Capsid-expressing DNA in AAV vectors and its elimination by use of an oversize capsid gene for vector production.

Adeno-associated virus (AAV) vectors have been shown to mediate persistent transduction in animal models of gene therapy. However, clinical trials with AAV vectors have shown that an immune response to AAV capsid protein can result in clearance of transduced cells. One source of capsid antigen is from the delivered vector virions, but expression of cap DNA impurities in AAV vector preparations might provide an alternative and persistent source of capsid antigen. Here we show that DNA without any AAV sequences can be packaged in AAV virions, and that both cap and rep DNA are packaged into AAV vectors produced by standard methods. Using a sensitive complementation assay, we also observed significant expression of capsid in cultured cells transduced with AAV vectors. In an attempt to solve this problem, we inserted a large intron into the cap gene to generate a capsid expression cassette (captron) that is too large for packaging into AAV virions. Both complementation assays and quantitative reverse-transcription PCR analysis showed that cultured cells infected with AAV vectors made with the captron plasmid expressed no detectable capsid. Elimination of transfer of capsid-expressing DNA may reduce immune responses to AAV vector-transduced cells and promote long-term expression of therapeutic proteins.

Pregnancy luteoma presenting as ovarian torsion with rupture and intra-abdominal bleeding.

We report a case of pregnancy luteoma, which had undergone torsion in a 33-year-old Indian woman, who presented with severe abdominal pain and decreasing haemoglobin levels at 33 weeks gestation. Ultrasonography showed a right adnexal mass, probably ovarian in origin, with suspicious intratumoral bleed. The pain was treated symptomatically, and the symptoms improved. A successful induction of labour was then performed at 36 weeks gestation. The pain recurred almost immediately after the delivery, and she experienced another intra-abdominal bleed. A diagnostic laparotomy and a right salpingo-oophorectomy were performed, and the diagnosis of luteoma was made based on histology. We discuss the clinical presentation of this unusual tumour, though often asymptomatic, can rarely present with severe abdominal pain from complications like torsion with rupture, leading to massive intra-abdominal bleeding. We also discuss the possible radiological investigations which can be done during pregnancy.

Clinics in diagnostic imaging (110). Right-sided tubal ligation clips complicated by the formation of an infected retention cyst.

A 35-year-old Chinese woman presented with a five-day history of right iliac fossa pain and mass. She had no significant past medical history apart from laparoscopic tubal ligation performed years ago. Pelvic ultrasonography demonstrated a well-rounded cystic mass with homogeneous internal echoes and a brightly echogenic component, compatible with tubal ligation clips. Right adnexal infected retention cyst secondary to tubal ligation clips was diagnosed. Complications of female sterilisation are rare but nevertheless have been reported and accounted for symptoms of lower abdominal pain, and should be considered as a differential diagnosis.

Clinics in diagnostic imaging (106). Viable left tubal twin ectopic pregnancy.

Live twin ectopic gestations are extremely rare. There are more than 100 reported twin tubal pregnancies but less than ten have foetal cardiac motions demonstrated in both embryos. We describe an additional patient with live twin ectopic gestation. A 32-year-old woman presented with increasing left-sided abdominal pains. She had a high beta-hCG level and a significant history of subfertility with previous surgical intervention. Transvaginal ultrasonography showed viable left tubal twin ectopic pregnancy. The diagnosis was confirmed at surgery. Factors that contribute to the risk of ectopic pregnancy, diagnosis and the management of this condition are described.

Paratubal cyst complicated by tubo-ovarian torsion: computed tomography features.

Paratubal cysts complicated by tubo-ovarian torsion are an uncommon cause of acute right iliac fossa pain in children and very rarely diagnosed accurately preoperatively. We report a constellation of diagnostic CT features in a 12-year-old pubertal girl.

Imaging findings in acute neck infection due to pyriform sinus fistula.

INTRODUCTION: Pyriform sinus fistula is a congenital branchial pouch abnormality that is often overlooked as a cause of acute neck infection in children. Our aim is to demonstrate the value of various imaging modalities (ultrasound, computed tomography [CT], barium oesophagraphy) in its diagnosis. MATERIALS AND METHODS: The preoperative imaging findings of 5 patients with surgically proven pyriform sinus fistula who presented with acute neck infection between September 2001 and March 2003 were retrospectively reviewed. CT was performed in all patients, 4 patients had barium oesophagraphy and 3 had an ultrasound scan. RESULTS: All 5 patients suffered from upper respiratory tract infection within a week of developing a tender swelling on the left side of the neck. Four patients had a history of recurrent neck infections. CT depicted inflammation of the left perithyroid soft tissue and adjacent left thyroid lobe in every case. In 2 cases, CT demonstrated the presence of a pyriform sinus fistula. Ultrasound, performed in 3 patients, correlated strongly with the CT findings. It also showed gas within a fistula in 1 case. Barium oesophagraphy clearly delineated the fistula in 3 out of 4 cases. CONCLUSION: Ultrasound and CT accurately showed the presence of acute neck infection and could demonstrate the pyriform sinus fistula. Barium oesophagraphy most clearly depicted the presence and course of the fistula. Recurrent left-sided neck infection in a child should alert the physician to the possibility of an underlying pyriform sinus fistula and imaging should be performed to confirm its presence.

Clinics in diagnostic imaging (98). Heterotopic pregnancy with an intrauterine blighted ovum and a live tubal ectopic pregnancy.

A 26-year-old woman who was six weeks amenorrhoeic presented with vaginal spotting and mild abdominal pain. She had been coming to the 24 hours women's clinic at two-day intervals for the past week with the same symptoms. Ultrasonography two days ago showed one intrauterine gestational sac (IUGS) with a yolk sac within. Her pregnancy was clomiphene-related. Repeat transvaginal ultrasonography during this admission showed an IUGS, together with a left live tubal ectopic pregnancy. Heterotopic pregnancies used to be a rare entity. Now, given the increasing rise in assisted reproduction techniques, there should be a high index of suspicion for heterotopic pregnancy as this would impact upon clinical management. The ultrasonographical features of heterotopic pregnancy and its differential diagnoses are discussed.

Clinics in diagnostic imaging (92).

A 43-year-old woman presented with a 10-day history of lower abdominal pain and two days of vaginal bleeding. Ultrasonography showed a sac-like structure within the endocervical canal. The structure had a thick echogenic rim. No foetal heartbeat was seen. Intramuscular methotrexate was given and evacuation of the uterus yielded a small amount of tissue which was revealed histologically as products of conception, confirming the diagnosis of a cervical ectopic pregnancy. The clinical and imaging features of cervical ectopic pregnancy are highlighted.

A countrywide approach to the control of non-communicable diseases--the Singapore experience.

INTRODUCTION: Many developed and developing countries are grappling with recent epidemics of non-communicable diseases and how to effectively control them. Singapore as a small, compact and highly urbanized country has similarly experienced a rapid increase in its chronic disease load and has adopted a national approach to control them. METHODS: This paper traces the strategies taken in the 80s and the 90s to control non-communicable diseases and evaluates the effectiveness of the two approaches. RESULTS: In the 80s, the control programme was largely a Ministry of Health responsibility using a persuasive approach to reach out to the target groups. This produced some results through the lowering of hypertension and cholesterol. For the 90s, the approach was one of leadership by government working with relevant agencies to reach out to all relevant sectors in the population. This National Healthy Lifestyle Programme was given top political support. The integrated and comprehensive approach used showed some improvements in health and gave the direction where efforts should be channelled. CONCLUSION: The countrywide approach taken in Singapore for the control of non-communicable diseases illustrates a model where a national framework was adopted, harnessing health promotion and disease prevention and involving personal responsibility as a key success factor. This rides on a well-developed continuum of healthcare which aims to become an integrated and seamless one.

Subungual squamous cell carcinoma: report of 2 cases.

Subungual squamous cell carcinoma (SCC) is a rare malignant tumor with an indolent course. Its etiology is unknown. It often involves the distal phalanx of the thumb or the index finger, and often presents as a chronic ulcer that is commonly misdiagnosed as chronic paronychia, pyogenic granuloma or verruca vulgaris. Approximately 150 cases of subungual SCC, including one in a Taiwanese patient, have been reported. Here, we report two cases of subungual SCC presenting as a chronic ulcer of the nail bed refractory to antibiotic treatment. One case involved the right thumb, while the other involved the right index finger. Radiographs of both cases showed an osteolytic bone lesion involving the distal phalanx. Excision biopsy revealed SCC in both cases. Both patients received amputation of the involved distal phalanx and showed no signs of reoccurrence during 5 years of follow-up. These findings suggest that all recalcitrant ulcers of the nail bed should be biopsied to facilitate early diagnosis and treatment of subungual SCC.

Ultrasonography and computed tomography in a clinical algorithm for the evaluation of suspected acute appendicitis in children.

AIM: To evaluate the roles and effectiveness of US and CT in a clinical algorithm for the evaluation of children with suspected appendicitis. METHODS: Patients with suspected appendicitis were prospectively evaluated with ultrasound (US), and in some cases with CT, after they were graded to have high, intermediate or low clinical likelihood for appendicitis. Imaging findings were made known to clinicians who then decided on a line of management. Patho-histological examination and clinical follow-up established the final diagnoses, which were correlated with the imaging findings. The effect of imaging on the management of patients was examined. RESULTS: Overall, the sensitivity of US was 92.9%, specificity 96.9%, accuracy 96.0%, positive predictive value 89.7% and negative predictive value 97.9%. Imaging did not affect the decision to operate in 13/14 (92.9%) patients in the high likelihood subgroup. Imaging guided the clinicians to the right management pathway in 26/30 (86.7%) patients in the intermediate group. 77/82 (93.9%) of US was truly negative in the low likelihood group. CT was performed in 12 patients because of unsatisfactory US scans or incompatibility between the US and the clinical findings. CT correctly diagnosed the presence or absence of appendicitis in all 12 patients. CONCLUSION: US and CT are accurate modalities in the diagnosis of acute appendicitis in children. US is most useful in patients with equivocal clinical findings. US should be the first modality used to evaluate children with suspected appendicitis. CT should be reserved for cases where US is sub-optimal or where the findings are inconsistent with the clinical findings.

HealthLine--a telephone health information and promotion service in Singapore.

HealthLine is a toll-free telephone information service on healthy living. Provided by Singapore's Ministry of Health, it augments national health promotion programmes and simultaneously feels the pulse of the common health concerns of the population. The development of the telephone information service is described and the ease of the use of telecommunication in the provision of health information and promotion is highlighted in the paper.

Sequence-specific local structural variations in solution structures of d(CGXX'CG)2 and d(CAXX'TG)2 self-complementary deoxyribonucleic acids.

In this study, the solution structures of eight self-complementary deoxyribonucleic acid molecules, d(CGXX'CG)2 and d(CAXX'TG)2 (where X = C, G, T or A and X' is complementary to X), have been determined using NMR interproton distances, endocyclic sugar torsion angles, backbone torsion angles and hydrogen bond constraints. Based on the structural data obtained in solution, a novel sequence-specific local structure function, sigma LS, composed of the sum of the contributions from the helix twist omega, base roll rho, base-pair slide delta delta and propeller twist omega, is introduced to describe their sequence-specific local structures. Sigma LS is found to produce an acceptable correlation (r = 0.96) with the relative local stability (delta G0 10) of a base-pair-step. This result demonstrates that in addition to the inter-strand purine-purine clashes, the base morphology of nearest-neighbor base-pairs is also important in defining the local geometry of base-pairs. Thus, in analyzing the base-pair structural parameter blocks of trimers are used as the basic unit whereas for the base-pair-step structural parameters, the basic unit is composed of blocks of tetramers. The omega LS-delta G(zero) 10 correlation is the first experimental evidence demonstrating the relationship between the relative local stabilities and the sequence-specific local structures of DNA duplexes. It also forms the basis for using the trimer-tetramer model for the prediction of sequence-specific local structures of deoxyribonucleic acid molecules.

The J-coupling restrained molecular mechanics (JrMM) protocol--an efficient alternative for deriving DNA endocyclic torsion angle constraints. Part I: Correlation of endocyclic torsion angles and vicinal torsion angle phi 1'2'.

An efficient alternative which makes use of the reliable 3J1'2' value to derive the endocyclic torsion angle constraints is proposed in this study. Based on the information embedded in the two plots, (i) the vicinal proton-proton J-couplings, 3J1'2', 3J1'2", 3J2'3', 3J2"3' and 3J3'4' against the pseudorotation phase angle, and (ii) 3J1'2", 3J2'3', 3J2"3' and 3J3'4' against 3J1'2'; using the calculated J-couplings obtained for a range of sugar geometries of deoxyribose ring in nucleosides and nucleotides encountered along the pseudorotation itinerary [J. van Wijk, B.D. Huckriede, J.H. Ippel and C. Altona, Methods Enzymol. 211, 286-306 (1992)], it is suggested that the vicinal 3J1'2' possesses structural information other than the vicinal torsion angle phi 1'2'. This study is divided into two parts. In Part I, a correlation diagram between the endocyclic torsion angles nu(i) (i = 0,1,2,3,4) and the restrained vicinal torsion angle phi 1'2' is obtained through the use of the J-coupling restrained molecular mechanics (JrMM) protocol. The established phi 1'2'-nu(i) correlation shows nu(i) can be deduced from the reliable 3J1'2' value and it forms the basis for developing an alternative protocol to derive endocyclic torsion angle constraints. In Part II of this series, extensive testing demonstrating the validity of the JrMM protocol to derive nu(i) for defining the sugar geometry of solution DNA molecules is presented.

The J-coupling restrained molecular mechanics (JrMM) protocol--an efficient alternative for deriving DNA endocyclic torsion angle constraints. Part II: Experimental application of the JrMM protocol.

The J-coupling restrained molecular mechanics (JrMM) protocol, which correlates deoxyribose endocyclic torsion angles and vicinal proton-proton torsion angle phi 1'2' in Part I of this study, is demonstrated to be a viable alternative to efficiently derive the endocyclic torsion angle constraints for the determination of the solution structures of DNA molecules. Extensive testing demonstrating the validity of the JrMM-derived torsion angle constraints in the restrained molecular dynamics and energy minimization structural refinement processes is performed theoretically using an energy-minimized B-DNA model and experimentally using a DNA hexamer d(CGTACG)2. The results show that only a 0.2 A difference exists between the RMSD values of the refined structures using the ideal and the JrMM-derived endocyclic torsion angle constraints. The JrMM-derived torsion angles are also determined to be in good agreement with the torsion angles derived through the use of the vicinal J-derived torsion angles. These results show that through the use of reliably measured J1'2' values and computer simulation method, the endocyclic torsion angle constraints can be derived reliably and efficiently. Thus the JrMM method serves as an alternative strategy to generate endocyclic torsion angle constraints for the determination of the solution structures of DNA molecules.

Secular trend of growth in pre-school children in Singapore.

An anthropometric study was conducted on more than 13 500 children aged 04 years in 1988. Percentile charts were plotted for height for age, weight for age, weight for height and head circumference for age. When compared to an earlier study done in 1972, it was found that Singapore pre-school children had increased in height by 4.4 %; weight by 10.9 %; and head circumference by 4.1 %. When the median height for age, weight for age and head circumference for age were plotted against those of the NCHS charts (1978), Singapore's pre-school children were generally shorter, lighter and had smaller head circumferences than their American counterparts of the same age. But, the median weight-for-height for each age group was comparable to the Americans. The results indicate the secular trend of growth in Singapore children and a trend towards reaching their full genetic potential for growth.

Quantitative determination of glucose in blood plasma and in fruit juices by combined WATR-CPMG 1H NMR spectroscopy.

The quantitative analysis of pure glucose solution < or = 225 mM (< or = 40.8 mg/mL) in 90/10 H2O/D2O was successfully completed in dilute aqueous solution by the WATR-CPMG method whereby the T2 of the water resonance is manipulated by the WATR method followed by elimination of the water peak by the CPMG pulse sequence. The method was applied to the quantitative analysis of total glucose in blood plasma from human subjects undergoing the oral glucose tolerance test in the teaching hospital, and the results were compared to those obtained using a standard glucose oxidase method in a hospital chemical pathology laboratory. The accuracy of the results obtained using the WATR-CPMG method were generally within 5% of the glucose oxidase method. The coefficient of variation was determined to be better than 4% using plasma samples of diabetic subjects. Application to the quantitative analysis of orange and guava juice was also successfully demonstrated.

Air in the cervical annulus--the lucent cleft sign.

The lucent cleft sign is a sign of soft tissue injury. It was first described as a sign of acute cervical injury but it is neither confined to cases of trauma nor the cervical region. The sign is also seen in degenerative disease and in the lumbar spine. The lucent cleft sign is seen anteriorly, as an area of hyperlucency above the superior end plate of the vertebra. It is the result of a partial tear of the annulus. In complete avulsion, the sign may be absent and the radiograph, completely normal. We report our experience of 15 patients with the lucent cleft sign. Our patients are relatively young (commonest age group 30-35 years) and the majority (10 patients) had a history of acute trauma. Four patients had non-specific (3 cervical and one lumbar) aches while in the last patient, the lucent cleft was an incidental finding. Although we only documented the disappearance of the sign in 4 patients, we believe that with healing, the sign will eventually disappear. Although the sign is transient, it is useful as it may be the only sign present in cases of trauma.

Knowledge and concerns of newly diagnosed NIDDM patients in Singapore.

This paper describes the findings of a questionnaire survey of the knowledge and concern of 152 newly diagnosed NIDDM patients presenting at the Government Polyclinic (primary health care centre). This study is part of a project on outpatient diabetic care in Singapore. It was found that 23.7% of the subjects had no education and 51.3% had only primary school education. More than eighty percent of the subjects understood the importance of diet control and weight control in their condition. More than half of the subjects, 61% of males and 52.7% of females were aware that diabetes could not be cured and that once diabetes is controlled, that medication cannot be stopped and that one must still take the medication even if one feels well. Less than 30% knew about the interpretation of blood and urine sugar findings. The two major concerns amongst the subjects were complications and the need for long-term medication. These findings will be incorporated into the next phase of the project where a health education programme for NIDDM patients will be developed and evaluated.