Unraveling the potential clinical utility of circulating tumor DNA detection in colorectal cancer-evaluation in a nationwide Danish cohort.

BACKGROUND: Increasingly, circulating tumor DNA (ctDNA) is proposed as a tool for minimal residual disease (MRD) assessment. Digital PCR (dPCR) offers low analysis costs and turnaround times of less than a day, making it ripe for clinical implementation. Here, we used tumor-informed dPCR for ctDNA detection in a large colorectal cancer (CRC) cohort to evaluate the potential for post-operative risk assessment and serial monitoring, and how the metastatic site may impact ctDNA detection. Additionally, we assessed how altering the ctDNA-calling algorithm could customize performance for different clinical settings. PATIENTS AND METHODS: Stage II-III CRC patients (N = 851) treated with a curative intent were recruited. Based on whole-exome sequencing on matched tumor and germline DNA, a mutational target was selected for dPCR analysis. Plasma samples (8 ml) were collected within 60 days after operation and-for a patient subset (n = 246)-every 3-4 months for up to 36 months. Single-target dPCR was used for ctDNA detection. RESULTS: Both post-operative and serial ctDNA detection were prognostic of recurrence [hazard ratio (HR) = 11.3, 95% confidence interval (CI) 7.8-16.4, P < 0.001; HR = 30.7, 95% CI 20.2-46.7, P < 0.001], with a cumulative ctDNA detection rate of 87% at the end of sample collection in recurrence patients. The ctDNA growth rate was prognostic of survival (HR = 2.6, 95% CI 1.5-4.4, P = 0.001). In recurrence patients, post-operative ctDNA detection was challenging for lung metastases (4/21 detected) and peritoneal metastases (2/10 detected). By modifying the cut-off for calling a sample ctDNA positive, we were able to adjust the sensitivity and specificity of our test for different clinical contexts. CONCLUSIONS: The presented results from 851 stage II-III CRC patients demonstrate that our personalized dPCR approach effectively detects MRD after operation and shows promise for serial ctDNA detection for recurrence surveillance. The ability to adjust sensitivity and specificity shows exciting potential to customize the ctDNA caller for specific clinical settings.

Inadequate safety reporting in the publications of randomised clinical trials in irritable bowel syndrome: drug versus probiotic interventions.

Randomised controlled clinical trials (RCTs) offer a unique opportunity to obtain controlled efficacy and safety data to support clinical decisions. However, most RCT reporting has a stronger focus on efficacy rather than safety. This study aimed to identify the safety profile of both probiotic and drug interventions in irritable bowel syndrome (IBS). In connection to this paper, an accompanying paper was published in which a meta-analysis was conducted to evaluate the efficacy of probiotic interventions compared to that of drug interventions in IBS. Together, these two studies provide a first assessment regarding the feasibility to determine a burden to benefit ratio for both probiotic and drug interventions in IBS. RCTs including participants (>18 years old) with IBS and comparing probiotic or drugs interventions with control groups were identified by a systematic search of MEDLINE (January 2015 - Jan 2021). Reported safety profiles in drug studies were completer and more detailed as compared with studies on probiotics. Several inconsistencies in safety reporting were identified between and within drug and probiotic studies, such as: didn't report on safety; only reported adverse reactions (ARs) or adverse events (AEs) with a certain severity; didn't report the total number of AEs; didn't split in the control- or experimental arm; didn't specify AEs; and used different thresholds for 'common' AEs. Hence, it is difficult to compare safety data from drug and probiotic RCTs across and between different studies. On the current approaches to safety reporting, we could not establish an unambiguous safety profile for neither probiotic and drug interventions in IBS. These shortcomings hamper a critical comparison of the burden to benefit ratio for IBS intervention.

Comparing probiotic and drug interventions in irritable bowel syndrome: a meta-analysis of randomised controlled trials.

Clinical decisions made by health professionals to recommend either drug or probiotic interventions for irritable bowel syndrome (IBS) should be supported by proper knowledge of the efficacy rates of both types of interventions. In this article, we performed a systematic review and meta-analysis to examine the efficacy of both probiotic- and drug interventions in IBS. Medline was searched between January 2015 - January 2021. Randomised controlled trials (RCT) recruiting participants > 18 years old with IBS and examining the effect of probiotics or drugs were eligible for inclusion. The data of the primary outcome, i.e. the persistence of IBS symptoms (dichotomous symptom data), were pooled to obtain a relative risk (RR), with a 95% confidence interval (CI). Secondary outcomes, abdominal pain- and bloating scores (continuous data), were pooled using a standardised mean difference with a 95% CI. The search identified 269 citations of which 32 RCTs were eligible. Our meta-analysis indicated that both probiotic and drug interventions are able to improve the persistence of IBS symptoms (RR 0.60 [0.51; 0.92] versus 0.87 [0.81; 0.92], respectively) and abdominal pain scores (standardised mean difference (SMD) -0.35 [-0.56; -0.14] versus -0.10 [-0.20; 0.00], respectively). However, determining the overall efficacy of both intervention types is inherently complex and such results should be interpreted with care, due to the large diversity of probiotic- and drug types and doses, which is also complicated by variety in IBS subtypes. Hence, as a first step, more large scale randomised double blind placebo-controlled trials focussing on a specific IBS subtype targeted with specific probiotic strains or specific pharmaceutical modalities should be executed, enabling a more proper comparison between trials.

On the importance of intraindividual variation in nutritional research.

Nutritional intervention studies, like those with pre- and probiotics, are often hampered by low effect sizes, reducing the power to demonstrate potential efficacy. Here, we perform computer simulations of a hypothetical clinical trial using such an intervention in order to elucidate determining factors that can be influenced in order to optimise the statistical power. Our simulations demonstrate that steering the study population towards a low intraindividual variation dramatically improves statistical power. A more than 10-fold decrease of number-to-treat could be reached. Also, a careful balancing between the number of subjects and measurements per subject, in combination with possible stratification of the subjects into responders and non-responders, based on inherent intraindividual variation, improves the likelihood to reach statistically significant results. Our results also show that traditional dogmas, with respect to clinical trials, i.e. aiming at low interindividual variation and a high number (n) of study participants, should be re-evaluated in favour of reducing intraindividual variation. This reduction in intraindividual variation could be achieved by maintaining a steady lifestyle, including dietary habits among others, within the timeframe of the intervention study.

Protein engineering of the chemokine CCL20 prevents psoriasiform dermatitis in an IL-23-dependent murine model.

Psoriasis is a chronic inflammatory skin disease characterized by the infiltration of T cell and other immune cells to the skin in response to injury or autoantigens. Conventional, as well as unconventional, γδ T cells are recruited to the dermis and epidermis by CCL20 and other chemokines. Together with its receptor CCR6, CCL20 plays a critical role in the development of psoriasiform dermatitis in mouse models. We screened a panel of CCL20 variants designed to form dimers stabilized by intermolecular disulfide bonds. A single-atom substitution yielded a CCL20 variant (CCL20 S64C) that acted as a partial agonist for the chemokine receptor CCR6. CCL20 S64C bound CCR6 and induced intracellular calcium release, consistent with G-protein activation, but exhibited minimal chemotactic activity. Instead, CCL20 S64C inhibited CCR6-mediated T cell migration with nominal impact on other chemokine receptor signaling. When given in an IL-23-dependent mouse model for psoriasis, CCL20 S64C prevented psoriatic inflammation and the up-regulation of IL-17A and IL-22. Our results validate CCR6 as a tractable therapeutic target for psoriasis and demonstrate the value of CCL20 S64C as a lead compound.

Identification and Characterization of a Nationwide Danish Adult Common Variable Immunodeficiency Cohort.

In this study, we identified all adults living in Denmark diagnosed with common variable immunodeficiency (CVID) and characterized them according to clinical presentation and EUROclass classification. Using a retrospective, cross-sectional design, possible CVID patients were identified in the Danish National Patient Register and Centers in Denmark treating patients with primary immunodeficiencies. The CVID diagnosis was verified by review of medical records. One-hundred-seventy-nine adults with CVID were identified. This corresponds to a prevalence of 1:26,000. The median age at onset of symptoms was 29 years with no sex difference. The median age at diagnosis was 40 years. Males were diagnosed earlier with a peak in the fourth decade of life, whereas females were diagnosed later with a peak in the sixth decade. The median diagnostic delay was seven years. Recurrent sinopulmonary infections were seen in 92.7% of the patients. The prevalence of non-infectious complications was similar to that of previously reported cohorts: bronchiectasis (35.8%), splenomegaly (22.4%), lymphadenopathy (26.3%), granulomatous inflammation (3.9%) and idiopathic thrombocytopenic purpura (14.5%). Non-infectious complications were strongly associated with B cell phenotype, with all having a reduced number of isotype-switched memory B cells. One-hundred-seventy (95%) were treated with immunoglobulin replacement therapy, primarily administered subcutaneously. According to international guidelines, diagnostic evaluation was inadequate in most cases. This study emphasizes the need for improved diagnostic criteria and more awareness of CVID as a differential diagnosis. Diagnosis and management of CVID patients is a challenge requiring specialists with experience in the field of PID.

Fibrinogen supplementation ex vivo increasesclot firmness comparable to platelet transfusion in thrombocytopenia.

BACKGROUND: Fibrinogen concentrate can improve clot firmness and offers a better safety profile than platelet concentrates. Reduction or avoidance of blood transfusions represents a strategy to reduce associated risks. We investigated whether supplementation of fibrinogen concentrate ex vivo can compensate for clot strength as compared with platelet transfusion in vivo METHODS: One hundred patients in need of platelet transfusion (PT) were enrolled. Blood samples were collected immediately before PT and at 1 h and 24 h after PT. Fibrinogen concentrate was added to these citrated whole blood samples at concentrations of 50, 100, 200 and 400 mg kg-1 and the maximum clot firmness (MCF) was analysed using ROTEM thromboelastometry. RESULTS: Fibrinogen supplementation increased MCF significantly and dose-dependently before and after PT. The effect of fibrinogen concentrate (equivalent to doses of 100 and 200 mg kg-1) ex vivo was comparable to that of PT in vivo, whereas 400 mg kg-1 fibrinogen significantly improved MCF compared with PT (P < 0.001). CONCLUSIONS: Fibrinogen concentrate can match the effect of PT on MCF in thrombocytopenia. This potential alternative haemostatic intervention should be evaluated in clinical trials.

Fibrinogen concentrate improves clot strength in patients with haematological malignancies requiring platelet transfusion.

BACKGROUND: Patients with bone marrow failure secondary to chemotherapy often develop thrombocytopenia and require platelet transfusion. Fibrinogen plays an important role in platelet aggregation and the establishment of the primary haemostatic plug. OBJECTIVES: To compare the effects of in vivo platelet transfusion on clot firmness in thrombocytopenic patients with in vitro-performed fibrinogen concentrate substitution. MATERIALS AND METHODS: Thirty patients with haematological malignancy admitted for platelet transfusion were included. Haemostatic effects from platelet transfusion and ex vivo addition of fibrinogen concentrate at three different doses were evaluated by thromboelastometry, with clot firmness as the primary endpoint (A30 ExTEM assay). Secondary endpoints were other thromboelastometry parameters, thrombin generation parameters, activated partial thromboplastin time (APTT), prothrombin time PT, fibrinogen and factor XIII levels and a clinical bleeding score. RESULTS: Twenty patients (66%) had clinical bleeding signs by prior to transfusion. Platelets increased from 17 (range, 1-109) to 40 (range 2-139) × 10(9) L(-1) following transfusion, with a median corrected count increment of 16·7 (range, 0·8-43·5). The A30 value increased significantly by platelet transfusion from 35 ± 11 to 47 ± 10 mm, with no changes in thrombin generation. Fibrinogen concentrate dose-dependently increased A 30 (to 43 ± 10, 49 ± 9 and 50 ± 9 mm, respectively) and reduced parameters of thrombin generation at high doses. Platelet transfusion, together with fibrinogen concentrate, further increased clot firmness. APTT and PT were within normal range, whereas fibrinogen levels were slightly elevated. CONCLUSION: Fibrinogen concentrate increased clot firmness to the same degree as platelet transfusion in patients with low platelet count requiring platelet transfusion.

Universal mechanisms of sound production and control in birds and mammals.

As animals vocalize, their vocal organ transforms motor commands into vocalizations for social communication. In birds, the physical mechanisms by which vocalizations are produced and controlled remain unresolved because of the extreme difficulty in obtaining in vivo measurements. Here, we introduce an ex vivo preparation of the avian vocal organ that allows simultaneous high-speed imaging, muscle stimulation and kinematic and acoustic analyses to reveal the mechanisms of vocal production in birds across a wide range of taxa. Remarkably, we show that all species tested employ the myoelastic-aerodynamic (MEAD) mechanism, the same mechanism used to produce human speech. Furthermore, we show substantial redundancy in the control of key vocal parameters ex vivo, suggesting that in vivo vocalizations may also not be specified by unique motor commands. We propose that such motor redundancy can aid vocal learning and is common to MEAD sound production across birds and mammals, including humans.

Improving the bowel habits of elderly residents in a nursing home using probiotic fermented milk.

Our aim was to determine whether a fermented milk drink containing probiotics could improve the bowel habits of frail elderly individuals living in a nursing home. A total of 135 participants were enrolled in this pilot study. The bowel habits (stool quality and bowel movements) were recorded by nursing staff during a baseline period of 3 weeks. After this period participants received daily a fermented milk drink containing minimally 6.5×10(9) colony forming units of Lactobacillus casei Shirota (LcS) for 6 weeks. During this period, bowel habits were recorded and compared to baseline period. Forty-four participants (74-99 years old) were compliant and used for analysis. Consumption of fermented milk containing LcS significantly increased the percentage of ideal stool types per week (P<0.01), lowered the percentage of constipation stool types per week (P<0.01) and significantly lowered the percentage of diarrhoea stool types per week (P=0.016) as compared to the baseline period. The study product had no significant effect on bowel movements. During the study, no changes in laxative usage or adverse events associated with the study product were reported. Our results suggest that a fermented milk containing LcS significantly improves the bowel habits of frail elderly residents in a nursing home. These promising results should be further substantiated by a confirmatory study.

Duties performed by occupational physicians in Norway.

BACKGROUND: Occupational health care for all is a global aim but has not yet been achieved. Further development should be based on knowledge of specific alternative models of occupational health services (OHS). Little is published on different OHS models and work as a physician in these services. AIMS: To describe duties for Norwegian physicians working in two different OHS models: internal and external. In the internal model, the physician is employed in an OHS located at the company served, whereas in the external model, OHS time is shared between several companies and the physician is often located outside the companies. METHODS: A web-based survey was sent to all members of the Norwegian Occupational Medicine Association. RESULTS: There were 206 responses (response rate of 73%). Only those working as OHS physicians were included (54%). Physicians in external OHS performed individual health examinations to a greater extent, otherwise few differences between physicians working in internal and external OHS were found. Changes in the priority of the physicians' duties through a period of 20 years seem to be related to changes in legislation and official guidelines related to OHS practice. CONCLUSIONS: In this study, OHS physicians in Norway performed a large number of individual-based health examinations but this was seen more in the external OHS model. Otherwise physicians' duties had similar priority in the external and internal models. Legislation and official guidelines seem to be of major importance to the duties performed.

Platelet activation and aggregation: the importance of thrombin activity--a laboratory model.

This study introduces a new laboratory model of whole blood platelet aggregation stimulated by endogenously generated thrombin, and explores this aspect in haemophilia A in which impaired thrombin generation is a major hallmark. The method was established to measure platelet aggregation initiated by tissue factor evaluated by means of impedance aggregometry. Citrated whole blood from healthy volunteers and haemophilia A patients with the addition of inhibitors of the contact pathway and fibrin polymerization was evaluated. In healthy persons, a second wave of platelet aggregation was found to coincide with the thrombin burst and to be abolished by thrombin inhibitors. In this system, platelet aggregation in severe haemophilia A (n = 10) was found to be significantly decreased as compared with healthy individuals (912 ± 294 vs. 1917 ± 793 AU × min, P = 0.003), most probably due to the weak level of thrombin generation. For the first time, analysis of platelet aggregation as induced by endogenously generated thrombin was demonstrated. The new method makes it possible to explore the influence of the coagulation system on platelet function. In contrast to the general understanding, the data suggest that the impaired thrombin generation in haemophilia may affect platelet activation. Future studies will address whether our results may contribute to understanding differences in bleeding phenotypes and response to haemostatic substitution observed among patients.

Identification of a bacterial di-haem cytochrome c peroxidase from Methylomicrobium album BG8.

The nucleotide sequence of an open reading frame (corB) downstream of the copper-repressible CorA-encoding gene of the methanotrophic bacterium Methylomicrobium album BG8 was obtained by restriction enzyme digestion and inverse PCR. The amino acid sequence deduced from this gene showed significant sequence similarity to the surface-associated di-haem cytochrome c peroxidase (SACCP) previously isolated from Methylococcus capsulatus (Bath), including both c-type haem-binding motifs. Homology analysis placed this protein, phylogenetically, within the subfamily containing the M. capsulatus SACCP of the bacterial di-haem cytochrome c peroxidase (BCCP) family of proteins. Immunospecific recognition confirmed synthesis of the M. album CorB as a protein non-covalently associated with the outer membrane and exposed to the periplasm. corB expression is regulated by the availability of copper ions during growth and the protein is most abundant in M. album when grown at a low copper-to-biomass ratio, indicating an important physiological role of CorB under these growth conditions. corB was co-transcribed with the gene encoding CorA, constituting a copper-responding operon, which appears to be under the control of a sigma(54)-dependent promoter. M. album CorB is the second isolated member of the recently described subfamily of the BCCP family of proteins. So far, these proteins have only been described in methanotrophic bacteria.

Phase II study of short-time oxaliplatin, capecitabine and epirubicin (EXE) as first-line therapy in patients with non-resectable gastric cancer.

Epirubicin, cisplatin and continuous infusion of 5-FU is a widely used palliative regimen in patients with gastric cancer. If cisplatin is substituted by oxaliplatin and 5-FU by capecitabine this regimen can be administered in the outpatient setting. Dose-limiting toxicity of oxaliplatin is peripheral sensory neuropathy and it is recommended to give oxaliplatin as a 120 min infusion. However, in patients with colorectal cancer a 30 min infusion of oxaliplatin can safely be administered without increasing neurotoxicity, standard infusion time is 30 min at our departments. In our phase I study the recommended doses of EXE was established (Dupont et al, 2006). Patients with non-resectable gastric adenocarcinoma were eligible. Patients received EXE (epirubicin 50 mg m(-2) day 1; capecitabine 1000 mg m(-2) day(-1) continuously and oxaliplatin 130 mg m(-2) day 1) as outpatient therapy every third week for a maximum of 8 cycles. From June 2004 to September 2005, we enroled 54 patients. Median age was 60 years (31-74 years) Median number of courses was 6 (1-8). Response rate was 45%. Median PFS was 6.8 (5.2-7.9) months and median survival was 10.1 (7.9-1.1) months. Most important grade 3 toxicities were as follows: nausea, vomiting, and diarrhoea (6%). Neurotoxicity grade 2 was seen in 36.5%. We therefore conclude, that EXE every third week is a convenient regimen that easily can be administrated in the outpatient setting but the regimen needs further evaluation in a phase III study.

Human immunodeficiency virus (HIV)-2-specific T lymphocyte proliferative responses in HIV-2-infected and in HIV-2-exposed but uninfected individuals in Guinea-Bissau.

Human immunodeficiency virus (HIV)-2-specific T lymphocyte proliferative responses were determined in cultures of peripheral blood mononuclear cells from HIV-2-exposed uninfected individuals, HIV-2-infected individuals and HIV-negative controls in Guinea-Bissau. Increased HIV-2-specific T lymphocyte proliferative responses were detected in both groups compared to HIV-negative controls (healthy HIV-uninfected individuals without known exposure to an HIV-infected person); five out of 29 of the HIV-2-exposed uninfected and half (16 of 32) of the HIV-2-infected individuals had stimulation indexes >2, compared to one out of 49 of the HIV-negative controls (P = 0.003 and P < 0.0001, respectively). The exposed uninfected individuals had reactivity to a HIV-2 V3-peptide corresponding to amino acids 311-326 of the envelope glycoprotein, while the HIV-2-infected people reacted mainly to HIV-2 whole viral lysate. Thus, this study demonstrates a high degree of HIV-2-specific T helper cell activity, as measured by lymphocyte proliferation, in HIV-2-exposed uninfected individuals as well as in HIV-2-infected subjects. These immune responses could be important for resistance to the infection and for the control of established infection and, thus, play a role in the lower transmission and progression of HIV-2 compared to HIV-1.

Football incident analysis: a new video based method to describe injury mechanisms in professional football.

OBJECTIVES: To develop and test a new video based method for match analysis that combines football specific and medical information to achieve a better understanding of the injury mechanisms and events leading up to high risk situations. METHODS: Football incident analysis (FIA) is a video based method describing incidents that may result in an injury using 19 variables and categories modified from match analysis. Videos from 35 of 76 (46%) official Norwegian under 21 matches played from 1994 to 1998 were analysed. Two football experts classified each incident on the basis of predetermined criteria, and their results were compared using interobserver and intraobserver reliability tests. RESULTS: kappa correlation coefficients for interobserver and intraobserver agreement were very good for 63% and 95% and good for 37% and 5% of the variables respectively. Fifty two incidents were recorded (1.6 incidents per team per match or 94 per 1000 player hours), and 16 (31%) led to injuries (0.5 injuries per match or 29 injuries per 1000 player hours). FIA results showed that 28 incidents occurred while attacking in midfield zone 2 or the attacking zone, and 24 took place while defending in the defensive zone or midfield zone 1. Midfielders were exposed in 67% of the incidents, mainly in breakdown attacks or during long attacks by the opposing team. Of the 28 incidents during offence, only one was classified as having great potential to score a goal. Most incidents (70%) were the result of tackling duels both in the offensive and defensive playing phases. Of the 21 offensive incidents resulting from tackling duels, in 19 cases the exposed player was unaware of the tackling (passive duellist). CONCLUSIONS: This study shows that football incident analysis is a potentially valuable tool for understanding the events leading up to injuries in football.

New perspectives on mechanisms of sound generation in songbirds.

The physical mechanisms of sound generation in the vocal organ, the syrinx, of songbirds have been investigated mostly with indirect methods. Recent direct endoscopic observation identified vibrations of the labia as the principal sound source. This model suggests sound generation in a pulse-tone mechanism similar to human phonation with the labia forming a pneumatic valve. The classical avian model proposed that vibrations of the thin medial tympaniform membranes are the primary sound generating mechanism. As a direct test of these two hypotheses we ablated the medial tympaniform membranes in two species (cardinal and zebra finch) and found that both were still able to phonate and sing without functional membranes. Small changes in song structure (harmonic emphasis, frequency control) occurred after medial tympaniform membrane ablation and suggest that the medial tympaniform membranes play a role in adjusting tension on the labia. Such a role is consistent with the fact that the medial tympaniform membranes are directly attached to the medial labia. There is no experimental support for a third hypothesis, proposing an aerodynamic model for generation of tonal sounds. Indirect tests (song in heliox atmosphere) as well as direct (labial vibration during tonal sound) measurements of syringeal vibrations support a vibration-based sound-generating mechanism even for tonal sounds.

Using the internet in patient-centred diabetes care for communication, education, and decision support.

The New England Journal of Medicine 329 (1993) 977- The present paper describes two systems for communication, education, and decision support in patient-centred diabetes care. Both systems are developed under the assumption that while the clinical resources in the health care sector are limited, patients' resources and new information technology may be able to play a much more central role. With DiasNet patients can experiment with their own data. They can, retrospectively, adjust insulin doses or meals sizes, and thereby learn how to cope with various situations. DiabVision, brings together algorithms for detection of retinal lesions in digital images and for aligning time series of retinal images. While the former algorithms are aimed at automated procedures for screening for diabetic retinopathy, the latter can be used to enable motivated patients to see the actual retinal lesions in their own eyes, illustrating how the changes appear, and perhaps disappear, as a consequence of changes in lifestyle and glycaemic control. One of the long-term goals of our work is to assess the potential of integrating all health related information for patients with diabetes and other major chronic diseases. One solution to these problems would be to organise data and information as one virtual database, which then could be accessed by both health professionals and by patients. This structure would facilitate easy access, a clear overview, and quality control. A web site taking the first step in this process has been launched.

Probing the structure and dynamics of a DNA hairpin by ultrafast quenching and fluorescence depolarization.

DNA hairpins have been investigated in which individual adenines were replaced by their fluorescent analog 2-aminopurine (2AP). The temperature dependence of the time evolution of polarized emission spectra was monitored with picosecond time resolution. Four isotropic decay components for each oligonucleotide indicated the coexistence of at least four conformations. The fluorescence for three of these was significantly quenched, which is explained by hole transfer from 2AP to guanine(s). An approximately 8-ps component is ascribed to direct hole transfer, the approximately 50-ps and approximately 500-ps components are ascribed to structural reorganization, preceding hole transfer. At room temperature, a fraction remains unquenched on a 10-ns timescale, in contrast to higher temperatures, where the flexibility increases. Besides quenching due to base stacking, a second quenching process was needed to describe the data. Evidence for both intrastrand and interstrand hole transfer was found. The extracted probability for stacking between neighboring bases in double-stranded regions was estimated to be approximately 75% at room temperature and approximately 25% at 80 degrees C, demonstrating structural disorder of the DNA. Fluorescence depolarization revealed both local dynamics of the DNA and overall dynamics of the entire oligonucleotide. Upon raising the temperature, the C-N terminus of the hairpin appears to melt first; the rest of the hairpin denatures above the average melting temperature.