Beam steering by liquid crystal elastomer fibres.

The problem of utilizing a laser beam as an information vehicle and dividing it into different channels is an open problem in the telecommunication field. The switching of a signal into different ports has been demonstrated, to date, by employing complex devices and mechanisms such as the electro optic effect, microelectromechanical system (MEMS) mirrors, or liquid crystal-based spatial light modulators (SLMs). We present here a simple device, namely a mirror held by a liquid crystal elastomer (LCE) fibre, as an optically and remotely driven beam steerer. In fact, a considered signal (laser beam) can be addressed in every in-plane direction by controlling the fibre and mirror rotation, i.e., the deflected probe beam angle. Such movement is possible due to the preparation of LCE fibres able to rotate and contract under a selective light stimulus. By adjusting the irradiation stimulus power, elastic fibres are able to rotate with a specific angle, performing more than one complete revolution around their axis. The described movement is perfectly reversible as soon as the stimulus is removed.

A NTCP approach for estimating the outcome in radioiodine treatment of hyperthyroidism.

Radioiodine has been in use for over 60 years as a treatment for hyperthyroidism. Major changes in clinical practice have led to accurate dosimetry capable of avoiding the risks of adverse effects and the optimization of the treatment. The aim of this study was to test the capability of a radiobiological model, based on normal tissue complication probability (NTCP), to predict the outcome after oral therapeutic 131I administration. Following dosimetric study, 79 patients underwent treatment for hyperthyroidism using radioiodine and then 67 had at least a one-year follow up. The delivered dose was calculated using the MIRD formula, taking into account the measured maximum uptake of administered iodine transferred to the thyroid, U0, and the effective clearance rate, Teff and target mass. The dose was converted to normalized total dose delivered at 2 Gy per fraction (NTD2). Furthermore, the method to take into account the reduction of the mass of the gland during radioiodine therapy was also applied. The clinical outcome and dosimetric parameters were analyzed in order to study the dose-response relationship for hypothyroidism. The TD50 and m parameters of the NTCP model approach were then estimated using the likelihood method. The TD50, expressed as NTD2, resulted in 60 Gy (95% C.I.: 45-75 Gy) and 96 Gy (95% C.I.: 86-109 Gy) for patients affected by Graves or autonomous/multinodular disease, respectively. This supports the clinical evidence that Graves' disease should be characterized by more radiosensitive cells compared to autonomous nodules. The m parameter for all patients was 0.27 (95% C.I.: 0.22-0.36). These parameters were compared with those reported in the literature for hypothyroidism induced after external beam radiotherapy. The NTCP model correctly predicted the clinical outcome after the therapeutic administration of radioiodine in our series.

Experimental determination of calibration settings of a commercially available radionuclide calibrator for various clinical measurement geometries and radionuclides.

Following the approach of the National Primary Laboratory of the UK (NPL) for the calibration of radionuclide calibrators, but using a commercially available instrument with no data available in the literature, the radionuclide calibrator response was investigated as a function of different measurement geometries at the "Regina Elena" National Cancer Institute (IRE) in Rome. Working with Italian National Metrology Institute for ionising radiation quantities (ENEA-INMRI), specific calibration factors with traceability to national primary standards were determined for different types of glass vials, solid capsules and plastic syringes, investigating three radionuclides with different energy spectra (Tc-99m, In-111, I-131). For each kind of syringe, calibration correction factors for different filling volumes were calculated. For Tc-99m and I-131 the difference between measured and true activity was in the range 2-7%, depending on measurement geometry. For In-111 a large percentage deviation from the true activity value was found in each geometry considered, reaching 35%. The magnitude of this difference is particularly dependent on the energies of the emitted photons.

Psoralen interstrand cross-link repair is specifically altered by an adjacent triple-stranded structure.

Targeting DNA-damaging agents to specific DNA sites by using sequence-specific DNA ligands has been successful in directing genomic modifications. The understanding of repair processing of such targeted damage and the influence of the adjacent complex is largely unknown. In this way, directed interstrand cross-links (ICLs) have already been generated by psoralen targeting. The mechanisms responsible for ICL removal are far from being understood in mammalian cells, with the proposed involvement of both mutagenic and recombinogenic pathways. Here, a unique ICL was introduced at a selected site by photoactivation of a psoralen moiety with the use of psoralen conjugates of triplex-forming oligonucleotides. The processing of psoralen ICL was evaluated in vitro and in cells for two types of cross-linked substrates, either containing a psoralen ICL alone or with an adjacent triple-stranded structure. We show that the presence of a neighbouring triplex structure interferes with different stages of psoralen ICL processing: (i) the ICL-induced DNA repair synthesis in HeLa cell extracts is inhibited by the triplex structure, as measured by the efficiency of 'true' and futile repair synthesis, stopping at the ICL site; (ii) in HeLa cells, the ICL removal via a nucleotide excision repair (NER) pathway is delayed in the presence of a neighbouring triplex; and (iii) the binding to ICL of recombinant xeroderma pigmentosum A protein, which is involved in pre-incision recruitment of NER factors is impaired by the presence of the third DNA strand. These data characterize triplex-induced modulation of ICL repair pathways at specific steps, which might have implications for the controlled induction of targeted genomic modifications and for the associated cellular responses.

Exacerbating effect of vitamin E supplementation on DNA damage induced in cultured human normal fibroblasts by UVA radiation.

The effects of vitamin E supplementation were evaluated in cultured human normal fibroblasts exposed to ultraviolet A radiation (320-380 nm) (UVA). Cells were incubated in medium containing alpha-tocopherol, alpha-tocopherol acetate or the synthetic analog Trolox for 24 h prior to UVA exposure. DNA damage in the form of frank breaks and alkali-labile sites, collectively termed single-strand breaks (SSB), was assayed by the technique of single cell gel electrophoresis (comet assay), immediately following irradiation or after different repair periods. The generation of hydrogen peroxide (H2O2) and superoxide ion (O2.-) was measured by flow cytometry through the oxidation of indicators into fluorescent dyes. It was observed that pretreatment of cells with any form of vitamin E resulted in an increased susceptibility to the photoinduction of DNA SSB and in a longer persistence of damage, whereas no significant change was observed in the production of H2O2 and O2.- reactive oxygen species, compared to untreated controls. These findings indicate that in human normal fibroblasts, exogenously added vitamin E exerts a promoting activity on DNA damage upon UVA irradiation and might lead to increased cytotoxic and mutagenic risks.

Effects of the lipophilic biscation, bis-pyridinium oxime BP12, on bioenergetics and induction of permeability transition in isolated mitochondria.

In order to further investigate the mechanism of action of bridged lipophilic bis-pyridinium oximes previously observed to interfere with mitochondrial metabolism and to induce growth arrest and apoptosis in HeLa cells (Nocentini et al., Biochem Pharmacol 53: 1543-1552, 1997), we studied the effects of a bis-pyridinium oxime with a polymethylene chain N = 12 (BP12) on isolated rat liver mitochondria. Respiration in the absence of ADP with succinate plus rotenone as substrate was not affected after treatment with various concentrations of BP12 up to 10 microM, while the ADP-stimulated respiration was slowed down, with a parallel decrease in ATP synthesis. No effects of BP12 were detected on membrane potential, ATPase activity, and inorganic phosphate transport, but the adenine nucleotide translocase was inactivated and a permeability transition of the inner membrane was induced in the presence of calcium. These data suggest that mitochondrial impairment of ATP synthesis and the formation of the permeability transition pore may be responsible for apoptotic cell death already observed in cells treated with BP12.

Rejoining kinetics of DNA single- and double-strand breaks in normal and DNA ligase-deficient cells after exposure to ultraviolet C and gamma radiation: an evaluation of ligating activities involved in different DNA repair processes.

The repair kinetics for rejoining of DNA single- and double-strand breaks after exposure to UVC or gamma radiation was measured in cells with deficiencies in DNA ligase activities and in their normal counterparts. Human 46BR cells were deficient in DNA ligase I. Hamster EM9 and EM-C11 cells were deficient in DNA ligase III activity as a consequence of mutations in the XRCC1 gene. Hamster XR-1 cells had mutation in the XRCC4 gene, whose product stimulates DNA ligase IV activity. DNA single- and double-strand breaks were assessed by the comet assay in alkaline conditions and by the technique of graded-field gel electrophoresis in neutral conditions, respectively. 46BR cells, which are known to re-ligate at a reduced rate the DNA single-strand breaks incurred during processing of damage induced by UVC but not gamma radiation, were shown to have a normal repair of radiation-induced DNA double-strand breaks. EM9 cells exhibited a reduced rate of rejoining of DNA single-strand breaks after exposure to ionizing radiation, as reported previously, as well as UVC radiation. EM-C11 cells were deficient in the repair of radiation-induced-DNA single-strand breaks but, in contrast to EM9 cells, demonstrated the same kinetics as the parental cell line in the resealing of DNA breaks resulting from exposure to UVC radiation. Both EM9 and EM-C11 cells displayed a significant defect in rejoining of radiation-induced-DNA double-strand breaks. XR-1 cells were confirmed to be highly deficient in the repair of radiation-induced DNA double-strand breaks but appeared to rejoin DNA single-strand breaks after UVC and gamma irradiation at rates close to normal. Taken together these results indicate that: (1) DNA ligase I is involved only in nucleotide excision repair; (2) DNA ligase IV plays an important role only in repair of DNA double-strand breaks; and (3) DNA ligase III is implicated in base excision repair and in repair of DNA double-strand breaks, but probably not in nucleotide excision repair.

[Use of radionuclides in the evaluation of intestinal transit time in children with idiopathic constipation]. / Impiego dei radionuclidi nella valutazione dei tempi di transito intestinale in bambini affetti da stipsi idiopatica.

Colonic transit times, in patients with chronic idiopathic constipation, in the past were estimated using radiopaque markers. Currently they are evaluated with colonic scintigraphy, which employs 111In DTPA orally, added to the usual children's breakfast in a 0.05 mCi dose. Anterior views of the abdomen are obtained at 6th, 24th, 30th, 48th, 54th, 72nd hour using a gamma camera on a 128 x 128 matrix and stored on hard disk. These images are processed in successive times, and the colon is divided in three main segments: right-, left- and recto-sigmoid-colon. Total and segmental percentage retentions are evaluated in each interval time. 58 children (35 males and 23 females), aged 1-12 years (mean 8.13), referred for chronic idiopathic constipation at Pediatric Surgery Department of Siena, were studied between January 1990 and September 1996. This group was compared with a control group formed by 15 patients (9 males and 6 females) aged 3-14 years (mean 8.53). Cutoff values, obtained in this control group, allowed us to distinguish, among the 58 children with idiopathic constipation, 6 symptomatic patients with normal colonic transit times and 52 symptomatic patients with pathologic ones. In this last group the evaluation of segmentary colonic transit times allowed us to identify 13 patients (25%) with increased right colonic transit time, 19 (36.5%) with increased left colonic transit time and 20 (38.5%) with increased recto-sigmoidal colonic transit time. Statistical survey allowed to distinguish significantly pathological subjects from control group ones.

Low catalase activity in xeroderma pigmentosum fibroblasts and SV40-transformed human cell lines is directly related to decreased intracellular levels of the cofactor, NADPH.

We have previously shown that fibroblasts from ultra-violet (UV) hypersensitive xeroderma pigmentosum patients (XP) are markedly deficient in catalase activity resulting in high intracellular levels of hydrogen peroxide (H2O2) following UV irradiation. No direct correlation between catalase activity and repair ability was found since XP variant cells which are proficient in nucleotide excision repair (NER) showed activities as low as those found in NER deficient classical XP groups A and D. However, in contrast to the skin cancer prone XP patients, another NER deficient syndrome, trichothiodystrophy (TTD), which does not exhibit any cancer predisposition, was found to present normal catalase activity. Moreover, it was found that a variety of SV40 transformed human cell lines also showed decreased catalase activities. Our previous data showed that a molecular analysis of the normal, XP, TTD or transformed human fibroblast cell lines did not reveal any differences in levels of catalase transcription or amount of catalase protein subunits. These results incited us to examine the structure/function relationship of the tetrameric active enzyme form of catalase (which is the only one able to carry out H2O2 dismutation) with its cofactor NADPH. In the present study, we have measured the effects on catalase activity after adding NADPH either to acellular extracts or during cell culture of the different cell types. The NADPH levels were also quantified directly in intact cells using flow cytometry. Our results show a clear relationship between low catalase activity and striking decrease in intracellular NADPH levels.

[Adhesion of the labia minora in children]. / L'agglutinazione delle piccole labbra nelle bambine.

Vulvar agglutination or vulva connivens in childhood is a common disease and an important source of anxiety for parents. The Authors report on the anatomic classification, the etiology and the prevalence of this genital anomaly in the pediatric age. In the 2021 patients of our series examined in the Department of Pediatrics in Poggibonsi and in the Department of Pediatric Surgery of the University of Siena during a period of 7 years and revisited in the period between January 1995 and December 1996 (follow-up: max 7 years) no cases were observed in the first month of life (793 newborn infants) and this fact might help to exclude a congenital origin. The incidence of vulvar agglutination in 1228 children was 9.8%; in the 3.6% of cases vulvar agglutination was complete, while in the 6.1% it was incomplete. 60% of complete vulvar adhesions were in 3-6 years old patients, while the incomplete one was prevalent during the first 3 years of life. In our series vulvar adhesion was more common than in other studies, but the Author report no cases of correlated urinary or genital infections. It is important to reassure parents and to maintain a conservative approach to this anomaly. Estrogen therapy in this study was applied only in a few cases, because almost all the patients healed before puberty; the surgical division was applied only in case of recurrence.

DNA damage recognition by XPA protein promotes efficient recruitment of transcription factor II H.

The human basal transcription factor IIH (TFIIH) is an essential component of the nucleotide excision repair machinery. TFIIH is required for reaction steps concomitant with or prior to the formation of dual incisions in the damaged DNA strand. To understand the mechanism underlying the recruitment of TFIIH to DNA damage sites we have analyzed i) the direct affinity of TFIIH for damaged or undamaged DNA and ii) the interaction of TFIIH with XPA.DNA complexes, formed using unirradiated or UV-irradiated DNA. Filter binding assays showed that TFIIH has some affinity for the DNA, but in contrast to XPA, does not show any preference for UV-irradiated DNA. Pull-down experiments demonstrated that TFIIH binds to XPA.DNA complexes in an UV damage-dependent manner by a direct protein-protein interaction with XPA. We propose that an enhancement of the affinity of XPA protein for TFIIH could arise from conformational changes of XPA when it binds to UV lesions on the DNA.

Transforming growth factor-beta1 enhances the lethal effects of DNA-damaging agents in a human lung-cancer cell line.

In tissue culture conditions, exogeneous active transforming growth factor-beta1 (TGF-beta1) enhances the lethal effect of DNA-damaging agents (UV-C, gamma rays, cisplatin, methotrexate and 5-fluorouracil) toward human A549 cells and mink Mv1Lu cells, as detected by the loss of their capacity to give rise to colonies; both these cell lines harbor a wild-type p53, as determined by immunoprecipitation. Contrastingly, the sole effect of the cytokine used alone is to inhibit reversibly the multiplication of the same cells without further impairing, once withdrawn from their environment, their capacity to divide and give rise to colonies. The lethal synergy between TGF-beta1 and UV-C was studied on mink and human cell lines, and the biomodulation by TGF-beta1 of cell killing by cisplatin, gamma rays, 5-fluorouracil or methotrexate was tested only on human cells. As investigated with UV-C-irradiated human A549 cells, TGF-beta1 appears to enhance apoptosis rather than to disturb the repair of DNA photolesions (mainly pyrimidine dimers) by the nucleotidic excision repair pathway according to results of nucleosomal ladder and comet tests. Our data raise the possibility that, in vivo, TGF-beta1 might affect the curative and/or undesirable secondary side effects of cancer therapy.

[Epididymitis in children: cause of acute scrotum]. / Epididimite in età pediatrica: una causa di scroto acuto.

The acute epididymitis in children is an uncommon disease which occurs mainly in adolescents. The pediatric surgeon must remember this illness whenever he observes a child with acute scrotum. The aim of this study is to review our experience at the Pediatric Surgery Department in Siena with 17 children (mean age: 7.53 years) underwent to surgery for acute scrotum and affected by primitive acute epididymitis without any other local or systemic associated disease. Our study shows that in children the acute epididymitis is more common than testicular torsion. It is often quite difficult to find the origin of epididymitis in children with no genito-urinary anomalies; so the acute scrotum and in particular the epididymitis require a standardized diagnostic and therapeutic approach based on an accurate physical exam, quickly feasible diagnostic procedures and a prompt surgical exploration of the scrotum.

[The role of transdermal anesthesia with EMLA in urologic surgery]. / Ruolo dell'anestesia transdermica con EMLA in chirurgia urologica.

The review of 50 cases of urologic diseases has allowed us to show the utility of EMLA; even if it has been limited by some practical factors, it has permitted alone or as a first step in local and loco-regional anesthesia, to carry out surgical urologic procedures, with good surgical conditions, both for the surgeon and for the patient.

Induction of mitochondrial dysfunction and apoptosis in HeLa cells by bis-pyridinium oximes, a newly synthesized family of lipophilic biscations.

When tested on HeLa cells, bis-pyridinium oximes (BPO), a family of newly synthesized molecules whose charged pyridinium moieties are linked by a linear polymethylene chain of variable length (N = 3 to 12) have been shown to possess an inhibitory effect on cell growth and finally to provoke cell death. BPO-affected cells displayed reduced mitochondrial oxygen consumption and ATP stores and were blocked in the G1 phase of the cell cycle. Mitochondrial membrane potential, as assayed with the dye 3,3'-diexyloxacarbocyanine iodide [DiOC6(3)], increased in BPO-treated cells with time of exposure. Cell growth inhibition as well mitochondrial dysfunction were observed only with derivatives having a long polymethylene linking chain (N > or = 6). Furthermore, the concentration of the compound eliciting such effects was inversely related to the number of methylene groups in the linking chain. None of the BPO with N = 6 to 12 modified the mitochondrial DNA content, relative to the nuclear DNA content. In BPO (N = 8 and N = 12)-treated cells, chromatin fragmentation and internucleosomal DNA cleavage occurred massively, indicating that the death mode induced by these compounds is apoptosis. The possible pathway of action and the potential pharmacological interest of these compounds are discussed.

6,4,4'-Trimethylangelicin photoadduct immunodetection in DNA: induction and repair in Fanconi's anemia and normal human fibroblasts.

6,4,4'-Trimethylangelicin (TMA)-photoinduced monoadducts (MAs) were detected and quantified on DNA of normal human and Fanconi's anemia (FA) fibroblasts (complementation groups A and D) by immuno-electron microscopy. TMA-modified DNA was extracted from the cells just after photoreaction, or after a subsequent 24 h repair period, for analysis of the MA processing capabilities of the different cell lines. Unmodified DNA was extracted from the control cells in parallel. The immunoreaction with antibody 7E3 was performed on single-stranded DNA fragments obtained by heat-formamide denaturation. On single-stranded DNA fragments scanned in the electron microscope, IgG-labeled MA sites appeared as isolated or clustered IgG molecules, which were not homogeneously distributed. The isolated IgG and the different clusters (doublets, triplets or near-neighbors (within a distance of 250 nucleotides)) were measured separately for induction frequency and removal. Few interstrand cross-links (CLs) were present on X-shape DNA fragments. At time zero, the distribution patterns of TMA-photoinduced IgG-labeled MA sites and CLs, and their amount per 10(6) nucleotides, were similar in the three cell lines. After the 24 h repair period, FA cells from two different genetic complementation groups demonstrated impaired incision-excision repair capabilities for both MAs (singlets or clusters) and CLs when compared with normal cells. In each cell line, the relative proportions of TMA-induced lesions remaining at time 24 h were similar to those initially induced. This implies analogous processing kinetics towards the TMA-photoinduced clusters of MAs and CLs in a given cell line.

[Acute-phase proteins in appendicitis in childhood: new findings]. / Le proteine di fase acuta nell'appendicite in età pediatrica: nuove acquisizioni.

The diagnosis of acute appendicitis in children can be sometimes difficult. The monitoring of acute phase protein response has been suggested as an accurate diagnostic procedure in these patients. This response is an aspecific event caused by phlogosis, infections and traumatisms; it is accomplished by the hepatic release of "endogenous leukocytic mediators" (L.E.M.) among which can be remembered IL-6 and IL-7. The acute phase proteins can be distinguished into positive and negative factors. Many authors used the acute phase protein response in order to stratify the severity of disease, to evaluate the efficacy of therapy and to find out any complication. They actually think that this response is useful to draw up a prognostic index in each patient. This second part of a study started in 1994 is based on the evaluation of the preoperative acute phase proteins values in pediatric patients affected by acute appendicitis underwent to surgery. The results of the statistic analysis show the utility of the evaluation of these parameters in the preoperative period; in particular G.B. count and P.C.R. rates very early with significant statistics, while the other values change later and are more difficult to interpret.

Comet assay analysis of repair of DNA strand breaks in normal and deficient human cells exposed to radiations and chemicals. Evidence for a repair pathway specificity of DNA ligation.

The induction and resealing of DNA strand breaks in a cell line with a proven defect in DNA ligase I, 46BR, and in two Bloom's syndrome cell lines, YBL6 and GM 1492, were compared to those observed in normal human 1BR/3 fibroblasts after treatment with a variety of genotoxic agents whose lesions are processed by different repair pathways. This analysis was performed using the single-cell gel electrophoresis assay. The three types of cells were found to have similar capabilities to recognize and incise ultraviolet photoproducts and also demonstrated similar amounts of DNA breaks immediately after gamma irradiation. During post-treatment incubation, 46BR cells showed a marked DNA re-ligation defect after ultraviolet radiation damage, GM 1492 cells demonstrated a highly reduced DNA joining ability after relatively high doses of ultraviolet radiation, and YBL6 cells were particularly affected in DNA re-ligation after damage by 4-nitroquinoline-1-oxide. The two Bloom's syndrome cell lines and 46BR cells had a nearly normal ability to reseal breaks resulting from gamma irradiation or treatment with xanthine plus xanthine oxidase. These findings suggest that different DNA ligases may be involved in different DNA repair pathways in human cells.

Biophysical and biological properties of newly synthesized dioxinocoumarin derivatives. II. Dark and photoinduced effects on T7 phage, yeast and HeLa cells.

The dioxinocoumarin derivatives 5H-[2]benzopyrano-[3,4-g][1,4]benzodioxin-5-one (I), 5H-[2]benzopyrano-[3,4-g][2,3]-dihydro-[1,4]benzodioxin-5-on e II, 6H-[2]benzopyrano[3,4-f]-1,4-benzodioxin-6-one (III) and 6H-[2]benzopyrano[3,4-f]-2,3-dihydro-1,4-benzodioxin-6-one (IV) were synthesized. Their biological effect was studied in the presence and absence of UVA radiation, and compared with that of 8-methoxypsoralen (8-MOP) and angelicin derivatives on T7 phage, diploid yeast (Saccharomyces cerevisiae) and HeLa cells. The photobiological activities of compounds I and III were stronger than that of 8-MOP in phage inactivation and DNA synthesis inhibition in HeLa cells, whereas compounds II and IV, with a saturated dioxin ring, showed very poor activity. The photosensitizing activity of dioxinocoumarins on phage inactivation decreased by a factor of two to three in the absence of oxygen. Treatments with compound I and UVA in the presence of oxygen modified the helical structure and stability of phage DNA and proteins. Compounds I and II were more active than IV for photoinduced cell killing in yeast, although always less active than 8-MOP. At comparable photocytotoxic levels, compounds I and III were as strong inducers of cytoplasmic "petite" mutants in yeast as angelicin, suggesting a possible monofunctional mode of action with cellular DNA.

Characterization and evaluation by 32P-postlabelling of psoralen-type DNA adducts in HeLa cells.

Samples of DNA irradiated at 405 and/or 365 nm in the presence of 8-methoxypsoralen (8-MOP) were analysed via a modified postlabelling assay using three hydrolysis enzymes other than those employed previously. These enzymes (deoxyribonucleaseI, venom phosphodiesterase and alkaline phosphatase) liberated 3'-adducted dinucleotide monophosphate instead of the 5'-modified dinucleotide monophosphate normally obtained. The first separation chromatography (D1) of samples irradiated in the presence of 8-MOP showed a single spot above the origin, and the next separation (D2) resolved this spot into two components (spots I and II). Double irradiation experiments in which samples of DNA were first irradiated at 405 nm before being irradiated at 365 nm showed that spot II could be transformed into spot I. The use of 6,4,4'-trimethylangelicin, which induced only photomonoadducts under UVA irradiation, gave only spot II. These two results indicated that spots I and II were respectively due to interstrand cross-links and monoadducts. Dose-effect experiments showed that spots I and II were dose dependent, and low-dose irradiations permitted us to measure one interstrand cross-link and two monoadducts per 10(8) base pairs.