Single-Base Lesions and Mismatches Alter the Backbone Conformational Dynamics in DNA.

DNA damage has been implicated in numerous human diseases, particularly cancer, and the aging process. Single-base lesions and mismatches in DNA can be cytotoxic or mutagenic and are recognized by a DNA glycosylase during the process of base excision repair. Altered local dynamics and conformational properties in damaged DNAs have previously been suggested to assist in recognition and specificity. Herein, we use solution nuclear magnetic resonance to quantify changes in BI-BII backbone conformational dynamics due to the presence of single-base lesions in DNA, including uracil, dihydrouracil, 1,N6-ethenoadenine, and T:G mismatches. Stepwise changes to the %BII and ΔG of the BI-BII dynamic equilibrium compared to those of unmodified sequences were observed. Additionally, the equilibrium skews toward endothermicity for the phosphates nearest the lesion/mismatched base pair. Finally, the phosphates with the greatest alterations correlate with those most relevant to the repair of enzyme binding. All of these results suggest local conformational rearrangement of the DNA backbone may play a role in lesion recognition by repair enzymes.

Alarming spread of vancomycin resistant enterococci in Sweden since 2007.

The total number of persons infected or colonised with vancomycin-resistant enterococci mandatorily reported to the Swedish Institute for Infectious Disease Control increased dramatically during 2007 and 2008. During a period of twenty months from 1 July 2007 to 28 February 2009, a total of 760 cases were reported compared with 194 cases reported during the entire period from 2000 to 2006. This rise was mainly attributed to a wide dissemination of vancomycin resistant enterococci which started in a number of hospitals in Stockholm in the autumn of 2007 and was followed by dissemination in various healthcare facilities (hospitals and homes for the elderly) in a further two Swedish counties in 2008. The majority of the cases (97%) were acquired in Sweden and among these, healthcare-acquired E. faecium vanB dominated (n=634). The majority of these isolates had identical or closely related pulsed-field gel electrophoresis patterns indicating clonal dissemination in the affected counties. The median minimum inhibitory concentration of vancomycin was 32 mg/L (ranging from 4 to >128 mg/L) and of teichoplanin 0.12 mg/L (ranging from 0.06 to 0.25 mg/L). Particular emphasis was placed on countermeasures such as screening, contact tracing, cleaning procedures, education in accurate use of infection control practices as well as increasing awareness of hygiene among patients and visitors. With these measures the dissemination rate decreased substantially, but new infections with the E. faecium vanB strain were still detected.

Regional cerebral metabolic rate (positron emission tomography) during inhalation of nitrous oxide 50% in humans.

BACKGROUND: Recent studies in man have shown that cerebral blood flow increases during inhalation of nitrous oxide (N2O), a finding which is believed to be a result of an increased cerebral metabolic rate (CMR). However, this has not previously been evaluated in man. METHODS: Regional CMR(glu) (rCMR(glu)) was measured three dimensionally with positron emission tomography (PET) after injection of 2-(18F)fluoro-2-deoxy-D-glucose in 10 spontaneously breathing men (mean age 31 yr) inhaling either N2O 50% in O2 30% or O2 30% in N2. RESULTS: Global CMR(glu) in young men was 27 (3) micromol 100 g(-1) min(-1) [mean (SD)]. Inhalation of N2O 50% did not change global CMR(glu) [30 (5) micromol 100 g(-1) min(-1)] significantly, but it changed the distribution of the metabolism in the brain (P<0.0001 analysis of variance). Compared with inhalation of O2 30% in N2, N2O 50% inhalation increased the metabolism in the basal ganglia [14 (17)%, P<0.05] and thalamus [22 (23) %, P<0.05]. There was a prolonged metabolic effect of N2O inhalation seen on a succeeding PET scan with oxygen-enriched air (P<0.0001) performed 1 h after the N2O administration. CONCLUSIONS: Inhalation of N2O 50% did not change global CMR(glu), but the metabolism increased in central brain structures, an effect that was still present 1 h after discontinuation of N2O.

High levels of hepatitis B virus DNA in body fluids from chronic carriers.

Chronic infection with hepatitis B virus (HBV) is a major global health problem. Transmission is mainly blood-borne, although the route of infection during horizontal transmission in childhood is unclear. Nosocomial outbreaks of HBV have been widely reported, but have mainly focused on blood-borne transmission. There is limited knowledge of the viral load levels in other body fluids. In the present study, chronic HBV carriers were tested for the presence of HBV DNA in serum, saliva, nasopharyngeal fluid, urine and tears by means of qualitative and quantitative polymerase chain reaction (PCR) methods. Twenty-five patients who were positive for HBV DNA with both PCRs were included. Low titres in real-time PCR corresponded with weak bands in the qualitative assay. HBV DNA was found in two urine samples, 10 saliva samples, five nasopharyngeal swabs and in tear fluid from four patients. One highly viraemic HBeAg-positive carrier with serum HBV DNA levels of 7 x 10(9) genome copies had high copy numbers detected in both saliva and nasopharyngeal fluid. These results demonstrate that highly viraemic HBV carriers may have high titres of HBV DNA in other body fluids. This has particular importance for infection control programmes and regulations, underlining the importance of aiming towards regular HBV DNA testing and thus infectivity assessment of chronic carriers in order to prevent transmission.

Novel mutation in Hepatitis B virus preventing HBeAg production and resembling primate strains.

Chronic carriers of hepatitis B infection often harbour virus strains with mutations in the precore region. These mutations are temporally associated with the development of HBeAg loss and seroconversion to anti-HBe. The most common precore mutation is a stop codon at position 1896, but other mutations leading to abolished HBeAg secretion have been described. Here, a novel precore mutation introducing a lysine in the precore position 28, a sequence shared by non-human primates but not by other human isolates, is described. However, the insertion causes a frame-shift preventing the expression of HBeAg by introducing a stop codon 5 aa downstream of the mutation. Analysis of the predicted RNA secondary structure indicates that the insertion could occur without fatally affecting the stability of the stem-loop encapsidation signal.

Effects on the nervous system in different groups of workers exposed to aluminium.

OBJECTIVE: To investigate possible neurotoxic effects in groups of aluminium pot room and foundry workers, aluminium welders, and a small group of workers exposed to aluminium in the production of flake powder. METHODS: Exposure to aluminium was evaluated with aluminium concentrations in blood and urine as well as a questionnaire. The groups exposed to aluminium were compared with a group of mild steel welders. Neurotoxic effects were studied with mood and symptom questionnaires and several psychological and neurophysiological tests. RESULTS: The pot room and foundry workers showed very low aluminium uptake as their aluminium concentrations in blood and urine were close to normal, and no effects on the nervous system were detected. The group of workers exposed to flake powder had high concentrations of aluminium in blood and urine, even higher than those of the aluminium welders. However, aluminium could not be shown to affect the functioning of the nervous system in flake powder producers. Although significant effects could not be shown in the present analysis of the data on welders, the performance of the welders exposed to high concentrations of aluminium was affected according to the analyses in the original paper from this group. CONCLUSIONS: For the pot room and foundry workers no effects related to the exposure to aluminium could be found. For the group of flake powder producers exposed for a short term no effects on the nervous systems were evident despite high levels of exposure. Due to the high concentrations of aluminium in the biological samples of this group, measures to reduce the exposure to aluminium are recommended, as effects on the central nervous system might develop after protracted exposures. However, this assumption needs to be verified in further studies.

Occult hepatitis B virus after acute self-limited infection persisting for 30 years without sequence variation.

BACKGROUND/AIMS: After acute self-limited hepatitis B virus (HBV) infection, serological loss of viral antigens and appearance of anti-HBs is generally believed to signify viral clearance. Latent and occult HBV infection appearing decades after self-limited hepatitis B has not been reported, nor has the evolutionary rate of HBV DNA over the same observation period. METHODS: DNA from serum and leukocytes from 16 patients with acute self-limited hepatitis B 30 years earlier was tested by polymerase chain reaction and positive samples were sequenced. Liver tissue from four patients was also tested. Additionally, another 10 HBV strains isolated from acute HBV cases in 1969-72 were compared to 11 strains isolated from acute cases in 1998-99 in the same community. RESULTS: HBV DNA was detected in liver from two patients, but not in serum or leukocytes. The HBV strains detected in liver showed complete homology, in the sequences analyzed, to the strains originally infecting these patients. Ten strains from 1998-99 were identical in pre-S and core promoter/precore regions to strains from the same community isolated 30 years earlier. CONCLUSIONS: HBV can persist as an occult infection three decades after acute, apparently self-limited hepatitis B, and both the mutation and evolutionary rates of HBV DNA are low.

The hepatitis B virus pregenome: prediction of RNA structure and implications for the emergence of deletions.

The terminally redundant pregenomic RNA of human hepatitis B virus (HBV) comprises some 3,330 nucleotides and is a replicative intermediate in the production of the circular DNA genome. Deletions are known to arise in the HBV genome during the course of chronic infection and are sometimes associated with interferon therapy. These deletions are limited to small parts of the genome such as the 357-nucleotide pre-S1 region. Long RNA molecules such as the HBV pregenome have considerable structural flexibility and will undergo secondary structure shifts between energetically favourable states in a continuous and semi-random fashion. Since prediction of structure elements that are highly conserved in different forms of one RNA molecule is now feasible by computer modelling, we have analysed the whole HBV pregenome by two different RNA structure prediction algorithms and by new methods that exploit these algorithms. Significantly, the ends of pregenomic RNA were predicted to undergo both short-range and long-range interactions, which has relevance to our knowledge of the virus replicative cycle. By incorporating phylogenetic information relating to the 6 recognised genotypes of HBV, it was possible to highlight short secondary structures that may be common to all HBV strains. For example, although the pre-S1 region was predicted to undergo local folding of a loosely defined nature, most observed pre-S1 deletions mapped to all or part of an arm carrying a better-defined structure. The loss of such sequences may be mechanistically attributable to polymerase skipping during reverse transcription, and the possible advantages of such deletions are considered.

Recent high incidence of fulminant hepatitis in Samara, Russia: molecular analysis of prevailing hepatitis B and D virus strains.

Until 1991, the Russian city of Samara was largely isolated from other parts of Russia and the rest of the world. Very recently, Samara has seen an alarming increase in the incidence of hepatitis. The proportion of fulminant cases is unusually high. We wanted to assess the roles of hepatitis B virus (HBV) and hepatitis D virus (HDV) in acute viral hepatitis in this region by analyzing the prevailing strains of both and by determining their genotypes and possible origin. Serum samples were screened for different serological markers and by PCR followed by direct sequencing. Of the 94 HBV-positive samples (80% of which were acute infections), 37 (39%) were also HDV positive. Sixty-seven percent of the patients had anti-HCV antibodies. Twenty-five percent of all patients in the study had fulminant hepatitis. Statistically significant sex differences were found among fulminant cases. For HBV, the core promoter sequences of 62 strains were determined and all but one were found to be of genotype D. None of these had any deletions. Only one strain, from a patient with fulminant fatal hepatitis, showed multiple mutations. The pre-S2 region sequences of 31 HBV strains were also compared. Phylogenetically, these fell into two distinct groups within genotype D, suggesting different origins. For HDV, part of the region encoding the delta-antigen was sequenced from four strains. All proved to be of genotype I and were similar to Far Eastern and Eastern European strains. The contribution of intravenous drug use to the sharp increase in viral hepatitis in this unique setting is discussed.

Differentiation of hepatitis B virus genotypes D and E by ELISA using monoclonal antibodies to epitopes on the preS2-region product.

An enzyme-linked immunosorbent assay (ELISA) has been described for serological determination of hepatitis B virus genotypes, using monoclonal antibodies (mAb) against seven distinct epitopes (b, m, k, s, u, f and g) on the preS2-region products of hepatitis B surface antigen (HBsAg). The usefulness of this method for serological detection of genotype E, however, was theoretical, because no HBsAg samples of this genotype were included in the original test panel. Moreover, the predicted serotype of genotype E (bksufg) closely resembled that of genotype D (bksu, bksuf or bksug). Four HBsAg samples of genotype E were tested by the original described ELISA. The epitope g, predicted to be present in these samples by amino acid sequences, was not detected when HBsAg of genotype E was captured on a solid phase by mAb to the common determinant 'a' of HBsAg and then reacted with mAb to g (5156) labeled with horseradish peroxidase. However, the four examples of HBsAg of genotype E were captured by mAb 5156 to g on a solid phase; they were then detected by labeled mAb to the common determinant 'a'. Since epitopes f and g co-occurred on HBsAg of genotype E, HBsAg samples of this genotype were also detected, by 'sandwiching' them between immobilized mAb to g and labeled mAb to f. By contrast, HBsAg of genotype D in 90 sera was not reactive when sandwiched between mAb to f and g. Thus, this modified ELISA enables the serological determination of all six genotypes of HBsAg and, by inference, of hepatitis B virus.

Hepatocellular carcinoma in Sweden: its association with viral hepatitis, especially with hepatitis C viral genotypes.

Viral markers of chronic hepatitis were tested for in 95 frozen serum samples from 299 patients from Malmö, Sweden, with hepatocellular carcinoma (HCC), diagnosed between 1977 and 1994. Hepatitis B analysis included anti-HBc, HBsAg and, if anti-HBc positive, HBV DNA. Hepatitis C infection analysis included anti-HCV screening, RIBA, HCV RNA and HCV genotyping. HCV genotyping was also carried out in 9 HCV-viraemic HCC-patients from Gothenburg. HCV genotype distribution in HCC cases was compared with Swedish HCV-infected blood donors. Among the 95 patients from Malmö, 28 (29%) had anti-HBc, but only 5 (5%) were chronic HBV carriers, compared with 16 (17%) with chronic hepatitis C (p = 0.021). HCV-related HCC was more common among immigrants (8/16 vs. 8/79; p < 0.001). Genotyping of 25 HCV-infected cases showed genotype 1a in 6 (24%), genotype 1b in 13 (52%), genotype 2b in 4 (16%), and genotype 3a in 2 (8.0%) patients. Genotype 1b was more common among HCC patients than among blood donors (p < 0.001), but 8 of 13 genotype 1b-infected patients were from countries where genotype 1b is predominant. Among native Swedes there was no difference between the HCV genotypes infecting blood donors and those found in HCC patients.

Long-term outcome of acute hepatitis B and C in an outbreak of hepatitis in 1969-72.

The objective of this study was to investigate the epidemiology, etiology, and long-term outcome of an extended outbreak of acute hepatitis that occurred in an area of Sweden between 1969 and 1972. The outbreak was analyzed retrospectively by retesting stored frozen serum samples for the presence of hepatitis A, B and C markers. The results were compared with the diagnoses that had been determined during the outbreak. Of 180 patients, 29 (16%) had acute hepatitis A, 126 (70%) had acute hepatitis B, and eight (4.4%) had acute hepatitis C. The Australia antigen test used during the outbreak had failed to identify 21 patients with acute hepatitis B virus infection. Genotyping of the hepatitis B virus strains showed that genotype D was the most prevalent, irrespective of the transmission route. An attempt was made to follow up patients with unresolved hepatitis B virus infection, 25-27 years after the acute infection. None of the 100 patients with acute hepatitis B infection who were traced had become chronic carriers. In ten patients with hepatitis C virus infection, the follow-up showed considerable variation in the outcome, ranging from spontaneous resolution to death through liver cirrhosis. Intravenous drug users had a high prevalence of hepatitis C virus infection, with 52% testing positive for hepatitis C antibodies.

Urine from chronic hepatitis B virus carriers: implications for infectivity.

Horizontal transmission of hepatitis B virus (HBV) without apparent sexual or parenteral exposure is common in hyperendemic areas. In most cases, the route of transmission is unknown. To investigate urine as a potential source of infection, serum and urine from 56 chronic hepatitis B surface antigen (HBsAg) carriers were examined for the presence of HBV DNA using the polymerase chain reaction (PCR). Thirty-four of the patients were anti-hepatitis B e antigen (anti-HBe) positive and 22 were hepatitis B e antigen (HBeAg) positive. HBV DNA was detected in serum from 46 patients (82%) and in urine from 28 patients (50%). Most HBeAg-positive patients had HBV DNA detectable in urine (91%), whereas urine samples from anti-HBe-positive patients were found to contain HBV DNA to a lesser extent (24%). When comparing HBV DNA from serum and urine by an end-point titration PCR, a titration difference averaging 10(3) was found between serum and urine. A significant female predominance was also noted among the positive urine samples (P < 0.05), which was not correlated to the presence of haematuria. Detection of HBV DNA may indicate active viral replication, and thereby infectivity. Because a high proportion of chronic HBV carriers were found to have HBV DNA in urine, it is suggested that irrespective of HBeAg/anti-HBe status, urine should be regarded as a potential route of transmission and therefore be investigated further as a means of horizontal and nosocomial transmission of HBV.

Genotypic differences in the hepatitis B virus core promoter and precore sequences during seroconversion from HBeAg to anti-HBe.

Hepatitis B virus (HBV) strains from anti-HBe positive patients often show specific mutations in the precore gene, the core promoter region, or both. The dynamics of seroconversion in relation to the appearance of these mutations has not been studied and compared between defined HBV genotypes. Samples from patients followed during seroconversion from HBeAg to anti-HBe were amplified by polymerase chain reaction (PCR), sequenced and genotyped. Among 16 sets of samples, 6 belonged to genotype A, 6 to genotype D, 2 to genotype B, 1 to genotype C, and 1 to genotype E. Whereas strains from genotypes B, C and E showed changes in the core promoter, precore codon 28 or both, genotype A and D strains displayed a different pattern. In 4 of 6 anti-HBe positive samples from genotype A, the precore had a wild-type sequence while the core promoter sequence showed a specific TGA mutation. In another genotype A strain a precore stop mutation was preceded by a mutation in codon 15, thus conserving base-pairing at the pregenomic RNA level in this region. In contrast, all genotype D strains showed wild-type sequences in both the core promoter and precore codon 28 in pre- and post-seroconversion samples. Thus, in 8 patients with a mean follow-up time of 17 months, wild-type sequences in both the core promoter and precore codon 28 were found after seroconversion to anti-HBe. This study also confirmed, for genotype D, that HBeAg seroconversion often occurs earlier than genomic conversion.

Synthesis of specifically deuterated nucleotides for NMR studies on RNA.

Abstract We propose a strategy for NMR structure determination of RNA based on deuteration and use of specific labeling patterns. This strategy involves the use of NTPs that are deuterated in the ribose ring except for specific positions, e.g. H2', and that are either unlabeled or uniformly labeled in (13)C and (15)N in either the ribose or the base or both. Incorporation of these NTPs into an RNA sequence reduces both resonance line-width and spectral overlap. A limited number of combinations of these differently labeled NTPs in an RNA sequence suffices to obtain all relevant proton resonance assignments and structure parameters necessary for structure determination of larger systems (≫ 50 nucleotides). We describe the in vitro synthesis of the deuterated and/or (13)C/(15)N-labeled NTPs from glucose via separate enzymatic reactions. First, enzymes from the pentose-phosphate pathway efficiently convert glucose into ribose and enzymes from nucleotide biosynthesis and salvage pathways subsequently convert the ribose into nucleosides triphosphates (NTPs). The enzymes from the pentosephosphate pathway are all commercially available; the remaining enzymes have been purified from over-expressing strains. Separate enzymatic reactions were used to convert (2)H(7)- (13)C(6)-glucose into [1',3',4',5',5″-(2)H(5)-1',2',3',4',5',2,4,5,6-(13)C(9)-1,3-(15)N(2)]UTP and (2)H(7)-glucose into [1',3',4',5',5″-(2)H(5)]ATP, [1',3',4',5',5″-(2)H(5)]GTP, and [1',3',4',5',5″-(2)H(5)] CTP. The synthesis yields up to 1 gram of NTPs from 1 gram of glucose, which is about 5 to 10 times as efficient extraction for E. Coli grown on glucose. The synthesis presented here, is a modification of the method described by Tolbert & Williamson (1,2). (1)D and (2)D NMR spectra were acquired to demonstrate the utility of the new labeling patterns. The enzymatically synthesized NTPs were used in the synthesis of a 31-nucleotide RNA derived from the primer binding site of Hepatitis B virus genomic RNA to asses their efficiency in transcription.

Nosocomial transmission of hepatitis B virus infection through multiple-dose vials.

The source of acute hepatitis B virus (HBV) infection in two women (55 and 72 years old) was investigated. They displayed no risk factors for acquiring HBV infection, other than treatment with local anaesthetic injections some months previously. The HBV strains were sequenced and showed distinct homology to strains seen in Swedish intravenous drug users (IVDU). Prior to these patients' acute infection, an outbreak of HBV had occurred among IVDU in the same county. Analysis of the HBV strains from six of these IVDUs showed their core promoter, precore and pre-S sequences (679 nucleotides) to be identical to those from the two patients. Cross-contamination between samples was excluded and the most likely source of infection was thought to be multiple-dose vials of local anaesthetic that had been contaminated with the HBV strain circulating among the IVDU population in the community. We believe that multiple-dose vials have no place in modern healthcare and recommend sequence homology analysis as an alternative or additional way to trace a source of HBV infection.

Hepatitis B virus X gene 1751 to 1764 mutations: implications for HBeAg status and disease.

A translational stop in the hepatitis B virus (HBV) precore codon 28 and specific changes in the core promoter region of the X gene have been suggested to influence the level of circulating HBeAg in patients. We analysed the core promoter region and precore sequences from 59 HBV strains (including 14 from the databank) of different genotypes and from patients with different HBeAg/anti-HBe patterns. The initiator and TATA elements for transcription of precore and pregenomic RNA were highly conserved. The majority of X gene deletions in the core promoter region would lead to translational frame-shifts and stops, truncating the C-terminal end of the X protein. We found significant associations between specific changes in core promoter positions 1762 to 1764, or in precore codon 28, and absence of circulating HBeAg. For the core promoter mutations alone, this association was related to the apparent degree of liver damage (as estimated by alanine aminotransferase levels) at the time of sampling. Mutations at nucleotides 1762 and/or 1764 were often accompanied by point mutations at positions 1751 to 1755. Since mutations at nucleotide positions 1762 and 1764 have recently been shown by in vitro studies to suppress HBeAg production with a concomitant enhancement of virus production, disappearance of the HBeAg-positive phenotype associated with 1762 to 1764 mutations may thus have at least as much significance for the course of infection as HBeAg absence associated with precore codon 28 stop mutations. These observations are considered against a secondary structural model for the 3' end of HBV pregenomic RNA which also predicts enhancement of virus replication after mutation at positions 1762 and 1764.

Biokinetics of indium-111-bleomycin complex in head and neck cancer--implementations for radiochemotherapy.

Bleomycin (BLM) has been used successfully for treatment of head and neck cancer. Combining radionuclide therapy with chemotherapy is a fascinating possibility. We have studied the biokinetics of BLM labeled with indium-111 (In-111). A complex formed at low pH had an activity of 100 MBq/mg BLM. This substance was intravenously injected into 10 head and neck cancer patients in escalating doses of 75, 175, and 375 MBq. Scintigraphic data from these patients were compared with tissue samples obtained at surgery. The activity distribution and penetration into tumor tissue was not affected by increasing the injected activity. In-111-BLMC uptake was directly proportional to Ki-67/MIB-1 activity and number of mitoses in tumor tissue. Based on the biokinetics, dosimetric calculations for In-111 and In-114m have been performed. S values for realistic geometry (a phantom designed from Patient CT) have been calculated. In-114m could deliver a 4-fold absorbed radiation dose into the tumor compared with In-111. We think that In-111-BLMC could be used for radiochemotherapy in head and neck cancer or adjuvant Auger-electron therapy using In-114m combined with BLM. Further studies on cellular dosimetry should be undertaken.