Spatial visualization of DNA in solution.

An image analysis method is presented which allows for the reconstruction of the three-dimensional path of filamentous objects from two of their projections. Starting with stereo pairs, this method is used to trace the trajectory of DNA molecules embedded in vitreous ice and leads to a faithful representation of their three-dimensional shape in solution. This computer-aided reconstruction is superior to the subjective three-dimensional impression generated by observation of stereo pairs of micrographs because it enables one to look at the reconstructed molecules from any chosen direction and distance and allows quantitative analysis such as determination of distances, curvature, persistence length, and writhe of DNA molecules in solution.

Long sequence duplications, repeats, and palindromes in HIV-1 gp120: length variation in V4 as the product of misalignment mechanism.

We have shown that indels in gp120 V4 are associated to the presence of duplicated and palindromic sequences, suggesting that they may be produced by strand-slippage misalignment mechanism. Indels in V4 involved region-specific duplications 9 to 15 bp long, and repeats of various lengths, associated to trinucleotides AAT. No duplications were found in V3 and C3. The frequency of palindromic sequences in individual genes was found to be significantly higher in gp120 (p < or = 3.00E-7), and significantly lower in Tat (p < or = 9.00E-7) than the average frequency calculated over the full genome. The finding of elements of misalignment in association with indels in V4 suggests that these mutations may occur in proviral DNA after integration of HIV into the host genome. It also implies that occurrence of large indels in gp120 is not random but is directed by the presence and distribution of elements of misalignment in the HIV genome.

Length variation in HIV-1 gp120 as the product of DNA misalignment mechanism

Background: To determine whether misalignment structures such as duplications, repeats, and palindromes are associated to insertions/deletions (indels) in gp120, indicating that indels are indeed frameshift mutations generated by DNA misalignment mechanism. Methods: Cloning and sequencing of a fragment of HIV-1 gp120 spanning C2-C4 derived from plasma RNA in 12 patients with early chronic disease and naïve to antiretroviral therapy. Results: Indels in V4 involved always insertion and deletion of duplicated nucleotide segments, and AAT repeats, and were associated to the presence of palindromic sequences. No duplications were detected in V3 and C3. Palindromic sequences occurred with similar frequencies in V3, C3 and V4; the frequency of palindromes in individual genes was found to be significantly higher in structural (gp120, p ≤ 3.00E-7) and significantly lower in regulatory (Tat, p ≤ 9.00E-7) genes, as compared to the average frequency calculated over the full genome. Discussion: Indels in V4 are associated to misalignment structures (i.e. duplications repeat and palindromes) indicating DNA misalignment as the mechanism underlying length variation in V4. The finding that indels in V4 are caused by DNA misalignment has some very important implications: 1) indels in V4 are likely to occur in proviral DNA (and not in RNA), after integration of HIV into the host genome; 2) they are likely to occur as progressive modifications of the early founder virus during chronic infection, as more and more cells get infected; 3) frameshift mutations involving any number of base pairs are likely to occur evenly across gp120; however, only those mutants carrying a functional gp120 (indels as multiples of three base pairs) will be able to perpetuate the virus cycle and to keep spreading through the population.

Energy deficit and length of hospital stay can be reduced by a two-step quality improvement of nutrition therapy: the intensive care unit dietitian can make the difference.

OBJECTIVE: Critically ill patients are at high risk of malnutrition. Insufficient nutritional support still remains a widespread problem despite guidelines. The aim of this study was to measure the clinical impact of a two-step interdisciplinary quality nutrition program. DESIGN: Prospective interventional study over three periods (A, baseline; B and C, intervention periods). SETTING: Mixed intensive care unit within a university hospital. PATIENTS: Five hundred seventy-two patients (age 59 ± 17 yrs) requiring >72 hrs of intensive care unit treatment. INTERVENTION: Two-step quality program: 1) bottom-up implementation of feeding guideline; and 2) additional presence of an intensive care unit dietitian. The nutrition protocol was based on the European guidelines. MEASUREMENTS AND MAIN RESULTS: Anthropometric data, intensive care unit severity scores, energy delivery, and cumulated energy balance (daily, day 7, and discharge), feeding route (enteral, parenteral, combined, none-oral), length of intensive care unit and hospital stay, and mortality were collected. Altogether 5800 intensive care unit days were analyzed. Patients in period A were healthier with lower Simplified Acute Physiologic Scale and proportion of "rapidly fatal" McCabe scores. Energy delivery and balance increased gradually: impact was particularly marked on cumulated energy deficit on day 7 which improved from -5870 kcal to -3950 kcal (p < .001). Feeding technique changed significantly with progressive increase of days with nutrition therapy (A: 59% days, B: 69%, C: 71%, p < .001), use of enteral nutrition increased from A to B (stable in C), and days on combined and parenteral nutrition increased progressively. Oral energy intakes were low (mean: 385 kcal*day, 6 kcal*kg*day ). Hospital mortality increased with severity of condition in periods B and C. CONCLUSION: A bottom-up protocol improved nutritional support. The presence of the intensive care unit dietitian provided significant additional progression, which were related to early introduction and route of feeding, and which achieved overall better early energy balance.

Telomere length dynamics in normal individuals and in patients with hematopoietic stem cell-associated disorders.

The telomere length in nucleated peripheral blood (PB) cells indirectly reflects the mitotic history of their precursors: the hematopoietic stem cells (HSCs). The average length of telomeres in PB leukocytes can be measured using fluorescence in situ hybridization and flow cytometry (flow FISH). We previously used flow FISH to characterize the age-related turnover of HSCs in healthy individuals. In this review, we describe results of recent flow FISH studies in patients with selected hematopoietic stem cell-associated disorders: chronic myelogenous leukemia (CML) and several bone marrow failure syndromes. CML is characterized by a marked expansion of myeloid Philadelphia chromosome positive (Ph+) cells. Nevertheless, nonmalignant (Ph-) HSCs typically coexist in the bone marrow of CML patients. We analyzed the telomere length in > 150 peripheral blood leukocytes (PBLs) and bone marrow samples of patients with CML as well as samples of Ph- T-lymphocytes. Compared to normal controls, the overall telomere fluorescence in PBLs of patients with CML was significantly reduced. However, no telomere shortening was observed in Ph- T-lymphocytes. Patients in late chronic phase (CP) had significantly shorter telomeres than those assessed earlier in CP. Our data suggest that progressive telomere shortening is correlated with disease progression in CML. Within the group of patients with bone marrow failure syndromes, we only found significantly shortened telomeres (compared to age-adjusted controls) in granulocytes from patients with aplastic anemia (AA). Strikingly, the telomere length in granulocytes from AA patients who had recovered after immunosuppressive therapy (recAA) did not differ significantly from controls, whereas untreated patients and nonresponders with persistent severe pancytopenia (sAANR) showed marked and significant telomere shortening compared to healthy donors and patients with recAA. Furthermore, an inverse correlation between age-adjusted telomere length and peripheral blood counts was found in support of a model in which the degree of cytopenia and the amount of telomere shortening are correlated. These results support the concept of extensive proliferation of HSCs in subgroups of AA patients and suggest a potential use of telomere-length measurements as a prognostic tool in this group of disorders as well.

Effect of hip flexion angle on hamstring optimum length after a single set of concentric contractions.

The eccentric contraction mode was proposed to be the primary stimulus for optimum angle (angle at which peak torque occurs) shift. However, the training range of motion (or muscle excursion range) could be a stimulus as important. The aim of this study was to assess the influence of the training range of motion stimulus on the hamstring optimum length. It was hypothesised that performing a single set of concentric contractions beyond optimal length (seated at 80° of hip flexion) would lead to an immediate shift of the optimum angle to longer muscle length while performing it below (supine at 0° of hip flexion) would not provide any shift. Eleven male participants were assessed on an isokinetic dynamometer. In both positions, the test consisted of 30 consecutive knee flexions at 4.19 rad · s⁻¹. The optimum angle was significantly shifted by ∼15° in the direction of longer muscle length after the contractions at 80° of hip flexion, while a non-significant shift of 3° was found at 0°. The hamstring fatigability was not influenced by the hip position. It was concluded that the training range of motion seems to be a relevant stimulus for shifting the optimum angle to longer muscle length. Moreover, fatigue appears as a mechanism partly responsible for the observed shift.

Histological description of seminiferous epithelium and cycle length of spermatogenesis in the water shrew Neomys fodiens (Mammalia: Soricidae).

Recently, we examined the spermatogenesis cycle length in two shrews species, Sorex araneus characterized by a very high metabolic rate and a polyandric mating system (sperm competition) resulting in a short cycle and Crocidura russula characterized by a much lower metabolic rate and a monogamous mating system showing a longer cycle. In this study, we investigated the spermatogenesis cycle in Neomys fodiens showing an intermediate metabolic rate. We described the stages of seminiferous epithelium according to the spermatid morphology method and we calculated the cycle length of spermatogenesis using incorporation of 5-bromodeoxyuridine into DNA of the germ cells. Twelve males were injected intraperitoneally with 5-bromodeoxyuridine, and the testes were collected. For cycle length determination, we applied a recently developed statistical method. The calculated cycle length is 8.69 days and the total duration of spermatogenesis based on 4.5 cycles is approximately 39.1 days, intermediate between the duration of spermatogenesis of S. araneus (37.6 days) and C. russula (54.5 days) and therefore congruent with both the metabolic rate hypothesis and the sperm competition hypothesis. Relative testes size of 1.4% of body mass indicates a promiscuous mating system.

Relationships of basal metabolic rate, relative testis size and cycle length of spermatogenesis in shrews (Mammalia, Soricidae).

The aim of the present study was to determinate the cycle length of spermatogenesis in three species of shrew, Suncus murinus, Sorex coronatus and Sorex minutus, and to assess the relative influence of variation in basal metabolic rate (BMR) and mating system (level of sperm competition) on the observed rate of spermatogenesis, including data of shrew species studied before (Sorex araneus, Crocidura russula and Neomys fodiens). The dynamics of sperm production were determined by tracing 5-bromodeoxyuridine in the DNA of germ cells. As a continuous scaling of mating systems is not evident, the level of sperm competition was evaluated by the significantly correlated relative testis size (RTS). The cycle durations estimated by linear regression were 14.3 days (RTS 0.3%) in Suncus murinus, 9.0 days (RTS 0.5%) in Sorex coronatus and 8.5 days (RTS 2.8%) in Sorex minutus. In regression and multiple regression analyses including all six studied species of shrew, cycle length was significantly correlated with BMR (r2=0.73) and RTS (r2=0.77). Sperm competition as an ultimate factor obviously leads to a reduction in the time of spermatogenesis in order to increase sperm production. BMR may act in the same way, independently or as a proximate factor, revealed by the covariation, but other factors (related to testes size and thus to mating system) may also be involved.

Respiratory failure in a mouse model of myotonic dystrophy does not correlate with the CTG repeat length.

Myotonic dystrophy (DM1) is a multisystemic disease caused by an expansion of CTG repeats in the region of DMPK, the gene encoding DM protein kinase. The severity of muscle disability in DM1 correlates with the size of CTG expansion. As respiratory failure is one of the main causes of death in DM1, we investigated the correlation between respiratory impairment and size of the (CTG)n repeat in DM1 animal models. Using pressure plethysmography the respiratory function was assessed in control and transgenic mice carrying either 600 (DM600) or >1300 CTG repeats (DMSXL). The statistical analysis of respiratory parameters revealed that both DM1 transgenic mice sub-lines show respiratory impairment compared to control mice. In addition, there is no significant difference in breathing functions between the DM600 and DMSXL mice. In conclusion, these results indicate that respiratory impairment is present in both transgenic mice sub-lines, but the severity of respiratory failure is not related to the size of the (CTG)n expansion.

Independent sources of condition dependency and multiple pathways determine a composite trait: lessons from carotenoid-based plumage colouration.

Many colour ornaments are composite traits consisting of at least four components, which themselves may be more complex, determined by independent evolutionary pathways, and potentially being under different environmental control. To date, little evidence exists that several different components of colour elaboration are condition dependent and no direct evidence exists that different ornamental components are affected by different sources of variation. For example, in carotenoid-based plumage colouration, one of the best-known condition-dependent ornaments, colour elaboration stems from both condition-dependent pigment concentration and structural components. Some environmental flexibility of these components has been suggested, but specifically which and how they are affected remains unknown. Here, we tested whether multiple colour components may be condition dependent, by using a comprehensive 3 × 2 experimental design, in which we carotenoid supplemented and immune challenged great tit nestlings (Parus major) and quantified effects on different components of colouration. Plumage colouration was affected by an interaction between carotenoid availability and immune challenge. Path analyses showed that carotenoid supplementation increased plumage saturation via feather carotenoid concentration and via mechanisms unrelated to carotenoid deposition, while immune challenge affected feather length, but not carotenoid concentration. Thus, independent condition-dependent pathways, affected by different sources of variation, determine colour elaboration. This provides opportunities for the evolution of multiple signals within components of ornamental traits. This finding indicates that the selective forces shaping the evolution of different components of a composite trait and the trait's signal content may be more complex than believed so far, and that holistic approaches are required for drawing comprehensive evolutionary conclusions.

Copeptin is associated with kidney length, renal function, and prevalence of simple cysts in a population-based study.

Arginine vasopressin (AVP) has a key role in osmoregulation by facilitating water transport in the collecting duct. Recent evidence suggests that AVP may have additional effects on renal function and favor cyst growth in polycystic kidney disease. Whether AVP also affects kidney structure in the general population is unknown. We analyzed the association of copeptin, an established surrogate for AVP, with parameters of renal function and morphology in a multicentric population-based cohort. Participants from families of European ancestry were randomly selected in three Swiss cities. We used linear multilevel regression analysis to explore the association of copeptin with renal function parameters as well as kidney length and the presence of simple renal cysts assessed by ultrasound examination. Copeptin levels were log-transformed. The 529 women and 481 men had median copeptin levels of 3.0 and 5.2 pmol/L, respectively (P<0.001). In multivariable analyses, the copeptin level was associated inversely with eGFR (β=-2.1; 95% confidence interval [95% CI], -3.3 to -0.8; P=0.002) and kidney length (β=-1.2; 95% CI, -1.9 to -0.4; P=0.003) but positively with 24-hour urinary albumin excretion (β=0.11; 95% CI, 0.01 to 0.20; P=0.03) and urine osmolality (β=0.08; 95% CI, 0.05 to 0.10; P<0.001). A positive association was found between the copeptin level and the presence of renal cysts (odds ratio, 1.6; 95% CI, 1.1 to 2.4; P=0.02). These results suggest that AVP has a pleiotropic role in renal function and may favor the development of simple renal cysts.

Changes of Root Length and Root-to-Crown Ratio after Apical Surgery: An Analysis by Using Cone-beam Computed Tomography.

INTRODUCTION: Apical surgery is an important treatment option for teeth with post-treatment periodontitis. Although apical surgery involves root-end resection, no morphometric data are yet available about root-end resection and its impact on the root-to-crown ratio (RCR). The present study assessed the length of apicectomy and calculated the loss of root length and changes of RCR after apical surgery. METHODS: In a prospective clinical study, cone-beam computed tomography scans were taken preoperatively and postoperatively. From these images, the crown and root lengths of 61 roots (54 teeth in 47 patients) were measured before and after apical surgery. Data were collected relative to the cementoenamel junction (CEJ) as well as to the crestal bone level (CBL). One observer took all measurements twice (to calculate the intraobserver variability), and the means were used for further analysis. The following parameters were assessed for all treated teeth as well as for specific tooth groups: length of root-end resection and percentage change of root length, preoperative and postoperative RCRs, and percentage change of RCR after apical surgery. RESULTS: The mean length of root-end resection was 3.58 ± 1.43 mm (relative to the CBL). This amounted to a loss of 33.2% of clinical and 26% of anatomic root length. There was an overall significant difference between the tooth groups (P < .05). There was also a statistically significant difference comparing mandibular and maxillary teeth (P < .05), but not for incisors/canines versus premolars/molars (P = .125). The mean preoperative and postoperative RCRs (relative to CEJ) were 1.83 and 1.35, respectively (P < .001). With regard to the CBL reference, the mean preoperative and postoperative RCRs were 1.08 and 0.71 (CBL), respectively (P < .001). The calculated changes of RCR after apical surgery were 24.8% relative to CEJ and 33.3% relative to CBL (P < .001). Across the different tooth groups, the mean RCR was not significantly different (P = .244 for CEJ and 0.114 for CBL). CONCLUSIONS: This CBCT-based study demonstrated that the RCR is significantly changed after root-end resection in apical surgery irrespective of the clinical (CBL) or anatomic (CEJ) reference levels. The lowest, and thus clinically most critical, postoperative RCR was observed in maxillary incisors. Future clinical studies need to show the impact of resection length and RCR changes on the outcome of apical surgery.