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Current prognostic tools cannot clearly distinguish indolent and aggressive prostate cancer (PC). We hypothesized that analyzing individual contributions of epithelial and stromal components in localized PC (LPC) could improve risk stratification, as stromal subtypes may have been overlooked due to the emphasis on malignant epithelial cells. Hence, we derived molecular subtypes of PC using gene expression analysis of LPC samples from prostatectomy patients (cohort 1, n = 127) and validated these subtypes in two independent prostatectomy cohorts (cohort 2, n = 406, cohort 3, n = 126). Stroma and epithelium-specific signatures were established from laser-capture microdissection data and non-negative matrix factorization was used to identify subtypes based on these signatures. Subtypes were functionally characterized by gene set and cell type enrichment analyses, and survival analysis was conducted. Three epithelial (E1-E3) and three stromal (S1-S3) PC subtypes were identified. While subtyping based on epithelial signatures showed inconsistent associations to biochemical recurrence (BCR), subtyping by stromal signatures was significantly associated with BCR in all three cohorts, with subtype S3 indicating high BCR risk. Subtype S3 exhibited distinct features, including significantly decreased cell-polarity and myogenesis, significantly increased infiltration of M2-polarized macrophages and CD8 + T-cells compared to subtype S1. For patients clinically classified as CAPRA-S intermediate risk, S3 improved prediction of BCR. This study demonstrates the potential of stromal signatures in identification of clinically relevant PC subtypes, and further indicated that stromal characterization may enhance risk stratification in LPC and may be particularly promising in cases with high prognostic ambiguity based on clinical parameters., (© 2024. The Author(s).)

The glaciers of North Greenland are hosting enough ice to raise sea level by 2.1 m, and have long considered to be stable. This part of Greenland is buttressed by the last remaining ice shelves of the ice sheet. Here, we show that since 1978, ice shelves in North Greenland have lost more than 35% of their total volume, three of them collapsing completely. For the floating ice shelves that remain we observe a widespread increase in ice shelf mass losses, that are dominated by enhanced basal melting rates. Between 2000 and 2020, there was a widespread increase in basal melt rates that closely follows a rise in the ocean temperature. These glaciers are showing a direct dynamical response to ice shelf changes with retreating grounding lines and increased ice discharge. These results suggest that, under future projections of ocean thermal forcing, basal melting rates will continue to rise or remain at high level, which may have dramatic consequences for the stability of Greenlandic glaciers., (© 2023. The Author(s).)

Background: Coarctation of the aorta (CoA), a congenital narrowing of the proximal descending thoracic aorta, is a relatively common form of congenital heart disease. Untreated significant CoA has a major impact on morbidity and mortality. In the past 3 decades, transcatheter intervention (TCI) for CoA has evolved as an alternative to surgery., Objectives: The authors report on all TCIs for CoA performed from 2000 to 2016 in 4 countries covering 25 million inhabitants, with a mean follow-up duration of 6.9 years., Methods: During the study period, 683 interventions were performed on 542 patients., Results: The procedural success rate was 88%, with 9% considered partly successful. Complications at the intervention site occurred in 3.5% of interventions and at the access site in 3.5%. There was no in-hospital mortality. During follow-up, TCI for CoA reduced the presence of hypertension significantly from 73% to 34%, but despite this, many patients remained hypertensive and in need of continuous antihypertensive treatment. Moreover, 8% to 9% of patients needed aortic and/or aortic valve surgery during follow-up., Conclusions: TCI for CoA can be performed with a low risk for complications. Lifetime follow-up after TCI for CoA seems warranted., Competing Interests: Funding Support and Author Disclosures This work was funded by the Swedish state under the ALF agreement (grant 236611) and the Swedish Heart-Lung Foundation (grant 20180644) and Forskningsstiftelsen vid kardiologiska sektionen SU/Ostra Hospital. This study received no financial or other industry support. The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2023 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

The Greenland ice sheet has been one of the largest sources of sea-level rise since the early 2000s. However, basal melt has not been included explicitly in assessments of ice-sheet mass loss so far. Here, we present the first estimate of the total and regional basal melt produced by the ice sheet and the recent change in basal melt through time. We find that the ice sheet's present basal melt production is 21.4 +4.4/-4.0 Gt per year, and that melt generated by basal friction is responsible for about half of this volume. We estimate that basal melting has increased by 2.9 ± 5.2 Gt during the first decade of the 2000s. As the Arctic warms, we anticipate that basal melt will continue to increase due to faster ice flow and more surface melting thus compounding current mass loss trends, enhancing solid ice discharge, and modifying fjord circulation.

High coverage is essential for the effectiveness of national screening programmes. Identifying non-screeners across different screening programmes may help inform strategies to improve uptake. This study aims to analyse the association between previous cervical cancer screening (CCS) coverage and participation in breast cancer screening (BCS). This historical register-based cohort study included 91,787 Danish women aged 50-64 years who were invited to participate in the first organised round of BCS in the Central Denmark Region (CDR) in 2008-09. CCS coverage was defined as having a smear registered in the 5 1/2 years preceding the BCS, and BCS participants were divided into participants and non-participants and further categorised as active non-participants (ANP) if they cancelled and passive non-participants (PNP) if they abstained from the appointment. Of all 91,787 women included in the study, 62,391 (68%) were covered both by CCS and participated in BCS. Women not covered by CCS were more likely to be non-participants in BCS than women covered by CCS (PRRadjusted = 2.80, 95% CI: 2.68-2.93). Both PNP (PRRadjusted = 3.99, 95% CI: 3.80-4.19) and ANP (PRRadjusted = 2.50, 95% CI: 2.34-2.68) were more likely not to be covered by the CCS. In conclusion, non-coverage by CCS was strongly associated with nonparticipation in BCS. Specific groups of women only participated in one screening programme. To increase uptake, future interventions may specifically target these groups.

Background: The treatment of congenital heart (CHD) has changed rapidly., Objectives: The authors reviewed CHD treatment through a 39-year nationwide population-based study on congenital heart surgery and catheter-based interventions, unbiased by referral patterns., Methods: Using medical registries, the authors identified children (<18 years of age) treated for CHD in Denmark from 1977 to 2015, their need for reinterventions, and their long-term survival. Ten controls per patient, matched by sex and year of birth, allowed comparison with the background population. Survival was described using Kaplan-Meier curves., Results: A total of 9,372 patients underwent 11,968 cardiac surgeries and 1,912 catheter-based interventions. Median age at first procedure decreased from 3.4 years (5th and 95th percentiles: 0.01 to 15.4 years) in 1977 to 1989 (period 1), 0.8 years (5th and 95th percentiles: 0.003 to 13.8 years) in 1990 to 2002 (period 2), and to 0.6 years (5th and 95th percentiles: 0.0 to 14.9 years) in 2003 to 2015 (period 3). More patients were born preterm (<37 weeks) in period 3 compared with those in period 1 (18.5% vs. 6.7%). Catheter-based interventions, not recorded before 1990, were increasingly used as the initial procedure in 5.8% of patients in period 2 and 25.9% of patients in period 3. An increasing part of the population did not undergo surgery at all (4.8% in period 2; 24.0% in period 3). Thirty-day survival increased from 97% (period 1) to 98% (period 2) to 100% (period 3). Ten-year survival increased from 80% (period 1) to 87% (period 2) to 93% (period 3). Compared with the background population, CHD was associated with lower survival in all 3 time periods., Conclusions: Interventional treatment of CHD has evolved from 1977 to 2015 and is now performed on younger and more preterm patients, often with catheter-based interventions. However, compared with the background population, survival remains significantly reduced., (Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objective: Few studies have reported procedure complications or adverse events for transcatheter interventions in unselected congenital heart disease cohorts. We report our 23-year experience with transcatheter interventions in congenital heart disease and examine predictors for adverse events., Design and Setting: This study was a population-based cohort study of children and adults with congenital heart disease, covering a population of 3 million inhabitants in western Denmark. Adverse events were subdivided into 5 levels according to their severity. Procedure-type risk category, age, weight, and year of procedure were included in multivariate logistic models to identify risk factors for adverse events., Results: Between 1990 and 2012, 1595 patients had 1878 catheter-based interventions performed. We identified 241 adverse events, corresponding to 13% of the procedures; 58 (3%) were considered to be of high severity. During the study period, there was an increase in number of procedures per year (P < .001) and a decrease in frequency of adverse events (P = .01). Procedure-type categories 3 and 4 had increased risk of adverse events when compared with category 2, with odds ratios of 1.7 (95% confidence interval [CI]: 1.2-2.3) for category 3 and 2.3 for category 4 (95% CI: 1.4-3.6). Age and weight at catheterization were not independently associated with adverse events., Conclusions: We found an increase in number of procedures over time and a decrease in frequency of adverse events. Higher procedure-type risk categories were associated with increased risk of adverse events., (© 2014 Wiley Periodicals, Inc.)

Objectives: We investigated whether open-heart surgery with the use of extracorporeal circulation has an impact on menstrual bleeding., Methods: The menstrual bleeding pattern was registered in fertile women undergoing open-heart surgery in 2010-12. Haematocrit and 24-h postoperative bleeding were compared with those of men undergoing cardiac surgery., Results: Women (n = 22), with mean age of 36 (range 17-60) years, were operated on and hospitalized for 4-5 postoperative days. The mean preoperative haematocrit was 40% (range 32-60%), and mean haematocrit at discharge was 32% (range 26-37%). Mean postoperative bleeding in the first 24 h was 312 (range 50-1442) ml. They underwent surgery for atrial septal defect (n = 5), composite graft/David procedure (n = 4), pulmonary or aortic valve replacement (n = 6), myxoma (n = 2), mitral valvuloplasty (n = 2), ascending aortic aneurysm (n = 1), aortic coarctation (n = 1) and total cavopulmonary connection (n = 1). Unplanned menstrual bleeding (lasting 2-5 days) was detected in 13 (60%) patients. Of them, 4 were 1-7 days early, 4 were 8-14 days early, 3 were 1-7 days late and 2 had menstruation despite having had menstrual bleeding within the last 2 weeks. None had unusually large or long-lasting menstrual bleeding. Ten women took oral contraceptives, 8 of whom had unexpected menstrual bleeding during admission. Men (n = 22), with a mean age of 35 (range 17-54) years, had mean bleeding of 331 (range 160-796) ml postoperatively, which was not statistically significantly different from the women's. The mean preoperative haematocrit was 40% (range 29-49%) among men, while haematocrit at discharge was 32% (28-41), not significantly different from that seen in the female subgroup., Conclusions: Menstrual bleeding patterns are disturbed by open-heart surgery in the majority of fertile women. Nevertheless, the unexpected menstrual bleeding is neither particularly long-lasting nor of large quantity, and the postoperative surgical bleeding is unaffected. We recommend that information about menstrual cycle disturbances related to cardiac surgery be provided, but no special precautions be taken, when operating on women of fertile age.

Progressive cyanosis often develops following Kawashima operation in patients with left atrial isomerism, interrupted inferior vena cava and hepatic veins draining to the atria. Knowledge on the timing and extend of resolution following hepatic venous redirection is sparse. A girl developed progressing cyanosis following Kawashima operation at the age of ten months. Arterial oxygen saturations at rest dropped to 60-65%. Surgical redirection of hepatic veins into the cavopulmonary circulation at the age of three years had no immediate effect. However, arterial oxygen saturations increased gradually over nine months to 90-93% and 95-100% after three years.

Background: We investigated the natural history, outcomes of myectomy, and impact on survival of obstructive hypertrophic cardiomyopathy (HCM) in childhood., Methods: All 120 children diagnosed with HCM between 1971 and 2006 were studied. Available echocardiograms (n=685) were incorporated in regression analyses adjusted for repeated measures. Multiphase parametric models and competing risks methodology were used to define outcomes., Results: Left ventricular outflow tract (LVOT) obstruction (peak gradient>30 mm Hg) developed in 61 (50%) of the cohort. Onset of obstructive features occurred during 2 discrete periods of risk; either during infancy (within 3 years of age), or otherwise later during early adolescence and beyond. Among children with obstructive HCM, the following 2 distinct groups were apparent: (1) those with peak LVOT gradients less than 65 mm Hg showed hemodynamic improvement (p<0.001) with medical strategies; and (2) those with peak gradients greater than 65 mm Hg instead had accelerated septal hypertrophy (p<0.001) with progression of peak gradients (p<0.01), and therefore underwent myectomy. Myectomy restored the gradient (mean 57 mm Hg reduction, 95% confidence interval 25 to 88, p<0.01) to nonobstructive levels. Furthermore, peak gradients and septal hypertrophy did not progress thereafter; they instead mirrored the natural history of nonobstructive HCM. Overall, in our experience, obstructive disease (or need for myectomy) did not influence late risk of death compared with children with nonobstructive HCM., Conclusions: Obstructive HCM is phenotypically heterogeneous in childhood. Peak gradients less than 65 mm Hg respond well to nonsurgical management. The patient subset with higher gradients (>65 mm Hg) instead responds poorly and early myectomy should be pursued. Reassuringly, late survival in this cohort was not compromised by development of obstructive symptoms., (Copyright © 2012 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Objectives: The Risk Adjusted Classification for Congenital Heart Surgery can predict early mortality. However, the relation to long-term outcome in terms of mortality and morbidity is unknown., Design: We did a population-based follow-up study of 801 children undergoing congenital heart surgery between 1996 and 2002. All patients were followed from surgery until death or January 1, 2008. Operations were classified according to the Risk Adjusted Classification for Congenital Heart Surgery. Each patient was matched by age and sex with 10 population controls. Cox regression analysis, area under the receiver operator curve and competing risk analysis were used for the analyses., Results: Overall follow-up was 99.6%. The distribution of the Risk Adjusted Classification for Congenital Heart Surgery was: Category one 20%, category two 37%, category three 27%, category four 8%, category five 0% and category six 2%. Overall survival after a median follow-up of 8.2 years was 86% (95% confidence interval: 83-88%), with 54 early deaths occurring within 30 days after surgery and 57 late deaths. Long-term survival in those who were alive 30 days after surgery was 92% (90-94%); ranging from 98% (93-100%) in risk category one to 33% (5-68%) in category six. Survival overall and beyond 30 days was lower in each risk category than in controls (P < .001). During follow-up, 124 (15%) patients had new operations and 106 (13%) catheter-based interventions. These events were more frequent in category three, four, and six compared with category one, with no difference between category one and two. The area under the receiver operator curve for long-term mortality was 0.81 (95% confidence interval 0.75-0.87)., Conclusions: Children operated for congenital heart disease have impaired survival and often undergo new operations or catheter-based interventions. The risk of these events is related to the surgical complexity according to the Risk Adjusted Classification for Congenital Heart Surgery., (© 2011 Copyright the Authors. Congenital Heart Disease © 2011 Wiley Periodicals, Inc.)

Background: Extracardiac total cavo-pulmonary connection (TCPC) bypasses the right atrium and has in theory better hemodynamics than intraatrial TCPC repair. Both are thought to have inferior hemodynamics compared with a normal circulation. Direct comparison of flow rates at rest and during exercise with magnetic resonance imaging technique have not been performed., Methods: The study comprised 20 children. Six children (median age, 9.3 years; interquartile range, 2.2) had undergone extracardiac TCPC. Eight children (median age, 8.9 years; interquartile range, 5.0) had an intraatrial TCPC, and 6 children (median age, 10.3 years; interquartile range, 2.6) were healthy control subjects. Blood flows in the aorta, inferior vena cava, and superior vena cava were measured at rest and during two levels of submaximal supine bicycle exercise (0.5 W/kg and 1.0 W/kg) using a magnetic resonance imaging scanner mounted with a bicycle., Results: Heart rate, respiratory rate, inspiratory fraction, and blood flow rates in the aorta and inferior vena cava increased equally in all three groups. If patients were grouped together, flow rates were significantly lower, and the inspiratory flow fraction in the inferior vena cava was significantly higher, than in control subjects. Retrograde flows were observed in all three groups at rest but tapered off with exercise., Conclusions: At submaximal levels of lower limb exercise, patients with extracardiac as well as intraatrial TCPC showed a similar increase in respiration, heart rate, and aortic and caval flow rates as healthy control subjects. This is in accordance with the observation that many patients with TCPC perform well during daily life activities.

Objective: To review the literature on the relationship between obesity and sexual function., Method: A search in the medical literature from 1966 and onwards was carried out through Medline and Embase for publications on obesity, in combination with Medical Subject Heading words related to sexual function and dysfunction., Comments: Four prospective and seven cross-sectional studies were found describing association between obesity and erectile dysfunction (ED). One cross-sectional study was found describing obesity and female sexual dysfunction (FSD). The prospective studies on ED all demonstrated a direct association and so did five of the seven cross-sectional studies. The single FSD study did not find any relationship. Eight intervention studies on weight loss and sexual difficulties were identified. All included few individuals and results were mixed even if most indicated an increase of sexual activity among both men and women after weight loss intervention., Conclusion: Support for the assumption that obesity is associated with ED was found in both prospective and cross-sectional studies. FSD was not adequately described in the literature and prospective studies are needed here. Results from weight loss intervention studies are less conclusive, but also point toward improvement in sexual dysfunction with reduced weight.

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by approximately 25-fold in WT MEFs, but only by approximately 4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency approximately 23-fold in WT MEFs, but only approximately 4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, approximately 59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only approximately 28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

Objective: The Risk Adjusted classification for Congenital Heart Surgery (RACHS-1) was created in order to compare in-hospital mortality for groups of children undergoing surgery for congenital heart disease. The method was evaluated with two large multi-institutional data sets-the Paediatric Cardiac Care Consortium (PCCC) and Hospital Discharge (HD) data from three states in the USA. The RACHS-1 classification was later applied to a large German paediatric cardiac surgery population in Bad Oeynhausen (BO), where it was found that the RACHS-1 categories were also associated with length of stay. We applied the RACHS-1 classification to the 957 operations performed during January 1996 to December 2002 at Skejby Sygehus, Denmark and we examined the association between the RACHS-1 categories, in-hospital mortality and length of stay in the Intensive Care Unit., Methods: The operations were classified according to the six RACHS-1 categories by matching the procedure of each patient with a risk category. The ability of the RACHS-1 classification to predict mortality in our population was examined by estimating the area under the receiver operator characteristic (ROC) curve. Likelihood ratio chi(2) tests were used to compare the distribution of RACHS-1 categories and the distribution of mortality with PCCC, HD and BO. Linear regression was used to examine the correlation between the RACHS-1 categories and length of stay in the Intensive Care Unit., Results: The RACHS-1 category frequencies in our population were: category 1: 18.4%, category 2: 37.4%, category 3: 34.6%, category 4: 8.2%, category 5: 0% and category 6: 1.5%. The overall ability of the RACHS-1 classification to predict in-hospital mortality (area under the ROC curve 0.741; 95% confidence interval=0.690; 0.791) was equal to the findings from larger populations. We found no differences in the category specific mortality when comparing with the larger reported series. There was a positive association between RACHS-1 category and length of stay in the Intensive Care Unit., Conclusions: The RACHS-1 classification can also be used to predict in-hospital mortality and length of stay in the Intensive Care Unit in a small volume centre.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

Chronic alveolar hypoxia is the major cause of pulmonary hypertension. The cellular mechanisms involved in hypoxia- induced pulmonary arterial remodeling are still poorly understood. Mitogen-activated protein kinase (MAPK) is a key enzyme in the signaling pathway leading to cellular growth and proliferation. The purpose of this investigation was to determine the roles that MAPKs, specifically Jun-N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 kinase, play in the hypoxia-induced pulmonary arterial remodeling. Rats were exposed to normobaric hypoxia (10% O(2)) for 1, 3, 7, or 14 d. Hypoxia caused significant remodeling in the pulmonary artery characterized by thickening of pulmonary arterial wall and increases in tissue mass and total RNA. JNK, ERK, and p38 kinase tyrosine phosphorylations and their activities were significantly increased by hypoxia. JNK activation peaked at Day 1 and ERK/p38 kinase activation peaked after 7 d of hypoxia. The results from immunohistochemistry show that hypoxia increased phospho-MAPK staining in both large and small intrapulmonary arteries. Hypoxia also upregulated vascular endothelial growth factor messenger RNA (mRNA) and platelet-derived growth factor receptor mRNA levels in pulmonary artery with a time course correlated to the activation of ERK and p38 kinase. The gene expressions of c-jun, c-fos, and egr-1, known as downstream effectors of MAPK, were also investigated. Hypoxia upregulated egr-1 mRNA but downregulated c-jun and c-fos mRNAs. These data suggest that hypoxia-induced activation of JNK is an early response to hypoxic stress and that activation of ERK and p38 kinase appears to be associated with hypoxia-induced pulmonary arterial remodeling.

In current systems for molecular cloning of eukaryotic genes, bacterial cells are routinely utilized as intermediate hosts. We investigated the possibility of using a viral system for cloning DNA fragments independent of bacterial cell usage. In this report, we provide an alternative approach for molecular cloning of DNA fragments in eukaryotic cells by utilizing the inverted terminal repeats (ITRs) of the genome of a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). We constructed a series of chimeric linear duplex DNA molecules, ranging in length from 1.8 to 7.2 kb, containing the cruciform structures of AAV-ITRs at both ends. These 'no-end' (NE) DNA structures, when transfected into adenovirus-infected human cells in the presence of AAV replication proteins (Rep), underwent DNA replication. Furthermore, in the presence of AAV capsid proteins (Cap), all replicated DNA molecules of less than 5.0 kb were packaged into mature, biologically active AAV progeny virions. When a chimeric NE DNA (NE-neo) containing a gene (neo) encoding resistance to neomycin was transfected into human cells, neoR clones could be readily isolated in the presence of G418 (Geneticin). Southern-blot analysis of genomic DNA of several independently isolated neoR clones suggested stable integration of the NE-neo DNA into the host chromosomal DNA. AAV-ITRs, therefore, offer an alternative system for molecular cloning, as well as packaging of DNA fragments in mammalian cells independent of bacterial cell usage.

A polymerase chain reaction was used to amplify a 137-base-pair sequence of DNA from a Chlamydia trachomatis plasmid. Various parameters of the polymerase chain reaction were explored, and it was found that two short steps per reaction cycle were sufficient to achieve 10(12)-fold amplification in less than 1 h. By use of this procedure, 10(-18) g of a sequence of plasmid DNA, representing the amount of that sequence found in one C. trachomatis bacterium, was amplified to the point where it was clearly visible on an ethidium bromide-stained polyacrylamide gel under UV light. DNA from intact cells from each of the 15 serovars of C. trachomatis could also be amplified for visualization. With this procedure, the presence or absence of C. trachomatis DNA in a sample could be established in less than 1.5 h. The speed and extreme sensitivity of this detection procedure may make it a useful method for the detection of C. trachomatis, and similar techniques should be possible for any type of bacteria.

Human proline-rich proteins (PRPs) are polymorphic, homologous in sequence, and linked in a cluster called the human salivary protein complex (SPC). Recently this complex was localized to human chromosome band 12p13.2 (Mamula et al., Cytogenet. Cell Genet. 39:279, 1985). We have isolated a PRP cDNA, EO27, from a human parotid gland library, identified it by DNA sequencing, and used it to study the molecular and cellular biology of PRP production. Cell-free translation and mRNA characterization with EO27 indicate that the numerous PRPs seen in saliva are produced from relatively few, large precursors, probably by posttranslational cleavage. This supports an hypothesis originally proposed by Friedman and Karn in 1977 (Am. J. Hum. Genet. 29:44 A; Biochem. Genet. 15:549) and later supported by biochemical studies (Karn et al., Biochem Genet. 17:1061, 1979) and molecular studies (Mamula et al., Fed. Proc. 43:1522, 1984; Maeda et al., J. Biol. Chem. 260:1123, 1985). EO27 was also used in this study to localize PRP mRNA production to the acinar cells of the parotid gland by in situ hybridization.

Analysis of two agnogene mutants, dl2304 deleted over the entire agnogene and in2379 carrying a 2-base insert, indicated that the mutant phenotype of small plaque formation must be the result of a defect late in the maturation pathway. Both mutants were removed from the pool of molecules available for replication with wild-type kinetics. Whereas dl2304 was somewhat reduced in its rate of progression from chromatin to previrions-virions, in2379, which produced even smaller plaques than dl2304 did, progressed with wild-type kinetics. Therefore, the agnoprotein was not required for progression from chromatin to previrions.

Elementary bodies of lymphogranuloma venereum (LGV) strains of Chlamydia trachomatis contain, in addition to the genomic DNA, a 6.7 kb plasmid. The plasmid from serovar L2 (434-B) was cloned at the BamHI site of pBR327 into Escherichia coli and a restriction cleavage map of this pLGV125 recombinant plasmid determined. All 15 C. trachomatis serovars contained DNA sequences that hybridized with pLGV125. When total DNA from L2 elementary bodies was used as a probe in Southern blotting and spot hybridization, serovars L1, L2 and L3 exhibited significant homology. The detection level of homologous DNA was 100 pg and LGV DNA was detectable in infected cells when total L2 probe was used in the nucleic acid hybridization test. These DNA probes may be useful as investigative and diagnostic reagents for C. trachomatis.

A protein methylation reaction involved in chemotaxis of Escherichia coli has been identified. The involvement of this reaction in chemotaxis in indicated by four lines of evidence. (a) The methylation reaction is altered in several classes of generally nonchemotactic mutants and is coreverted with the chemotaxis defects. (b) The methylation level of the protein is affected by chemotactic stimuli. (c) The transferred methyl group is derived from methionine and is labile, in accord with the known fact that chemotaxis requires a continuous supply of methionine. (d) Methylation is abnormal in various mutants having defective or missing flagella.

To facilitate genetic analysis of the human pathogenic parvovirus B19, we constructed a hybrid B19 viral genome in which the defective B19 inverted terminal repeats were replaced with the full-length inverted terminal repeats from a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The hybrid AAV-B19 genome was rescued from a recombinant plasmid and then the DNA was replicated upon transfection into adenovirus 2-infected human KB cells in the presence of AAV genes coding for proteins required for AAV DNA replication (AAV-Rep proteins). In addition, in the presence of AAV genes coding for the viral capsid proteins (AAV-Cap proteins), the rescued/replicated hybrid AAV-B19 genomes were packed into mature AAV progeny virions, which were subsequently released into culture supernatants. The recombinant AAV-B19 progeny virions were infectious for normal human bone marrow cells and strongly suppressed erythropoiesis in vitro. The availability of an infectious recombinant B19 virus should facilitate the mutational analysis of the viral genome, which, in turn, may yield information on individual viral gene functions in B19-induced pathogenesis. The hybrid AAV-B19 genome may also prove to be a useful vector for gene transfer in human bone marrow cells.

The source of energy for bacterial motility is the intermediate in oxidative phosphorylation, not ATP directly. For chemotaxis, however, there is an additional requirement, presumably ATP. These conclusions are based on the following findings. (i) Unlike their parents, mutants of Escherichia coli and Salmonella typhimurium that are blocked in the conversion of ATP to the intermediate of oxidative phosphorylation failed to swim anaerobically, even when they produced ATP. When respiration was restored to the mutants, motility was simultaneously restored. (ii) Carbonylcyanide m-chlorophenylhydrazone, which uncouples oxidative phosphorylation, completely inhibited motility even though ATP remained present. (iii) Arsenate did not inhibit motility in the presence of an oxidizable substrate, though it did reduce ATP levels to less than 0.3% (iv) Arsenate completely inhibited chemotaxis under conditions where motility was normal.

The omc gene, encoding the outer membrane protein-macromolecular complex (OMP-MC), was cloned in two pieces from Neisseria gonorrhoeae 2686. The 5' fragment of the omc gene included a promoter sequence, as indicated by its unregulated expression in Escherichia coli. Attempts to reconstruct an intact omc gene were unsuccessful, suggesting that expression of the complete OMP-MC protein was toxic to E. coli. Complete sequence determination revealed a coding sequence of 2,133 nucleotides; the deduced amino acid sequence indicated a mature protein of 687 amino acids with an NH2-terminal signal peptide of 24 amino acids. Analysis of the deduced amino acid sequence revealed that the NH2-terminal half of OMP-MC is generally hydrophilic, while the COOH-terminal portion contains alternating hydrophobic and hydrophilic regions. Serological analyses demonstrated that the NH2-terminal portion of OMP-MC is exposed on the gonococcal surface and the COOH-terminal portion is membrane associated.

Pulse-labeled simian virus 40 (SV40) DNA is removed from the pool of molecules available for replication (i.e., it ceases to reenter replication) a few hours after synthesis. We studied this cessation of reentry with mutants containing different deletions in the structural genes of SV40. The DNAs of two independent deletion mutants, dl-1007 (24% deletion) and dl-1003 (8% deletion), were used as templates for further DNA synthesis (i.e., they reentered replication) to a greater extent than was wild-type DNA. The alteration in reentry kinetics was not because the DNAs were smaller; other deletion mutations that were from 76 to 85% of the length of wild-type DNA (dl-BE and dl-1133 with a deletion in the late region and F8dl with a deletion in the early region) did not reenter replication to a greater extent than the wild type did. Cotransfection experiments showed that the mutant phenotypes of dl-1007 and dl-1003 were poorly complemented, if at all, by the wild type. Thus, we propose that there is a cis-acting sequence located in the HindIII E fragment of SV40, not present in either of these mutants, that promotes the efficient removal of DNA from the replication pathway.

A nucleic acid spot hybridization assay was used to detect Chlamydia trachomatis DNA. The hybridization probes included DNA isolated from elementary bodies of lymphogranuloma venereum (LGV) strains and cloned fragments of both chromosomal and plasmid DNA. The sensitivity of the test was in the range 10 to 100 pg homologous DNA and 10 in vitro infected cells. Cross-reactivity with bacterial DNA was avoided when purified chlamydia-specific DNA fragments were used as probes. C. trachomatis was detectable in most of the clinical specimens with large amounts of infectious particles. Also some isolation-negative specimens gave a positive signal in the test.

Chemotactic responses are mediated by modulation of the frequency of tumbling. Studies with methionine auxotrophs of wild-type Escherichia coli and four mutants which tumble continuously show that methionine or one of its metabolites is involved in the tumbling process. Following removal of methionine, the wild type and two mutants, after various periods of time, became unable to tumble. The presence of constant levels of chemical attractants considerably shortened these periods in the three strains and eliminated tumbling in another mutant. This effect of attractants considerably shortened these periods in the three strains and eliminated tumbling in another mutant. This effect of attractants implies that methionine or some derivative of methionine is also involved in transducing chemical stimuli to bacterial responses.