Predicting Complications in 153 Lumbar Pedicle Subtraction Osteotomies by a Single Surgeon Over a 6-Year Period.

INTRODUCTION: Pedicle subtraction osteotomy (PSO) is a complex surgical procedure that provides correction of moderate sagittal imbalance. Surgical complications have adverse effects on patient outcomes and healthcare costs, making it imperative for clinical researchers to focus on minimizing complications. However, when it comes to risk modeling of PSO surgery, there is currently no consensus on which patient characteristics or measures should be used. This study aimed to describe complications and compare the performance of various sociodemographic characteristics, surgical variables, and established risk indices in predicting postoperative complications, infections, and readmissions after lumbar PSO surgeries. METHODS: A review was conducted on 191 patients who underwent PSO surgery at a single institution by a single fellowship-trained orthopaedic spine surgeon between January 1, 2018, and December 31, 2021. Demographic, intraoperative, and postoperative data within 30 days, 1 year, and 2 years of the index procedure were evaluated. Descriptive statistics, t -test, chi-squared analysis, and logistic regression models were used. RESULTS: Intraoperative complications were significantly associated with coronary artery disease (odds ratios [OR] 3.95, P = 0.03) and operating room time (OR 1.01, P = 0.006). 30-day complications were significantly cardiovascular disease (OR 2.68, P = 0.04) and levels fused (OR 1.10, P = 0.04). 2-year complications were significantly associated with cardiovascular disease (OR 2.85, P = 0.02). 30-day readmissions were significantly associated with sex (4.47, 0.04) and length of hospital stay (χ 2 = 0.07, P = 0.04). 2-year readmissions were significantly associated with age (χ 2 = 0.50, P = 0.03), hypertension (χ 2 = 4.64, P = 0.03), revision surgeries (χ 2 = 5.46, P = 0.02), and length of hospital stay (χ 2 = 0.07, P = 0.03). DISCUSSION: This study found that patients with coronary vascular disease and longer fusions were at higher risk of postoperative complications and patients with notable intraoperative blood loss were at higher risk of postoperative infections. In addition, physicians should closely follow patients with extended postoperative hospital stays, with advanced age, and undergoing revision surgery because these patients were more likely to be readmitted to the hospital.

Causes of Intraoperative Neuromonitoring Events in Adult Spine Deformity Surgery: A Systematic Review.

STUDY DESIGN: Systematic review. OBJECTIVES: Intraoperative neuromonitoring (IOMN) has become a standard practice in the detection and prevention of nerve damage and postoperative deficit. While multicenter studies have addressed this inquiry, there have been no systematic reviews to date. This systematic review identifies the leading causes of IONM alerts during adult spinal deformity (ASD) surgeries. METHODS: Following PRISMA guidelines, a literature search was performed in PubMed and Embase. IONM alert causes were grouped by equivalent terms used across different studies and binned into larger categories, including surgical maneuver, Changes in blood pressure/temperature, Oxygenation, Anesthesia, Patient position, and Unknown. RESULTS: Inclusion criteria were studies on adult patients receiving ASD correction surgery using IONM with documented alert causes. 1544 references were included in abstract review, 128 in full text review, and 16 studies qualified for data extraction. From those studies, there was a total of 3945 adult patients with 299 IONM alerts. Surgical maneuver led the alert causes (258 alerts/86.3%), with signal loss most commonly occurring at correction or osteotomy (101/33.8% and 95/31.8% respectively). Pedicle screw placement caused 35 alerts (11.7%). Changes in temperature and blood pressure were the third largest category (34/11.4%). CONCLUSIONS: The most frequent causes of IONM alerts in ASD surgery were surgical maneuvers such as correction, osteotomy, and pedicle screw placement. This information provides spine surgeons with a quantitative perspective on the causes of IONM changes and show that most occur at predictable times during ASD surgery.

Lumbar multiunit activity power spectrum during air stepping in the spinal cat: evidence for a flexor-dominated rostrocaudally distributed locomotor center.

Clues about the organization of spinal networks responsible for rhythmic motor behaviors have come from the examination of reflex circuitry, lesioning studies, and single-cell recordings. Recently, more attention has been paid to extracellularly recorded multiunit signals thought to represent the general activity of local cellular potentials. Focusing on the gross localization of spinal locomotor networks, we used multiunit signals of the lumbar cord to classify the activation and organization of those networks. We employed power spectral analysis to compare multiunit power across rhythmic conditions and locations and to infer patterns of activation based on coherence and phase measures. We found greater multiunit power in midlumbar segments during stepping, supportive of previous lesioning studies isolating rhythm-generating capabilities to these segments. We also found much greater multiunit power during the flexion phase of stepping than during the extension phase for all lumbar segments. Greater multiunit power at flexion indicates increased neural activity during this phase and is suggestive of previously reported asymmetries between flexor- and extensor-related interneuronal populations of the spinal rhythm-generating network. Finally, the multiunit power showed no phase lag at coherent frequencies throughout the lumbar enlargement indicative of a longitudinal standing wave of neural activation. Our results suggest that the multiunit activity may be representative of the spinal rhythm-generating activity that is distributed in a rostrocaudal gradient. Additionally, our results indicate that this multiunit activity may operate as a flexor-dominant standing wave of activation that is synchronized throughout the rostrocaudal extent of the lumbar enlargement.NEW & NOTEWORTHY We report on the power spectral analysis of multiunit activity (MUA) of lumbar spinal interneurons during a locomotor task. In line with prior studies, we found evidence of greater power at the frequency of locomotion in high lumbar segments and during the flexion phase. Our results also confirm prior observations from our laboratory that the rhythmically active MUA behaves as a longitudinal standing wave of neural activation that is flexor dominant.

Single-cell and ensemble activity of lumbar intermediate and ventral horn interneurons in the spinal air-stepping cat.

We explored the relationship between population interneuronal network activation and motor output in the adult, in vivo, air-stepping, spinal cat. By simultaneously measuring the activity of large numbers of spinal interneurons, we explored ensembles of coherently firing interneurons and their relation to motor output. In addition, the networks were analyzed in relation to their spatial distribution along the lumbar enlargement for evidence of localized groups driving particular phases of the locomotor step cycle. We simultaneously recorded hindlimb EMG activity during stepping and extracellular signals from 128 channels across two polytrodes inserted within lamina V-VII of two separate lumbar segments. Results indicated that spinal interneurons participate in one of two ensembles that are highly correlated with the flexor or the extensor muscle bursts during stepping. Interestingly, less than half of the isolated single units were significantly unimodally tuned during the step cycle whereas >97% of the single units of the ensembles were significantly correlated with muscle activity. These results show the importance of population scale analysis in neural studies of behavior as there is a much greater correlation between muscle activity and ensemble firing than between muscle activity and individual neurons. Finally, we show that there is no correlation between interneurons' rostrocaudal locations within the lumbar enlargement and their preferred phase of firing or ensemble participation. These findings indicate that spinal interneurons of lamina V-VII encoding for different phases of the locomotor cycle are spread throughout the lumbar enlargement in the adult spinal cord.NEW & NOTEWORTHY We report on the ensemble organization of interneuronal activity in the spinal cord during locomotor movements and show that lumbar intermediate zone interneurons organize in two groups related to the two major phases of walking: stance and swing. Ensemble organization is also shown to better correlate with muscular output than single-cell activity, although ensemble membership does not appear to be somatotopically organized within the spinal cord.

Forelimb force direction and magnitude independently controlled by spinal modules in the macaque.

Modular organization of the spinal motor system is thought to reduce the cognitive complexity of simultaneously controlling the large number of muscles and joints in the human body. Although modular organization has been confirmed in the hindlimb control system of several animal species, it has yet to be established in the forelimb motor system or in primates. Expanding upon experiments originally performed in the frog lumbar spinal cord, we examined whether costimulation of two sites in the macaque monkey cervical spinal cord results in motor activity that is a simple linear sum of the responses evoked by stimulating each site individually. Similar to previous observations in the frog and rodent hindlimb, our analysis revealed that in most cases (77% of all pairs) the directions of the force fields elicited by costimulation were highly similar to those predicted by the simple linear sum of those elicited by stimulating each site individually. A comparable simple summation of electromyography (EMG) output, especially in the proximal muscles, suggested that this linear summation of force field direction was produced by a spinal neural mechanism whereby the forelimb motor output recruited by costimulation was also summed linearly. We further found that the force field magnitudes exhibited supralinear (amplified) summation, which was also observed in the EMG output of distal forelimb muscles, implying a novel feature of primate forelimb control. Overall, our observations support the idea that complex movements in the primate forelimb control system are made possible by flexibly combined spinal motor modules.

Inhibition of HGF/MET signaling decreases overall tumor burden and blocks malignant conversion in Tpl2-related skin cancer.

Tumor progression locus 2 (Tpl2) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of serine/threonine kinases. Deletion of the Tpl2 gene is associated with a significantly higher number of papillomas and cutaneous squamous cell carcinomas (cSCCs). Overexpression of hepatocyte growth factor (HGF) and its receptor MET is abundant in cSCC and can lead to increased proliferation, migration, invasion or resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. The aim of this study was to address whether the increased tumor burden in Tpl2 -/- mice is due to aberrant HGF/MET signaling. C57Bl/6 wild type (WT) and Tpl2 -/- mice were subjected to a two-stage chemical carcinogenesis protocol for one year. At the time of promotion half of the mice received 44 mg/kg capmatinib (INC 280), a pharmacological inihibitor of MET, in their diet. Tpl2-/- mice had signficantly higher tumor incidence and overall tumor burden compared to WT mice. Further, carcinogen-intiated Tpl2 -/- mice could bypass the need for promotion, as 89% of Tpl2 -/- mice given only DMBA developed papillomas. v-rasHa -transduced keratinocytes and SCCs from Tpl2 -/- mice revealed an upregulation in HGF and p-MET signaling compared to WT animals. Long-term capmatinib treatment had no adverse effects in mice and capmatinib-fed Tpl2 -/- mice had a 60% reduction in overall tumor burden. Further, no tumors from Tpl2 -/- mice fed capmatinib underwent malignant conversion. In summary targeting MET may be a potential new strategy to combat cutaneous squamous cell carcinomas that result from dysregulation in MAPK signaling.

Transverse occiput position: Using manual Rotation to aid Normal birth and improve delivery OUTcomes (TURN-OUT): A study protocol for a randomised controlled trial.

BACKGROUND: Fetal occiput transverse position in the form of deep transverse arrest has long been associated with caesarean section and instrumental vaginal delivery. Occiput transverse position incidentally found in the second stage of labour is also associated with operative delivery in high risk cohorts. There is evidence from cohort studies that prophylactic manual rotation reduces the caesarean section rate. This is a protocol for a double blind, multicentre, randomised, controlled clinical trial to define whether this intervention decreases the operative delivery (caesarean section, forceps or vacuum delivery) rate. METHODS/DESIGN: Eligible participants will be ≥37 weeks pregnant, with a singleton pregnancy, and a cephalic presentation in the occiput transverse position on transabdominal ultrasound early in the second stage of labour. Based on a background risk of operative delivery of 49%, for a reduction to 35%, an alpha value of 0.05 and a beta value of 0.2, 416 participants will need to be enrolled. Participants will be randomised to either prophylactic manual rotation or a sham procedure. The primary outcome will be operative delivery. Secondary outcomes will be caesarean section, significant maternal mortality and morbidity, and significant perinatal mortality and morbidity. Analysis will be on an intention-to-treat basis. Primary and secondary outcomes will be compared using a chi-squared test. A logistic regression for the primary outcome will be undertaken to account for potential confounders. This study has been approved by the Ethics Review Committee (RPAH Zone) of the Sydney Local Health District, Sydney, Australia, (protocol number: X110410). DISCUSSION: This trial addresses an important clinical question concerning a commonly used procedure which has the potential to reduce operative delivery and its associated complications. Some issues discussed in the protocol include methods of assessing risk of bias due to inadequate masking of a procedural interventions, variations in intervention efficacy due to operator experience and the recruitment difficulties associated with intrapartum studies. TRIAL REGISTRATION: This trial was registered with the Australian New Zealand Clinical Trials Registry (identifier: ACTRN12613000005752 ) on 4 January 2013.

Modulation of spinal motor output by initial arm postures in anesthetized monkeys.

Proper execution of voluntary movement requires a sensorimotor transformation based on the initial limb state. For example, successfully reaching to a stable target requires the recruitment of different muscle groups depending on limb position at movement initiation. To test whether this transformation could occur at the spinal level, we stimulated the cervical spinal cord of anesthetized monkeys while systematically changing initial posture and examined the modulation of the twitch response induced in the upper limb muscles. In three monkeys, a multichannel microelectrode array was implanted into the C6 segment of the spinal cord and electromyographic electrodes were implanted in 12 limb muscles (five hand, four elbow, and three shoulder muscles). The magnitude and onset latency of the evoked response in each electrode-muscle pair were examined by systematically changing the hand position through nine positions in a horizontal plane with the monkey prone. Among 330 electrode-muscle pairs examined, 61% of pairs exhibited significant modulation of either magnitude or latency of twitch responses across different hand/arm configurations (posture dependency). We found that posture dependency occurred preferentially in the distal rather than proximal muscles and was not affected by the location of the electrode within the stimulated spinal segment. Importantly, this posture dependency was not affected by spinalization at the C2 level. These results suggest that excitability in the cervical spinal cord is affected by initial arm posture through spinal reflex pathways. This posture dependency of spinal motor output could affect voluntary arm movement by adjusting descending motor commands relative to the initial arm posture.

Persistent Occiput Posterior position - OUTcomes following manual rotation (POP-OUT): study protocol for a randomised controlled trial.

BACKGROUND: Occiput posterior position is the most common malpresentation in labour, contributes to about 18% of emergency caesarean sections and is associated with a high risk of assisted delivery. Caesarean section is now a major contributing factor to maternal mortality and morbidity following childbirth in developed countries. Obstetric intervention by forceps and ventouse delivery is associated with complications to the maternal genital tract and to the neonate, respectively. There is level 2 evidence that prophylactic manual rotation reduces the caesarean section rate and assisted vaginal delivery. But there has been no adequately powered randomised controlled trial. This is a protocol for a double-blinded, multicentre, randomised controlled clinical trial to define whether this intervention decreases the operative delivery (caesarean section, forceps or vacuum delivery) rate. METHODS/DESIGN: Eligible participants will be (greater than or equal to) 37 weeks' with a singleton pregnancy and a cephalic presentation in the occiput posterior position on transabdominal ultrasound early in the second stage of labour. Based on a background risk of operative delivery of 68%, then for a reduction to 50%, an alpha value of 0.05 and a beta value of 0.2, 254 participants will need to be enrolled. This study has been approved by the Ethics Review Committee (RPAH Zone) of the Sydney Local Health District, Sydney, Australia, and protocol number X110410. Participants with written consent will be randomised to either prophylactic manual rotation or a sham procedure. The primary outcome will be operative delivery (defined as vacuum, forceps and/or caesarean section deliveries). Secondary outcomes will be caesarean section, significant maternal mortality/morbidity and significant perinatal mortality/morbidity. Analysis will be by intention-to-treat. Primary and secondary outcomes will be compared using a chi-squared test. A logistic regression for the primary outcome will be undertaken to account for potential confounders. The results of the trial will be presented at one or more medical conferences. The trial will be submitted to peer review journals for consideration for publication. There will be potential to incorporate the results into professional guidelines for obstetricians and midwives. TRIAL REGISTRATION: The Australian New Zealand Clinical Trials Registry ACTRN12612001312831 . Trial registered 12 December 2012.

Grafted neural progenitors integrate and restore synaptic connectivity across the injured spinal cord.

Transplantation of neural progenitor cells (NPC) is a promising therapeutic strategy for replacing neurons lost after spinal cord injury, but significant challenges remain regarding neuronal integration and functional connectivity. Here we tested the ability of graft-derived neurons to reestablish connectivity by forming neuronal relays between injured dorsal column (DC) sensory axons and the denervated dorsal column nuclei (DCN). A mixed population of neuronal and glial restricted precursors (NRP/GRP) derived from the embryonic spinal cord of alkaline phosphatase (AP) transgenic rats were grafted acutely into a DC lesion at C1. One week later, BDNF-expressing lentivirus was injected into the DCN to guide graft axons to the intended target. Six weeks later, we observed anterogradely traced sensory axons regenerating into the graft and robust growth of graft-derived AP-positive axons along the neurotrophin gradient into the DCN. Immunoelectron microscopy revealed excitatory synaptic connections between regenerating host axons and graft-derived neurons at C1 as well as between graft axons and DCN neurons in the brainstem. Functional analysis by stimulus-evoked c-Fos expression and electrophysiological recording showed that host axons formed active synapses with graft neurons at the injury site with the signal propagating by graft axons to the DCN. We observed reproducible electrophysiological activity at the DCN with a temporal delay predicted by our relay model. These findings provide the first evidence for the ability of NPC to form a neuronal relay by extending active axons across the injured spinal cord to the intended target establishing a critical step for neural repair with stem cells.

Replication fork stalling by bulky DNA damage: localization at active origins and checkpoint modulation.

The integrity of the genome is threatened by DNA damage that blocks the progression of replication forks. Little is known about the genomic locations of replication fork stalling, and its determinants and consequences in vivo. Here we show that bulky DNA damaging agents induce localized fork stalling at yeast replication origins, and that localized stalling is dependent on proximal origin activity and is modulated by the intra-S-phase checkpoint. Fork stalling preceded the formation of sister chromatid junctions required for bypassing DNA damage. Despite DNA adduct formation, localized fork stalling was abrogated at an origin inactivated by a point mutation and prominent stalling was not detected at naturally-inactive origins in the replicon. The intra-S-phase checkpoint contributed to the high-level of fork stalling at early origins, while checkpoint inactivation led to initiation, localized stalling and chromatid joining at a late origin. Our results indicate that replication forks initially encountering a bulky DNA adduct exhibit a dual nature of stalling: a checkpoint-independent arrest that triggers sister chromatid junction formation, as well as a checkpoint-enhanced arrest at early origins that accompanies the repression of late origin firing. We propose that the initial checkpoint-enhanced arrest reflects events that facilitate fork resolution at subsequent lesions.

Multiple Rad5 activities mediate sister chromatid recombination to bypass DNA damage at stalled replication forks.

DNA damage that blocks replication is bypassed in order to complete chromosome duplication and preserve cell viability and genome stability. Rad5, a PCNA polyubiquitin ligase and DNA-dependent ATPase in yeast, is orthologous to putative tumor suppressors and controls error-free damage bypass by an unknown mechanism. To identify the mechanism in vivo, we investigated the roles of Rad5 and analyzed the DNA structures that form during damage bypass at site-specific stalled forks present at replication origins. Rad5 mediated the formation of recombination-dependent, X-shaped DNA structures containing Holliday junctions between sister chromatids. Mutants lacking these damage-induced chromatid junctions were defective in resolving stalled forks, restarting replication, and completing chromosome duplication. Rad5 polyubiquitin ligase and ATPase domains both contributed to replication fork recombination. Our results indicate that multiple activities of Rad5 function coordinately with homologous recombination factors to enable replication template switch events that join sister chromatids at stalled forks and bypass DNA damage.

Dysregulation of purine nucleotide biosynthesis pathways modulates cisplatin cytotoxicity in Saccharomyces cerevisiae.

We found previously that inactivation of the FCY2 gene, encoding a purine-cytosine permease, or the HPT1 gene, encoding the hypoxanthine guanine phosphoribosyl transferase, enhances cisplatin resistance in yeast cells. Here, we report that in addition to fcy2Delta and hpt1Delta mutants in the salvage pathway of purine nucleotide biosynthesis, mutants in the de novo pathway that disable the feedback inhibition of AMP and GMP biosynthesis also enhanced cisplatin resistance. An activity-enhancing mutant of the ADE4 gene, which constitutively synthesizes AMP and excretes hypoxanthine, and a GMP kinase mutant (guk1), which accumulates GMP and feedback inhibits Hpt1 function, both enhanced resistance to cisplatin. In addition, overexpression of the ADE4 gene in wild-type cells, which increases de novo synthesis of purine nucleotides, also resulted in elevated cisplatin resistance. Cisplatin cytotoxicity in wild-type cells was abolished by low concentration of extracellular purines (adenine, hypoxanthine, and guanine) but not cytosine. Inhibition of cytotoxicity by exogenous adenine was accompanied by a reduction of DNA-bound cisplatin in wild-type cells. As a membrane permease, Fcy2 may mediate limited cisplatin transport because cisplatin accumulation in whole cells was slightly affected in the fcy2Delta mutant. However, the fcy2Delta mutant had a greater effect on the amount of DNA-bound cisplatin, which decreased to 50 to 60% of that in the wild-type cells. Taken together, our results indicate that dysregulation of the purine nucleotide biosynthesis pathways and the addition of exogenous purines can modulate cisplatin cytotoxicity in Saccharomyces cerevisiae.

Small ubiquitin-related modifier pathway is a major determinant of doxorubicin cytotoxicity in Saccharomyces cerevisiae.

Development of drug resistance is a major challenge in cancer chemotherapy using doxorubicin. By screening the collection of Saccharomyces cerevisiae deletion strains to identify doxorubicin-resistant mutants, we have discovered that the small ubiquitin-related modifier (SUMO) pathway is a major determinant of doxorubicin cytotoxicity in yeast. Mutants lacking UBA2 (SUMO activating enzyme; E1), UBC9 (conjugating enzyme; E2), and ULP1 and ULP2 (desumoylation peptidases) are all doxorubicin resistant, as are mutants lacking MLP1, UIP3, and NUP60, which all interact with ULP1. Most informatively, mutants lacking the SUMO E3 ligase Siz1 are strongly doxorubicin resistant, whereas mutants of other SUMO ligases are either weakly resistant (siz2) or hypersensitive (mms21) to doxorubicin. These results suggest that doxorubicin cytotoxicity is regulated by Siz1-dependent sumoylation of specific proteins. Eliminating SUMO attachment to proliferating cell nuclear antigen or topoisomerase II does not affect doxorubicin cytotoxicity, whereas reducing SUMO attachment to the bud neck-associated septin proteins has a modest effect. Consistent with these results, doxorubicin resistance in the siz1Delta strain does not seem to involve an effect on DNA repair. Instead, siz1Delta cells accumulate lower intracellular levels of doxorubicin than wild-type (WT) cells, suggesting that they are defective in doxorubicin retention. Although siz1Delta cells are cross-resistant to daunorubicin, they are hypersensitive to cisplatin and show near WT sensitivity to other drugs, suggesting that the siz1Delta mutation does not cause a general multidrug resistance phenotype. Cumulatively, these results reveal that SUMO modification of proteins mediates the doxorubicin cytotoxicity in yeast, at least partially, by modification of septins and of proteins that control the intracellular drug concentration.

Genome-wide screen identifies genes whose inactivation confer resistance to cisplatin in Saccharomyces cerevisiae.

To identify novel genes that mediate cellular resistance to cisplatin, we have screened the collection of Saccharomyces cerevisiae deletion strains. We have found reproducibly 22 genes/open reading frames (ORF), which when deleted, confer resistance to cisplatin at a concentration that is lethal to wild-type cells. Complementation of individual deletion strains with the corresponding wild-type gene abolished cisplatin resistance, confirming that specific gene deletions caused the resistance. Twenty of the genes/ORFs identified have not been previously linked to cisplatin resistance and belong to several distinct functional groups. Major functional groups encode proteins involved in nucleotide metabolism, mRNA catabolism, RNA-polymerase-II-dependent gene regulation and vacuolar transport systems. In addition, proteins that function in ubiquitination, sphingolipid biogenesis, cyclic AMP-dependent signaling, DNA repair, and genome stability are also associated with cisplatin resistance. More than half of the identified genes are known to have sequences or functional homology to mammalian counterparts. Some deletion strains are cross-resistant to selected cytotoxic agents whereas hypersensitive to others. The sensitivity of certain resistant strains to other cytotoxic agents suggests that our findings may point to particular drug combinations that can overcome resistance caused by inactivation of specific genes.

SV40 DNA replication inhibition by the monofunctional DNA alkylator Et743.

Ecteinascidin 743 (Et743) is a highly cytotoxic anticancer agent isolated from the squirt Ecteinascidia turbinate, which alkylates DNA in the minor groove at GC-rich sequences resulting in an unusual bending toward the major groove. The ability of Et743 to block DNA replication was studied using the well-established simian virus (SV40) model for mammalian DNA replication in cells and cell-free extracts. Intracellular SV40 DNA isolated from Et743-treated BSC-1 cells was analyzed by native, two-dimensional agarose gel electrophoresis. A low frequency of Et743 adducts detected at 30-100 nM drug concentrations inhibited SV40 origin activity and induced formation of unusual DNA replication intermediates. Under cell-free conditions, only a high Et743 adduct frequency reduced SV40 DNA synthesis. Comparative studies involving related DNA alkylators, tomamycin and saframycin A, revealed inhibition of SV40 DNA replication in cells at concentrations approximately 10 times higher than Et743. Under cell-free conditions tomamycin- or saframycin-A-adducted DNA templates inhibited DNA synthesis similarly to Et743. Et743 appears to be unusual among other alkylators, because its adducts strongly inhibit intracellular SV40 DNA replication but are relatively weak as cis inhibitors as measured under cell-free conditions.

PATTERNFINDER: combined analysis of DNA regulatory sequences and double-helix stability.

BACKGROUND: Regulatory regions that function in DNA replication and gene transcription contain specific sequences that bind proteins as well as less-specific sequences in which the double helix is often easy to unwind. Progress towards predicting and characterizing regulatory regions could be accelerated by computer programs that perform a combined analysis of specific sequences and DNA unwinding properties. RESULTS: Here we present PATTERNFINDER, a web server that searches DNA sequences for matches to specific or flexible patterns, and analyzes DNA helical stability. A batch mode of the program generates a tabular map of matches to multiple, different patterns. Regions flanking pattern matches can be targeted for helical stability analysis to identify sequences with a minimum free energy for DNA unwinding. As an example application, we analyzed a regulatory region of the human c-myc proto-oncogene consisting of a single-strand-specific protein binding site within a DNA region that unwindsin vivo. The predicted region of minimal helical stability overlapped both the protein binding site and the unwound DNA region identified experimentally. CONCLUSIONS: The PATTERNFINDER web server permits localization of known functional elements or landmarks in DNA sequences as well as prediction of potential new elements. Batch analysis of multiple patterns facilitates the annotation of DNA regulatory regions. Identifying specific pattern matches linked to DNA with low helical stability is useful in characterizing regulatory regions for transcription, replication and other processes and may predict functional DNA unwinding elements.PATTERNFINDER can be accessed freely at: http://wings.buffalo.edu/gsa/dna/dk/PFP/

WEB-THERMODYN: Sequence analysis software for profiling DNA helical stability.

WEB-THERMODYN analyzes DNA sequences and computes the DNA helical stability, i.e. the free energy required to unwind and separate the strands of the double helix. A helical stability profile across a selected DNA region or the entire sequence is generated by sliding-window analysis. WEB-THERMODYN can predict sites of low helical stability present at regulatory regions for transcription and replication and can be used to test the influence of mutations. The program can be accessed at: http://wings.buffalo.edu/gsa/dna/dk/WEBTHERMODYN/.