically detectable AsCF mechanism was angular deformity (increase in kyphus) in 29 patients. Increasing age (p<0.02) and primary breast (13/41, 31.7%) (p<0.01) tumors were associated with higher AsCF rates. There was a non-significant trend towards AsCF in patients with a spinal instability neoplastic score ≥7, instrumentation across junctional regions, and construct lengths of 6–9 levels. None of the patients with AsCF underwent revision surgery. @*Conclusions@#AsCF after MSTS is a distinct entity. Most patients with early AsCF did not require intervention. Patients who survived and maintained ambulation for longer periods had late failure. Increasing age and tumors with a better prognosis have a higher likelihood of developing AsCF. AsCF is not necessarily an indication for aggressive/urgent intervention.

In the Extraordinary Times of Coronavirus Disease 2019: Clinical Strategies for Performing Spinal Surgery.

The coronavirus disease 2019 (COVID-19) pandemic has caused pronounced strain on global healthcare systems, forcing the streamlining of clinical activities and conservation of health resources. There is a pressing need for institutions to present discipline-specific strategies for the management of COVID-19 patients. We present the comprehensive considerations at the National University Hospital, Singapore from the surgeon's and anesthetist's perspectives in the performance of spinal surgery in COVID-19 patients. These are based on national guidelines and overarching principles of protection for the healthcare workers (HCWs) and efficiency in surgical planning. The workflow begins with the emergency department screening that has been adapted to the local epidemiology of COVID-19 in order to identify suspected/confirmed cases. If patient history cannot be obtained, demographic, clinical, and imaging data are used. Designated orthopedic "contaminated teams" are available 24/7 with an activation time of <30 minutes for review. In cases where sub-specialty spine surgeons were required, these professionals were inducted into the "contaminated team" and quarantined until cleared to return to work. Indications for emergency spine surgery were determined pre-emptively. Preoperative surgical considerations included the minimization of manpower, limited dissection, reduced operative time, and judicious use of equipment, leading to reduced aerosolization. Anesthesia considerations include preoperative screening for COVID-19-related concerns that influence surgery, operating room process planning and induction, intraoperative, reversal, recovery, and resuscitation considerations. Focused multi-disciplinary preoperative briefing facilitates familiarization. Surgical, anesthetic, and postoperative workflows were designed to reduce the risk of transmission and protect HCWs while effectively performing spinal surgery. The COVID-19 pandemic has necessitated paradigm shifts in healthcare planning, hospital workflows, and operative protocols. The viral burden does not discriminate between surgeons and physicians, and it is crucial that we, as medical professionals, adapt practices to be malleable and fluid to address the ever-changing developments.

Is there an optimal timing between radiotherapy and surgery to reduce wound complications in metastatic spine disease? A systematic review.

PURPOSE: Surgery with radiation therapy (RT) is more effective in treating spinal metastases, than RT alone. However, RT when administered in close proximity to surgery may predispose to wound complications. There exist limited guidelines on the optimal timing between RT and surgery. The purpose of this systematic review is to: (1) address whether pre-operative RT (preop-RT) and/or post-operative RT (postop-RT) is associated with wound complications and (2) define the safe interval between RT and surgery or vice versa. METHODS: PubMed, Embase and Scopus databases were systematically searched for articles dealing with spinal metastases, treated with surgery and RT, and discussing wound status. RESULTS: We obtained 2332 articles from all databases, and after applying exclusion criteria, removing duplicates and reading the full text, we identified 27 relevant articles. Fourteen additional articles were identified by hand-search, leading to a total of 41 articles. All 41 mentioned wound complications/healing. Sixteen articles discussed preop-RT, 8 postop-RT, 15 both, and 2 mentioned intraoperative-RT with additional pre/postop-RT. Twenty studies mentioned surgery-RT time interval; one concluded that wound complications were higher when RT-surgery interval was ≤ 7 days. Seven studies reported significant association between preop-RT and wound complications. CONCLUSIONS: Evidence is insufficient to draw definitive conclusion about optimal RT-surgery interval. However, based on published literature and expert opinions, we conclude that an interval of 2 weeks, the minimum being 7 days, is optimum between RT-surgery or vice versa; this can be reduced further by postop-stereotactic body RT. If RT-surgery window is > 12 months, wound-complications rise. Postop-RT has fewer wound complications versus preop-RT.

In the Extraordinary Times of Coronavirus Disease 2019: Clinical Strategies for Performing Spinal Surgery

The coronavirus disease 2019 (COVID-19) pandemic has caused pronounced strain on global healthcare systems, forcing the streamlining of clinical activities and conservation of health resources. There is a pressing need for institutions to present discipline-specific strategies for the management of COVID-19 patients. We present the comprehensive considerations at the National University Hospital, Singapore from the surgeon’s and anesthetist’s perspectives in the performance of spinal surgery in COVID-19 patients. These are based on national guidelines and overarching principles of protection for the healthcare workers (HCWs) and efficiency in surgical planning. The workflow begins with the emergency department screening that has been adapted to the local epidemiology of COVID-19 in order to identify suspected/confirmed cases. If patient history cannot be obtained, demographic, clinical, and imaging data are used. Designated orthopedic “contaminated teams” are available 24/7 with an activation time of

Identification of modifications in microbial, native tRNA that suppress immunostimulatory activity.

Naturally occurring nucleotide modifications within RNA have been proposed to be structural determinants for innate immune recognition. We tested this hypothesis in the context of native nonself-RNAs. Isolated, fully modified native bacterial transfer RNAs (tRNAs) induced significant secretion of IFN-α from human peripheral blood mononuclear cells in a manner dependent on TLR7 and plasmacytoid dendritic cells. As a notable exception, tRNA(Tyr) from Escherichia coli was not immunostimulatory, as were all tested eukaryotic tRNAs. However, the unmodified, 5'-unphosphorylated in vitro transcript of tRNA(Tyr) induced IFN-α, thus revealing posttranscriptional modifications as a factor suppressing immunostimulation. Using a molecular surgery approach based on catalytic DNA, a panel of tRNA(Tyr) variants featuring differential modification patterns was examined. Out of seven modifications present in this tRNA, 2'-O-methylated G(m)18 was identified as necessary and sufficient to suppress immunostimulation. Transplantation of this modification into the scaffold of yeast tRNA(Phe) also resulted in blocked immunostimulation. Moreover, an RNA preparation of an E. coli trmH mutant that lacks G(m)18 2'-O-methyltransferase activity was significantly more stimulatory than the wild-type sample. The experiments identify the single methyl group on the 2'-oxygen of G(m)18 as a natural modification in native tRNA that, beyond its primary structural role, has acquired a secondary function as an antagonist of TLR7.

PARP-1 activity in normal and cancerous human endometrium and its relationship with quantity of abasic sites (AP).

OBJECTIVES: Poly (ADP-ribose) polymerase (PARP-1) is involved in the processes of DNA repair contributing to the maintenance of genomic stability. Recent data suggest that polymerase is involved in the development of endometrial adenocarcinomas and more advanced tumors displaying lowest enzyme protein expression. Data on PARP-1 activity regarding carcinogenesis in human endometrium are scarce. That was the reason why the authors of the present work wished to investigate the enzyme activity in human uterine hormone-dependent cancer and to compare the results with those obtained for normal endometrial tissue. The next aim was to check whether enzyme activity in normal and cancerous endometrium depends on the number of AP sites, which are widely known as oxidative stress DNA damage markers and PARP-1 activity stimulators. MATERIAL AND METHODS: Universal Colorimetric PARP Assay Kit was used to estimate the enzyme activity in units/ mg protein. Apurinic sites/105 base pairs (bp) were measured by Oxidative DNA Damage Kit Quantitative. Results were calculated for 47 endometrial samples and 15 uterine adenocarcinomas specimens. Finally the PARP-1 activity was analyzed for histological and some clinical features of neoplasms. RESULTS AND CONCLUSIONS: 1. no differences in PARP-1 activity were found in non-cancerous types of human endometrium; 2. mean enzyme activity was lower in sporadic endometrial cancers than in noncancerous endometrial specimens (2.89 +/- 0.55 vs 6.39 +/- 0.06; p < 0.005); 3. mean PARP-1 activity in lower grade neoplasms was higher than in G3 tumors and was lower in adenocarcinomas displaying deep uterine wall infiltration; 4. there was no relationship between PARP-1 activity and AP level.

DNA-adducts in subjects exposed to urban air pollution by benzene and polycyclic aromatic hydrocarbons (PAHs) in Cotonou, Benin.

Air pollution effect on humans represents a major public health problem. Exposure to genotoxic compounds in the ambient air is evaluated using different biomarkers. In the present study we assessed DNA-adducts levels in apparently healthy people living and working in the city of Cotonou (Benin) in which exposure to air pollutants such as benzene and polycyclic aromatic hydrocarbons (PAHs) mainly benzo(a)pyrene has been evidenced. Rural inhabitants were enrolled as control group. Taxi-motorbike drivers, street food vendors, and gasoline salesmen were recruited in Cotonou whereas suburban residents were recruited in Godomey, 12 km from Cotonou. We found that taxi-motorbike drivers, roadside residents, street vendors, taxi-motor-bike drivers and gasoline sellers had significantly higher levels of DNA-adducts than suburban and village inhabitants (P < 0.001; post hoc, LSD). Means values were 24.6 ± 6.4, 23.78 ± 6.9, 34.7 ± 9.8, and 37.2 ± 8.1 in the exposed groups versus 2.1 ± 0.6 and 3.1 ± 0.8 adducts/10(8) nucleotides, in the two control groups, respectively. We did not find any significant difference within the high exposure groups and inside low exposure subgroups (namely suburban residents and villagers) because the mean individual exposure values to both PAHs and benzene were similar among subjects exposed in the city of Cotonou and those in suburban and village areas. However, there is significant interindividual variations in adducts levels that may reflect variation of genetic susceptibility factors. Ranges of adduct level/10(8) nucleotides were: 1-69, 1-76, 3-169, 4-124, 0-9, 0-8 adducts/10(8) for taxi-motorbike drivers, roadside residents, street vendors, gasoline sellers, suburban and village inhabitants, respectively. Our study demonstrated a clear-cut elevated level of DNA adducts in city residents than in none exposed people (or very low exposure levels people) and designate these city residents groups as people at risks for the chronic diseases possibly caused by benzene and PAHs.

Detection of enzymatic activity of transfer RNA modification enzymes using radiolabeled tRNA substrates.

The presence of modified ribonucleotides derived from adenosine, guanosine, cytidine, and uridine is a hallmark of almost all cellular RNA, and especially tRNA. The objective of this chapter is to describe a few simple methods that can be used to identify the presence or absence of a modified nucleotide in tRNA and to reveal the enzymatic activity of particular tRNA-modifying enzymes in vitro and in vivo. The procedures are based on analysis of prelabeled or postlabeled nucleotides (mainly with [(32)P] but also with [(35)S], [(14)C] or [(3)H]) generated after complete digestion with selected nucleases of modified tRNA isolated from cells or incubated in vitro with modifying enzyme(s). Nucleotides of the tRNA digests are separated by two-dimensional (2D) thin-layer chromatography on cellulose plates (TLC), which allows establishment of base composition and identification of the nearest neighbor nucleotide of a given modified nucleotide in the tRNA sequence. This chapter provides useful maps for identification of migration of approximately 70 modified nucleotides on TLC plates by use of two different chromatographic systems. The methods require only a few micrograms of purified tRNA and can be run at low cost in any laboratory.

An efficient mammalian transfer RNA target for bleomycin.

The antitumor antibiotic bleomycin has long been believed to exert its therapeutic effects at the level of DNA cleavage. Recently, evidence has been presented to suggest that RNA cleavage may also be important and that one or more transfer RNAs may be involved. To define those tRNAs that may represent important loci for the action of bleomycin, we have fractionated chicken liver tRNAs and identified those isoacceptors most susceptible to oxidative cleavage by Fe(II).BLM. Two chicken liver tRNAs, tRNA3Lys and tRNAPhe, were found to be cleaved with exceptional facility by Fe(II).BLM, and both were cleaved predominantly at U66. The cleavage of tRNA3Lys was shown to be minimally affected by physiological concentrations of Mg2+. Chicken liver tRNA3Lys is identical in sequence with human tRNA3Lys. These findings support a possible role for a critical tRNA such as tRNA3Lys in the mechanism by which bleomycin mediates its antitumor activity.

Genotoxic effects of bitumen fumes in Big Blue transgenic rat lung.

Road paving workers are exposed to bitumen fumes (CAS No. 8052-42-4), a complex mixture of volatile compounds and particles containing carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons. However, epidemiological and experimental animal studies failed to draw unambiguous conclusions concerning their toxicity. In order to gain better insights on their genotoxic potential, we used an experimental design able to generate bitumen fumes at road paving temperature (temperature: 170 degrees C, total particulate matter: 100mg/m3) and perform a nose-only exposure of Big Blue transgenic rodents 6h/day for five consecutive days. The mutagenic properties of bitumen fumes were determined by analyzing the mutation frequency and spectrum of the neutral reporter gene cII inserted into the rodent genome. We previously observed in mouse lung, that bitumen fumes did not induce an increase of cII mutants, a modification of the mutation spectrum, nor the formation of DNA adducts. Since DNA adducts were found in the lungs of rats exposed to asphalt fumes in similar conditions, we decided to carry out an analogous experiment with Big Blue rats. A DNA adduct was detected 3 and 30 days after the end of treatment suggesting that these genetic alterations were quite steady. Thirty days after exposure, the cII mutant frequency was similar in control and exposed rats. In addition, a slight but not significant modification of the mutation spectrum associated with an increase of G:C to T:A and A:T to C:G transversions was noticeable in the treated animals. Then, these data failed to demonstrate a pulmonary mutagenic potential for bitumen fumes generated at road paving temperature in our experimental conditions despite the presence of a DNA adduct. These results may provide information concerning the pulmonary mechanism of action of this aerosol and may contribute to the occupational health hazard assessment.

[Asbestos and malignant pleural mesothelioma: molecular, cellular and physiopathological aspects]. / Amiante et mésothéliome pleural malin: aspects moléculaires, cellulaires et physiopathologiques.

Asbestos is known as mutagenic and carcinogenic for human and is responsible for many pulmonary diseases including asbestosis, bronchogenic carcinoma and malignant pleural mesothelioma. Occupational exposure to asbestos is involved in 70-80% of all malignant pleural mesothelioma. The later presents a growing challenge for both researcher and clinician. The diagnosis of malignant pleural mesothelioma is difficult and the current treatments did not show significant improvement of the survival. The increasing incidence of malignant pleural mesothelioma, its gravity and its human, social and financial consequences are of high concern in public health. In this paper we summarize the so far knowledge on cellular, molecular and pathophysiological events involved in genesis and development of malignant pleural mesothelioma. Finally, the paper also report recent data sourced from the study of malignant pleural mesothelioma transcriptome using high-throughput technologies such as gene expression array. These data should improve the accuracy of mesothelioma diagnosis and therapy.

A new yeast poly(A) polymerase complex involved in RNA quality control.

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNAMet (tRNAiMet). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNAiMet with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNAiMet by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover.