Challenging the current norm: Does health related quality of life data from reference populations accurately reflect baseline values in breast cancer patients? An observational cohort study comparing EORTC QLQ-C30 scores between the general Swedish population and baseline scores in breast cancer patients.

BACKGROUND: Increased overall survival in breast cancer patients has led to a growing recognition of long-term effects of cancer treatment of patients' quality of life. Health related quality of life (HRQoL) data, as measured by patient reported outcome measures (PROMs), is increasingly incorporated into clinical practice and research. A commonly used method current available to interpret HRQoL PROMs data is by comparison to reference values, often obtained from sampling of the general population. The aim of this study was to assess whether HRQoL reference values derived from the general population are an accurate representation of the baseline values of an outpatient breast clinic population. METHODS: A prospective observational cohort study was conducted by obtaining EORTC QLQ-C30 values for all patients offered an appointment in the outpatient breast clinic. These results were then compared to published baseline values in the general Swedish population, matched by gender and age. RESULTS: 568 questionnaires were returned with a response rate of 81,1 %. The outpatient breast clinic cohort reported a higher grade of symptoms, lower function and lower quality of life compared to the equivalent reference population. CONCLUSION: This study challenges the assumption that the reference values accurately reflect those of the study population which clinicians and researchers need to account for in study design and clinical practice.

Intensity-dependent effects of aerobic training on pressure pain threshold in overweight men: A randomized trial.

PURPOSE: To investigate the chronic and acute effects of high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) on pressure pain thresholds (PPT) in overweight men. METHODS: Twenty-eight participants performed stationary cycling exercise three times per week for 6 weeks. Participants were randomly allocated to HIIT (10 × 1-min intervals at 90% peak heart rate) or MICT (30 min at 65-75% peak heart rate). PPTs were assessed over the rectus femoris, tibialis anterior and upper trapezius before and after the 6-week training programme (chronic effect) as well as before and after the first, middle and final exercise sessions (acute effect). RESULTS: For chronic exercise, PPTs increased more after MICT compared to HIIT over the rectus femoris (p = 0.009, effect size r = 0.54) and tibialis anterior (p = 0.012, r = 0.54), but not the trapezius (p = 0.399, r = 0.29). The effect of acute exercise on PPT was more varied and ranged from moderate hypoalgesia to moderate hyperalgesia. Overall, however, there was no consistent change in PPT after acute exercise for HIIT or MICT (p ≥ 0.231, r ≥ -0.31 and ≤0.31). CONCLUSION: Six weeks of MICT cycling (chronic exercise) increased PPT for the lower body, but not upper body, in overweight men, whereas HIIT did not provide any hypoalgesic effect for chronic exercise. The acute effect of exercise on PPT was highly variable and negligible overall. SIGNIFICANCE: This study shows that aerobic training increases pressure pain threshold in pain-free adults. This effect was observed only for MICT over-exercised muscles, implying intensity- and site-specific effects of exercise training on pain threshold.

Perinatal health services organization for preterm births: a multinational comparison.

OBJECTIVE: To explore population characteristics, organization of health services and comparability of available information for very low birth weight or very preterm neonates born before 32 weeks' gestation in 11 high-income countries contributing data to the International Network for Evaluating Outcomes of Neonates (iNeo). STUDY DESIGN: We obtained population characteristics from public domain sources, conducted a survey of organization of maternal and neonatal health services and evaluated the comparability of data contributed to the iNeo collaboration from Australia, Canada, Finland, Israel, Italy, Japan, New Zealand, Spain, Sweden, Switzerland and UK. RESULTS: All countries have nationally funded maternal/neonatal health care with >90% of women receiving prenatal care. Preterm birth rate, maternal age, and neonatal and infant mortality rates were relatively similar across countries. Most (50 to >95%) between-hospital transports of neonates born at non-tertiary units were conducted by designated transport teams; 72% (8/11 countries) had designated transfer and 63% (7/11 countries) mandate the presence of a physician. The capacity of 'step-down' units varied between countries, with capacity for respiratory care available in <10% to >75% of units. Heterogeneity in data collection processes for benchmarking and quality improvement activities were identified. CONCLUSIONS: Comparability of healthcare outcomes for very preterm low birth weight neonates between countries requires an evaluation of differences in population coverage, healthcare services and meta-data.

Hypertensive disorders of pregnancy and outcomes of preterm infants of 24 to 28 weeks' gestation.

OBJECTIVE: To examine the relationship between hypertensive disorders of pregnancy (HDPs) and mortality and major morbidities in preterm neonates born at 24 to 28 weeks of gestation. STUDY DESIGN: Using an international cohort, we retrospectively studied 27 846 preterm neonates born at 240 to 286 weeks of gestation during 2007 to 2010 from 6 national neonatal databases. The incidence of HDP was compared across countries, and multivariable logistic regression analyses were conducted to examine the association of HDP and neonatal outcomes including mortality to discharge, bronchopulmonary dysplasia, severe brain injury, necrotizing enterocolitis and treated retinopathy of prematurity. RESULTS: The incidence of HDP in the entire cohort was 13% (range 11 to 16% across countries). HDP was associated with reduced odds of mortality (adjusted odds ratio (aOR) 0.77; 95% confidence interval (CI) 0.67 to 0.88), severe brain injury (aOR 0.74; 95% CI 0.62 to 0.89) and treated retinopathy (aOR 0.82; 95% CI 0.70 to 0.96), but increased odds of bronchopulmonary dysplasia (aOR 1.16; 95% CI 1.05 to 1.27). CONCLUSIONS: In comparison with neonates born to mothers without HDP, neonates of HDP mothers had lower odds of mortality, severe brain injury and treated retinopathy, but higher odds of bronchopulmonary dysplasia. The impact of maternal HDP on newborn outcomes was inconsistent across outcomes and among countries; therefore, further international collaboration to standardize terminology, case definition and data capture is warranted.

Low Apgar score, neonatal encephalopathy and epidural analgesia during labour: a Swedish registry-based study.

BACKGROUND: Maternal intrapartum fever (MF) is associated with neonatal sequelae, and women in labour who receive epidural analgesia (EA) are more likely to develop hyperthermia. The aims of this study were to investigate if EA and/or a diagnosis of MF were associated to adverse neonatal outcomes at a population level. METHODS: Population-based register study with data from the Swedish Birth Register and the Swedish National Patient Register, including all nulliparae (n=294,329) with singleton pregnancies who gave birth at term in Sweden 1999-2008. Neonatal outcomes analysed were Apgar score (AS)<7 at 5 min and ICD-10 diagnosis of neonatal encephalopathy (e.g. convulsions or neonatal cerebral ischaemia). Multivariate logistic regression was used to calculate adjusted odds ratios (AOR) with 95% confidence intervals (CI). RESULTS: EA was used in 44% of the deliveries. Low AS or encephalopathy was found in 1.26% and 0.39% of the children in the EA group compared with 0.80% and 0.29% in the control group. In multivariate analysis, EA was associated with increased risk with low AS, AOR 1.27 (95% CI 1.16-1.39), but not with diagnosis of encephalopathy, 1.11 (0.96-1.29). A diagnosis of MF was associated with increased risk for both low AS, 2.27 (1.71-3.02), and of neonatal encephalopathy, 1.97 (1.19-3.26). CONCLUSION: Diagnosis of MF was associated with low AS and neonatal encephalopathy, whereas EA was only associated with low AS and not with neonatal encephalopathy. The found associations might be a result of confounding by indication, which is difficult to assess in a registry-based population study.

Intrapartum GBS screening and antibiotic prophylaxis: a European consensus conference.

Group B streptococcus (GBS) remains worldwide a leading cause of severe neonatal disease. Since the end of the 1990s, various strategies for prevention of the early onset neonatal disease have been implemented and have evolved. When a universal antenatal GBS screening-based strategy is used to identify women who are given an intrapartum antimicrobial prophylaxis, a substantial reduction of incidence up to 80% has been reported in the USA as in other countries including European countries. However recommendations are still a matter of debate due to challenges and controversies on how best to identify candidates for prophylaxis and to drawbacks of intrapartum administration of antibiotics. In Europe, some countries recommend either antenatal GBS screening or risk-based strategies, or any combination, and others do not have national or any other kind of guidelines for prevention of GBS perinatal disease. Furthermore, accurate population-based data of incidence of GBS neonatal disease are not available in some countries and hamper good effectiveness evaluation of prevention strategies. To facilitate a consensus towards European guidelines for the management of pregnant women in labor and during pregnancy for the prevention of GBS perinatal disease, a conference was organized in 2013 with a group of experts in neonatology, gynecology-obstetrics and clinical microbiology coming from European representative countries. The group reviewed available data, identified areas where results were suboptimal, where revised procedures and new technologies could improve current practices for prevention of perinatal GBS disease. The key decision issued after the conference is to recommend intrapartum antimicrobial prophylaxis based on a universal intrapartum GBS screening strategy using a rapid real time testing.

Antimicrobial resistance in colonizing group B Streptococci before the implementation of a Swedish intrapartum antibiotic prophylaxis program.

The prevalence of antibiotic resistance and their genetic determinants in colonizing group B streptococci (GBS) sampled in a Swedish nationwide survey was examined. In five GBS isolates (1.3%), kanamycin/amikacin resistance and the presence of the aphA-3 gene was identified. Three of these isolates carried the aad-6 gene and were streptomycin-resistant. Screening with kanamycin and streptomycin 1,000-µg disks enabled a rapid and easy detection of these isolates. In all, 312/396 (79%) GBS were tetracycline-resistant and 95% of the examined isolates harbored the tetM gene. Among the 22 (5.5%) GBS resistant to erythromycin and/or clindamycin, the ermB gene was detected in nine isolates (41%) and erm(A/TR) in ten isolates (45%). A high level of erythromycin and clindamycin resistance with minimum inhibitory concentrations (MICs) >256 mg/L was found in four serotype V isolates that harbored ermB. The erythromycin/clindamycin resistance was distributed among all of the common serotypes Ia, Ib, II, III, IV, and V, but was not present in any of the 44 serotype III isolates associated to clonal complex 17. Screening for penicillin resistance with 1-µg oxacillin disks showed a homogenous population with a mean inhibition zone of 20 mm. A change in the present oxacillin breakpoints for GBS is suggested.

Differential effects of polymers PVP90 and Ficoll400 on storage stability and viability of Lactobacillus coryniformis Si3 freeze-dried in sucrose.

AIMS: To investigate the effect of freeze-dried Lactobacillus coryniformis Si3 on storage stability by adding polymers to sucrose-based formulations and to examine the relationship between amorphous matrix stability and cell viability. METHODS AND RESULTS: The resistance to moisture-induced sucrose crystallization and effects on the glass transition temperature (Tg) by the addition of polymers to the formulation were determined by different calorimetric techniques. Both polymers increased the amorphous matrix stability compared to the control, and poly(vinyl)pyrrolidone K90 was more effective in increasing amorphous stability than Ficoll 400. The viability of Lact. coryniformis Si3 after storage was investigated by plate counts following exposure to different moisture levels and temperatures for up to 3 months. The polymers enhanced the cellular viability to different degrees, dependent upon polymer and storage condition. CONCLUSIONS: Polymers can be used to enhance the stability of freeze-dried Lact. coryniformis Si3 products, but cell viability and matrix stability do not always correlate. The general rule of thumb to keep a highly amorphous product 50 degrees below its Tg for overall stability seemed to apply for this type of bacterial products. We showed that by combining thermal analysis with plate counts, it was possible to determine storage conditions where cell viability and matrix stability were kept high. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will aid in the rational formulation design and proper determination of storage conditions for freeze-dried and highly amorphous lactic acid bacteria formulations. We propose a hypothesis of reason for different stabilizing effects on the cells by the different polymers based on our findings and previous findings.

Impact and risk factors for early-onset group B streptococcal morbidity: analysis of a national, population-based cohort in Sweden 1997-2001.

OBJECTIVES: To study early-onset group B streptococcal (EOGBS) morbidity, mortality, and maternal risk factors. DESIGN: Observational, population-based, retrospective. Data from national registers and medical records. SETTING: Sweden, 1997-2001. POPULATION: Cohort of 640 infants with a diagnosis of GBS infection out of 435 070 live births. METHOD: Infants with diagnoses GBS sepsis (P36.0) and/or pneumonia (P23.3) were analysed. In cases with P36.0, register data were validated against infant and maternal medical records. Odds ratios (OR) were estimated by multiple logistic regression. MAIN OUTCOME MEASURES: Incidence of EOGBS morbidity, mortality, frequency of maternal risk factors and administration of intrapartum antibiotics. RESULTS: There were 319 cases with EOGBS sepsis. Blood culture verified 174 cases. There were 145 with clinical sepsis and 180 with pneumonia only. The incidences were 0.40, 0.33, and 0.41 per 1000 live births, respectively. The mortality was 7.5, 0.7, and 2.2% in respective groups. The frequencies of established maternal risk factors were: membrane rupture > or =18 hours, 44%; prematurity, 26%; temperature during labour > or =38 degrees C, 22%. Novel maternal risk factors identified in verified cases were gestational age (GA) of 37 completed weeks (OR 3.5, 1.8-6.5) and gestational diabetes (OR 3.7, 1.8-8.5). When including clinical sepsis, also epidural anaesthesia, infant large for GA, postmaturity, and high maternal age were significant risk factors. CONCLUSION: The incidence of verified EOGBS disease was 0.4 per 1000 live births with a total burden of EOGBS morbidity approximately three times higher. GA of 37 completed weeks and gestational diabetes were identified as additional significant risk factors.

Survival of the biocontrol yeast Pichia anomala after long-term storage in liquid formulations at different temperatures, assessed by flow cytometry.

AIMS: Investigate the survival of liquid formulations of the biocontrol yeast Pichia anomala J121 at different temperatures, and develop a system for comparative studies of different storage conditions and formulations. METHODS AND RESULTS: The survival of P. anomala in liquid formulations with lactose, starch and trehalose amendments was measured during prolonged storage at temperatures ranging from -20 to +30 degrees C. The relative survival of the stored cells was rapidly estimated by flow cytometry. After 4 weeks incubation at 4 and 10 degrees C, 75-90% of the cells were viable, with no significant differences between the various formulations. Supplementing the storage buffer with lactose or trehalose increased the survival after longer incubations (8 and 12 weeks) at all temperatures (-20 to 30 degrees C). Trehalose was the most effective protectant at 20 and 30 degrees C (>20% viable cells after 12 weeks at 20 degrees C). The biocontrol activity was maintained after formulation and prolonged storage of P. anomala. CONCLUSIONS: The storage potential of liquid formulated P. anomala cells can be increased by supplementation with lactose or trehalose. The combination of a custom made incubation chamber and flow cytometry was suitable to evaluate stability of P. anomala formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: Liquid formulated P. anomala have a long shelf life. The developed test system can be used to study different formulations of other biocontrol agents.

Extremely preterm cesarean delivery: a clinical study.

OBJECTIVES: To describe indications for cesarean section for extremely preterm delivery, peri- and postoperative complications and perinatal outcome. DESIGN: A case-referent study with clinical follow-up. SETTING: A tertiary perinatal center. POPULATION: All deliveries at gestational age <28 weeks at Umeå University Hospital in 1997-2003. For preterm cesarean section referents were women with elective first-time term cesarean section. METHODS: Indications for cesarean section delivery were assessed. Peri- and postoperative complications, asphyxia, and infant survival at discharge were described. RESULTS: The cesarean section rate was 75%, in one third the operation was considered as difficult. Indications for extremely preterm abdominal delivery were severe disease during pregnancy and delivery complications. Six out of ten cesarean sections were performed on fetal indication. Nonisthmic incision was performed in 20% of cases. No major postoperative complications and few minor postoperative complications were noted. Irrespective of mode of delivery, few of the infants had severe asphyxia. CONCLUSION: In balancing the risks of complications related to the surgical procedure against the purported benefits of the infant, this study adds support to the argument to deliver even extremely preterm infants by cesarean section.

Short-term outcome after active perinatal management at 23-25 weeks of gestation. A study from two Swedish tertiary care centres. Part 2: infant survival.

AIM: To determine neonatal survival rates based on both foetal (stillborn) and neonatal deaths among infants delivered at 23-25 wk, and to identify maternal and neonatal factors associated with survival. METHODS: The medical records of 224 infants who were delivered in two tertiary care centres in 1992-1998 were reviewed retrospectively. At these centres, policies of active perinatal and neonatal management were universally applied. Data were analysed by gestational age groups and considered in three time periods. Logistic regression models were used to identify factors associated with survival. RESULTS: The rate of foetal death was 5%. Of infants born alive, 63% survived to discharge. Survival rates including foetal deaths in the denominator at 23, 24 and 25 wk were 37%, 61% and 74%, respectively, and survival rates excluding foetal deaths were 43%, 63% and 77%, respectively. Of infants born with 1-min Apgar scores of 0-1, 43% survived. In the total cohort, survival rates including foetal deaths in the denominator increased from 52% in time period 1 to 61% in time period 2 and 74% in time period 3 (p < 0.02). On multivariate logistic regression analysis, higher birthweight (OR: 1.91 per 100 g increment; 95% CI: 1.45-2.52), female gender (OR: 3.33; 95% CI: 1.65-6.75), administration of antenatal steroids (OR: 2.95; 95% CI: 1.46-5.98) and intrauterine referral from a peripheral hospital (OR: 2.35; 95% CI: 1.18-4.68) were associated with survival. Apgar score < or = 3 at 1 min (OR: 0.46; 95% CI: 0.22-0.95) was associated with decreased survival. The use of antenatal steroids was protective at 23-24 wk (OR: 5.2; 95% CI: 2.0-13.7), but not at 25 wk. CONCLUSIONS: Active perinatal management that included universal initiation of neonatal intensive care virtually eliminated intrapartum stillbirths and delivery room deaths, and resulted in survival rates that compare favourably with those of recent studies. However, the policies of active care postponed death in non-survivors. Individual variations in outcome in relation to the infant's condition at birth as reflected by the Apgar scores preclude the making of treatment decisions in the delivery room.

Short-term outcome after active perinatal management at 23-25 weeks of gestation. A study from two Swedish perinatal centres. Part 3: neonatal morbidity.

AIM: To determine major neonatal morbidity in surviving infants born at 23-25 weeks, and to identify maternal and infant factors associated with major morbidity. METHODS: The medical records of 224 infants who were delivered at two tertiary care centres in 1992-1998 were reviewed retrospectively. At these centres, policies of active perinatal and neonatal management were universally applied. Of the 213 liveborn infants, 140 (66%) survived to discharge. Data were analysed by gestational age and considered in three time periods. Logistic regression models were used to identify factors associated with morbidity. RESULTS: Of the survivors, 6% had intraventricular haemorrhage grade > or = 3 (severe IVH) or periventricular leukomalacia (PVL), 15% retinopathy of prematurity > or = stage 3 (severe ROP) and 36% bronchopulmonary dysplasia (BPD). On logistic regression analysis, severe IVH or PVL was associated with duration of mechanical ventilation (odds ratio, OR: 1.53 per 1-wk increment in duration; 95% confidence interval, CI: 1.01-2.33). Severe ROP was associated with the presence of a patent ductus arteriosus (PDA) (OR: 3.31; 95% CI: 1.11-9.90) and birth in time period 3 versus time periods 1 and 2 combined (OR: 6.28; 95% CI: 2.10-18.74). BPD was associated with duration of mechanical ventilation (OR: 2.71 per 1-wk increment in duration; 95% CI: 1.76-4.18) and with the presence of any obstetric complication (OR: 2.67; 95% CI: 1.07-6.65). Gestational age and birthweight were not associated with major morbidity. Of all survivors, 81% were discharged home without severe IVH, PVL or severe ROP. CONCLUSIONS: Increased survival as a result of active perinatal and neonatal management was associated with favourable morbidity rates compared with those in recent studies. Among survivors born at 23-25 weeks, neither gestational age nor birthweight was a significant determinant of major morbidity.

Short-term outcome after active perinatal management at 23-25 weeks of gestation. A study from two Swedish tertiary care centres. Part 1: maternal and obstetric factors.

AIMS: To provide descriptive data on women who delivered at 23-25 wk of gestation, and to relate foetal and neonatal outcomes to maternal factors, obstetric management and the principal reasons for preterm birth. METHODS: Medical records of all women who had delivered in two tertiary care centres in 1992-1998 were reviewed. At the two centres, policies of active perinatal and neonatal management were universally applied. Logistic regression models were used to identify prenatal factors associated with survival. RESULTS: Of 197 women who delivered at 23-25 wk, 65% had experienced a previous miscarriage, 15% a previous stillbirth and 12% a neonatal death. The current pregnancy was the result of artificial reproduction in 13% of the women. In 71%, the pregnancy was complicated either by pre-eclampsia, chorioamnionitis, placental abruption or premature rupture of membranes. Antenatal steroids were given in 63%. Delivery was by caesarean section in 47%. The reasons for preterm birth were idiopathic preterm labour in 36%, premature rupture of membranes in 41% and physician-indicated deliveries in 23% of the mothers. Demographic details, use of antenatal steroids, caesarean section delivery and birthweight differed between mothers depending on the reason for preterm delivery. Of 224 infants, 5% were stillbirths and 63% survived to discharge. On multivariate logistic regression analysis comprising prenatally known variables, reasons for preterm birth were not associated with survival. Advanced gestational duration (OR: 2.43 per wk; 95% CI: 1.59-3.74), administration of any antenatal steroids (OR: 2.21; 95% Cl: 1.14-4.28) and intrauterine referral from a peripheral hospital (OR: 2.93; 95% CI: 1.5-5.73) were associated with survival. CONCLUSIONS: Women who deliver at 23-25 wk comprise a risk group characterized by a high risk of reproductive failure and pregnancy complications. Survival rates were similar regardless of the reason for preterm birth. Policies of active perinatal management virtually eliminated intrapartum stillbirths.

Caesarean section increases the risk of hospital care in childhood for asthma and gastroenteritis.

OBJECTIVE: To investigate if caesarean section (CS) increases the risk for childhood asthma and gastroenteritis with reference made to children born with vaginal delivery (VD). METHODS: Retrospective study of data from linked Swedish medical service registers--Medical Birth Registry (MBR) and Hospital Discharge Registry (HDR). Data were obtained from women without any background/perinatal morbidity noted, and from children without any neonatal complications. Children that had reached at least 1 year of age and were found in the HDR were considered as cases, whereas children not found in the HDR or hospitalized for other causes than asthma or gastroenteritis were defined as controls. Odds ratios (OR) stratified for year of birth, maternal age, parity and smoking in early pregnancy were calculated. Investigations were made comparing the risk for in hospital treatment for asthma or gastroenteritis in CS children and in VD siblings of CS children. The overall inpatient morbidity in CS and VD children were also investigated. RESULTS: The OR for asthma in CS children was 1.31 [95% confidence interval (CI) 1.23-1.40]. The same OR, 1.31, was found for gastroenteritis (95% CI 1.24-1.38). The OR for CS children having experienced both asthma and gastroenteritis was further increased (1.74, 95% CI 1.36-2.23). The risk for asthma in VD siblings of CS children was not significantly increased, whereas VD siblings experienced a slightly increased risk for gastroenteritis. CS children had an increased overall in hospital morbidity when compared to VD children. CONCLUSION: There is a significant increase of the risk for developing symptoms of asthma and/or gastroenteritis that motivates admission for hospital care in CS children older than 1 year. It is speculated that a disturbed intestinal colonization pattern in CS children may be a common pathogenic factor.

Actin filament polymerization regulates gliding motility by apicomplexan parasites.

Host cell entry by Toxoplasma gondii depends critically on actin filaments in the parasite, yet paradoxically, its actin is almost exclusively monomeric. In contrast to the absence of stable filaments in conventional samples, rapid-freeze electron microscopy revealed that actin filaments were formed beneath the plasma membrane of gliding parasites. To investigate the role of actin filaments in motility, we treated parasites with the filament-stabilizing drug jasplakinolide (JAS) and monitored the distribution of actin in live and fixed cells using yellow fluorescent protein (YFP)-actin. JAS treatment caused YFP-actin to redistribute to the apical and posterior ends, where filaments formed a spiral pattern subtending the plasma membrane. Although previous studies have suggested that JAS induces rigor, videomicroscopy demonstrated that JAS treatment increased the rate of parasite gliding by approximately threefold, indicating that filaments are rate limiting for motility. However, JAS also frequently reversed the normal direction of motility, disrupting forward migration and cell entry. Consistent with this alteration, subcortical filaments in JAS-treated parasites occurred in tangled plaques as opposed to the straight, roughly parallel orientation observed in control cells. These studies reveal that precisely controlled polymerization of actin filaments imparts the correct timing, duration, and directionality of gliding motility in the Apicomplexa.

Heparin binding by the HIV-1 tat protein transduction domain.

The protein transduction domain from the HIV-1 tat protein (termed PTD-tat) has been fused to the C-terminus of a model cargo protein, the IgG binding domain of streptococcal protein G. We demonstrate that PG-Ctat (PTD-tat fused to the C-terminus of protein G) binds to a heparin affinity column. PG-Ctat binds with relatively high affinity, as shown by its elution at 1.6 M NaCl. The heparin binding properties of PTD-tat are consistent with the idea that heparan sulfate, an analog of heparin found at the cell surface, plays a role in the translocation of PTD-tat fusions. We suggest that the heparin-binding properties of PTD-tat can be exploited for purification of PTD-tat fusions in the absence of affinity tags.

Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole.

Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry-derived secretory vesicles, we utilized cytochalasin D to prevent invasion, leading to accumulation of protein-rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite-containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endosomes and lysosomes. In contrast, evacuoles were recruited to pre-existing, parasite-containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two-step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by TOXOPLASMA: The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.

Genotyping of the capsule gene cluster (cps) in nontypeable group B streptococci reveals two major cps allelic variants of serotypes III and VII.

Forty group B Streptococcus (GBS) isolates obtained from Europe and the United States previously reported to be nontypeable (NT) by capsule serotype determination were subjected to buoyant density gradient centrifugation. From nearly half of the isolates capsule-expressing variants could be selected. For characterization of the remaining NT-GBS isolates, the capsule operon (cps) was amplified by the long-fragment PCR technique and compared by restriction fragment length polymorphism (RFLP) analysis. The patterns from serotype reference isolates (n = 32) were first determined and used as a comparison matrix for the NT-GBS isolates. Using two restriction enzymes, SduI and AvaII, cluster analysis revealed a high degree of similarity within serotypes but less than 88% similarity between serotypes. However, serotypes III and VII were each split in two distant RFLP clusters, which were designated III(1) and III(2) and VII(1) and VII(2), respectively. Among the isolates that remained NT after repeated Percoll gradient selections, two insertional mutants were revealed. Both were found in blood isolates and harbored insertion sequence (IS) elements within cpsD: one harbored IS1548, and the other harbored IS861. All other NT-GBS isolates could, by cluster analysis, be referred to different serotypes by comparison to the RFLP reference matrix. In pulsed-field gel electrophoresis of SmaI-restricted chromosomal DNA, patterns from allelic type 1 and 2 isolates were essentially distributed in separate clusters in serotypes III and VII. A covariation with insertion sequence IS1548 in the hylB gene was suggested for serotype III, since allelic type III(1) harboring IS1548 in hylB, clustered separately. The variation in serotype VII was not dependent on the presence of IS1548, which was not detected at any position in the type VII chromosome.