A systems biology approach to understand gut microbiota and host metabolism in morbid obesity: design of the BARIA Longitudinal Cohort Study.

INTRODUCTION: Prevalence of obesity and associated diseases, including type 2 diabetes mellitus, dyslipidaemia and non-alcoholic fatty liver disease (NAFLD), are increasing. Underlying mechanisms, especially in humans, are unclear. Bariatric surgery provides the unique opportunity to obtain biopsies and portal vein blood-samples. METHODS: The BARIA Study aims to assess how microbiota and their metabolites affect transcription in key tissues and clinical outcome in obese subjects and how baseline anthropometric and metabolic characteristics determine weight loss and glucose homeostasis after bariatric surgery. We phenotype patients undergoing bariatric surgery (predominantly laparoscopic Roux-en-Y gastric bypass), before weight loss, with biometrics, dietary and psychological questionnaires, mixed meal test (MMT) and collect fecal-samples and intra-operative biopsies from liver, adipose tissues and jejunum. We aim to include 1500 patients. A subset (approximately 25%) will undergo intra-operative portal vein blood-sampling. Fecal-samples are analyzed with shotgun metagenomics and targeted metabolomics, fasted and postprandial plasma-samples are subjected to metabolomics, and RNA is extracted from the tissues for RNAseq-analyses. Data will be integrated using state-of-the-art neuronal networks and metabolic modeling. Patient follow-up will be ten years. RESULTS: Preoperative MMT of 170 patients were analysed and clear differences were observed in glucose homeostasis between individuals. Repeated MMT in 10 patients showed satisfactory intra-individual reproducibility, with differences in plasma glucose, insulin and triglycerides within 20% of the mean difference. CONCLUSION: The BARIA study can add more understanding in how gut-microbiota affect metabolism, especially with regard to obesity, glucose metabolism and NAFLD. Identification of key factors may provide diagnostic and therapeutic leads to control the obesity-associated disease epidemic.

Distinct differences in gut microbial composition and functional potential from lean to morbidly obese subjects.

INTRODUCTION: The gut microbiome may contribute to the development of obesity. So far, the extent of microbiome variation in people with obesity has not been determined in large cohorts and for a wide range of body mass index (BMI). Here, we aimed to investigate whether the faecal microbial metagenome can explain the variance in several clinical phenotypes associated with morbid obesity. METHODS: Caucasian subjects were recruited at our hospital. Blood pressure and anthropometric measurements were taken. Dietary intake was determined using questionnaires. Shotgun metagenomic sequencing was performed on faecal samples from 177 subjects. RESULTS: Subjects without obesity (n = 82, BMI 24.7 ± 2.9 kg m-2 ) and subjects with obesity (n = 95, BMI 38.6 ± 5.1 kg m-2 ) could be clearly distinguished based on microbial composition and microbial metabolic pathways. A total number of 52 bacterial species differed significantly in people with and without obesity. Independent of dietary intake, we found that microbial pathways involved in biosynthesis of amino acids were enriched in subjects with obesity, whereas pathways involved in the degradation of amino acids were depleted. Machine learning models showed that more than half of the variance in body fat composition followed by BMI could be explained by the gut microbiome, composition and microbial metabolic pathways, compared with 6% of variation explained in triglycerides and 9% in HDL. CONCLUSION: Based on the faecal microbiota composition, we were able to separate subjects with and without obesity. In addition, we found strong associations between gut microbial amino acid metabolism and specific microbial species in relation to clinical features of obesity.

A multi-centred randomised trial of radical surgery versus adjuvant chemoradiotherapy after local excision for early rectal cancer.

BACKGROUND: Rectal cancer surgery is accompanied with high morbidity and poor long term functional outcome. Screening programs have shown a shift towards more early staged cancers. Patients with early rectal cancer can potentially benefit significantly from rectal preserving therapy. For the earliest stage cancers, local excision is sufficient when the risk of lymph node disease and subsequent recurrence is below 5 %. However, the majority of early cancers are associated with an intermediate risk of lymph node involvement (5-20 %) suggesting that local excision alone is not sufficient, while completion radical surgery, which is currently standard of care, could be a substantial overtreatment for this group of patients. METHODS/STUDY DESIGN: In this multicentre randomised trial, patients with an intermediate risk T1-2 rectal cancer, that has been locally excised using an endoluminal technique, will be randomized between adjuvant chemo-radiotherapylimited to the mesorectum and standard completion total mesorectal excision (TME). To strictly monitor the risk of locoregional recurrence in the experimental arm and enable early salvage surgery, there will be additional follow up with frequent MRI and endoscopy. The primary outcome of the study is three-year local recurrence rate. Secondary outcomes are morbidity, disease free and overall survival, stoma rate, functional outcomes, health related quality of life and costs. The design is a non inferiority study with a total sample size of 302 patients. DISCUSSION: The results of the TESAR trial will potentially demonstrate that adjuvant chemoradiotherapy is an oncological safe treatment option in patients who are confronted with the difficult clinical dilemma of a radically removed intermediate risk early rectal cancer by polypectomy or transanal surgery that is conventionally treated with subsequent radical surgery. Preserving the rectum using adjuvant radiotherapy is expected to significantly improve morbidity, function and quality of life if compared to completion TME surgery. TRIAL REGISTRATION: NCT02371304 , registration date: February 2015.

The use of Oxaliplatin or Mitomycin C in HIPEC treatment for peritoneal carcinomatosis from colorectal cancer: a comparative study.

BACKGROUND: Oxaliplatin and Mitomycin C (MMC) are both suitable as intraperitoneal chemotherapy agents in HIPEC for peritoneal carcinomatosis (PC) of colorectal cancer (CRC). METHODS: Patient cohorts from two different HIPEC-centers underwent cytoreductive surgery and HIPEC with Oxaliplatin (39 patients) and MMC (56 patients), respectively. They were compared for toxicity and survival data. The extent of PC was assessed using the Dutch 7-region count. RESULTS: The median 7-region count was 4 [range 0-7] for Oxaliplatin-patients versus 2.5 [range 1-6] for MMC-patients (P = 0.004). Median intra-operative blood loss was 650 ml [0-6,000 ml] in Oxaliplatin-patients versus 1,230 ml [range 0-5,300 ml] in MMC-patients (P < 0.001). Only MMC-patients developed neutropenia/leucopenia (26.8%, P < 0.001). After statistical correction for the extent of PC, the overall postoperative complication rate was significantly higher in MMC-patients (OR = 2.68 (95% CI: 1.04-6.91), P = 0.04), with a comparable intra-abdominal complication (IAC) rate (OR = 0.78 (95% CI: 0.30-2.03), P = 0.61), but a tendency towards more extra-abdominal complications (EAC) in MMC-patients (OR = 2.23 (95% CI: 0.91-5.43), P = 0.079). Median follow-up was significantly shorter for Oxaliplatin-patients (2.8 years) than for MMC-patients (5.1 years). Median RFS was 12.2 months [IQR: 7.2-undefined] in the Oxaliplatin-group and 13.8 months [IQR: 7.0-25.8] in the MMC-group (P = 0.87). Median OS is 37.1 months [IQR: 22.4-52.8] for Oxaliplatin-patients and 26.5 months [IQR: 16.9-64.8] for MMC-patients (P = 0.45). Logistic regression analysis (corrected for extent of PC) shows RFS (HR = 1.24 (95% CI: 0.75-2.05), P = 0.39) and OS (HR = 1.37 (95% CI: 0.74-2.54), P = 0.32) are not significantly different. CONCLUSIONS: No clear benefit in RFS and OS for HIPEC with Oxaliplatin or MMC could be demonstrated in patients with PC from CRC.

Gastric bypass and abdominal pain: think of Petersen hernia.

BACKGROUND: Laparoscopic Roux-en-Y gastric bypass (LRYGB) is one of the most commonly performed bariatric surgical procedures. A laparoscopic gastric bypass is associated with specific complications: internal herniation is one of these. CASE REPORT: A 47-year-old woman had undergone a laparoscopic Roux-en-Y gastric bypass (LRYGB) 18 months before presentation at our emergency department with mild abdominal complaints. Physical examination showed signs of an ileus in the absence of an acute abdomen. Laboratory investigations revealed no abnormalities (CRP 2.0 mg/L, white blood count 6.3 x 109/L). During admission, there was clinical deterioration on the third day. Emergency laparotomy was performed. An internal herniation through Petersen's space was found that strangulated and perforated the small bowel. A resection with primary anastomosis and closure of the defects was performed. CONCLUSION: Diagnosing an internal herniation through Petersen's space is difficult due to the nonspecific clinical presentation. The interpretation of the CT scan poses another diagnostic challenge. This sign is present in 74% of the cases with this herniation. A missed diagnosis of internal herniation may cause potentially serious complications. A patient with a gastric bypass who experiences intermittent abdominal complaints should undergo laparoscopy to rule out internal herniation.

Sulfated glycosaminoglycan-like polymers are present in an acidophilic biofilm from a sulfidic cave.

Sulfated glycosaminoglycans (sGAG) are negatively charged extracellular polymeric substances that occur in biofilms from various environments. Yet, it remains unclear whether these polymers are acquired from the external environment or produced by microbes in the biofilm. To resolve this, we analyzed the presence of sGAGs in samples of an acidophilic biofilm collected from Sulfur Cave in Puturosu Mountain (Romania), an environment that is largely inaccessible to contamination. A maximum of 55.16 ± 2.06 µg sGAG-like polymers were recovered per mg of EPS. Enzymatic treatment with chondroitinase ABC resulted in a decrease of the mass of these polymers, suggesting the structure of the recovered sGAG is similar to chondroitin. Subsequent FT-IR analysis of these polymers revealed absorbance bands at 1230 cm-1, 1167 cm-1 and 900 cm-1, indicating a possible presence of polysaccharides and sulfate. Analysis of genomic sequences closely related to those predominant in the acidophilic biofilm, contained genes coding for sulfotransferase (an enzyme needed for the production of sGAG), which supports the hypothesis of microbial synthesis of sGAGs within the biofilm.

The effects of laparoscopic Roux-en-Y gastric bypass and one-anastomosis gastric bypass on glycemic control and remission of type 2 diabetes mellitus: study protocol for a multi-center randomized controlled trial (the DIABAR-trial).

BACKGROUND: Metabolic surgery induces rapid remission of type 2 diabetes mellitus (T2DM). There is a paucity of high level evidence comparing the efficacy of the laparoscopic Roux-en-Y gastric bypass (RYGB) and the laparoscopic one-anastomosis gastric bypass (OAGB) in glycemic control. Also, the mechanisms that drive the conversion of T2DM in severe obese subjects to euglycemia are poorly understood. METHODS: The DIABAR-trial is an open, multi-center, randomized controlled clinical trial with 10 years follow-up which will be performed in 220 severely obese patients, diagnosed with T2DM and treated with glucose-lowering agents. Patients will be randomized in a 1:1 ratio to undergo RYGB or OAGB. The primary outcome is glycemic control at 12 months follow-up. Secondary outcome measures are diverse and include weight loss, surgical complications, psychologic status and quality of life, dietary behavior, gastrointestinal symptoms, repetitive bloodwork to identify changes over time, glucose tolerance and insulin sensitivity as measured by mixed meal tests, remission of T2DM, presence of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis in liver biopsy, oral and fecal microbiome, cardiovascular performance, composition of bile acids, and the tendency to develop gallstones. DISCUSSION: The DIABAR-trial is one of the few randomized controlled trials primarily aimed to evaluate the glycemic response after the RYGB and OAGB in severe obese patients diagnosed with T2DM. Secondary aims of the trial are to contribute to a deeper understanding of the mechanisms that drive the remission of T2DM in severe obese patients by identification of microbial, immunological, and metabolic markers for metabolic response and to compare complications and side effects of RYGB and OAGB. TRIAL REGISTRATION: ClinicalTrials.gov NCT03330756 ; date first registered: October 13, 2017.

Molecular alterations associated with liver metastases development in colorectal cancer patients.

BACKGROUND: Understanding the molecular biology of colorectal cancer (CRC) provides opportunities for effective personalised patient management. We evaluated whether chromosomal aberrations, mutations in the PI(3)K signalling pathway and the CpG-island methylator phenotype (CIMP) in primary colorectal tumours can predict liver metastases. METHODS: Formalin-fixed paraffin-embedded material from primary colorectal tumours of three different groups were investigated: patients with CRC without metastases (M0, n=39), patients who were treated with hyperthermal intraperitoneal chemotherapy for CRC metastases confined to the peritoneum (PM, n=46) and those who had isolated hepatic perfusion for CRC metastases confined to the liver (LM, n=48). RESULTS: All samples were analysed for DNA copy number changes, PIK3CA, KRAS, BRAF mutations, CIMP and microsatellite instability. The primary CRCs of the LM group had significantly higher frequency of amplified chromosome 20q (P=0.003), significantly fewer mutations in the PI(3)K signalling pathway (P=0.003) and fewer CIMP high tumours (P=0.05). There was a strong inverse correlation between 20q and the PI(3)K pathway mutations. CONCLUSION: The development of CRC liver metastases is associated with amplification of chromosome 20q and not driven by mutations in the PI(3)K signalling pathway.

Multidisciplinary discussion and management of rectal cancer: a population-based study.

BACKGROUND: The purpose of the present study was to evaluate the value of discussing rectal cancer patients in a multidisciplinary team (MDT). METHODS: All treated rectal cancer patients (>T1M0) diagnosed in 2006-2008 were included. According to the national guidelines, neoadjuvant (chemo)radiotherapy should be given to all rectal cancer patients. Patients were scored as "discussed" (MDT+) only if documented proof was available. The primary endpoint was the number of positive circumferential resection margins (CRM ≤ 1 mm). RESULTS: Of the 275 patients included, 210 were analyzed (exclusions: (recto)sigmoid tumor, acute laparotomy, and inoperability). Neoadjuvant treatment was applied in 174 (83%) patients and followed by total mesorectal excision in 171 (81%) patients. Patients considered not to require downstaging, received short-course radiotherapy (SCRT) (n = 116) or no radiotherapy (no RT) (n = 36), whereas 58 more advanced patients received chemoradiotherapy (CRT). The MDT discussion took place in 116 cases (55%). In the MDT+ group an MRI was used more often (p = 0.001) and TNM staging was more complete (p < 0.001). The proportion of patients with advanced disease was higher in the MDT+ group (88% ≥T3/N+ versus 68%; p = 0.001). The overall CRM+ rate was 13% and did not differ between the MDT+ and the MDT- group (p = 0.392). In patients receiving SCRT or no RT, the CRM+ rate was 10%, whereas the rate was 20% for patients receiving CRT. CONCLUSIONS: Although no difference in CRM+ rate was found for those patients who were discussed and those who were not, our results demonstrate room for improvement, especially in the selection of patients for SCRT or no RT. We advocate standardized documentation of treatment decisions and pathology reports.

Long-term Emergency Department Visits and Readmissions After Laparoscopic Roux-en-Y Gastric Bypass: a Systematic Review.

PURPOSE: There is considerable evidence on short-term outcomes after laparoscopic Roux-en-Y gastric bypass (LRYGB), but data on long-term outcome is scarce, especially on postoperative emergency department (ED) visits and readmissions. We aim to systematically review evidence on the incidence, indications, and risk factors of ED visits and readmissions beyond 30 days after LRYGB. MATERIALS AND METHODS: A systematic search in PubMed, Scopus, Embase.com , Cochrane Library, and PsycINFO was performed. All studies reporting ED visits and readmissions > 30 days after LRYGB, with ≥ 50 patients, were included. PRISMA statement was used and the Newcastle-Ottawa Scale for quality assessment. RESULTS: Twenty articles were included. Six studies reported on ED visits (n = 2818) and 19 on readmissions (n = 276,543). The rate of patients with an ED visit within 90 days after surgery ranged from 3.9 to 32.6%. ED visits at 1, 2, and 3 years occurred in 25.6%, 30.0%, and 31.1% of patients. Readmissions within 90 days and at 1-year follow-up ranged from 4.1 to 20.5% and 4.75 to 16.6%, respectively. Readmission was 29% at 2 years and 23.9% at 4.2 years of follow-up. The most common reason for ED visits and readmissions was abdominal pain. CONCLUSION: Emergency department visits and readmissions have been reported in up to almost one in three patients on the long-term after LRYGB. Both are mainly indicated for abdominal pain. The report on indications and risk factors is very concise. A better understanding of ED visits and readmissions after LRYGB is warranted to improve long-term care, in particular for patients with abdominal pains.

International point prevalence study of Intensive Care Unit transfusion practices-Pilot study in the Netherlands.

BACKGROUND: Anaemia and coagulopathy are common issues in critically ill patients. Transfusion can be lifesaving, however, is associated with potential life threatening adverse events. As an international transfusion guideline for this specific patient population is lacking, we hypothesize that a high heterogeneity in transfusion practices exists. In this pilot-study we assessed transfusion practice in a university hospital in the Netherlands and tested the feasibility of this protocol for an international multi-centre study. METHODS: A prospective single centre cohort study was conducted. For seven days all consecutive non-readmitted patients to the adult Intensive Care Unit (ICU) were included and followed for 28 days. Patients were prospectively followed until ICU discharge or up to day 28. Patient outcome data was collected at day 28. Workload for this study protocol was scored in hours and missing data. RESULTS: In total, 48 patients were included, needed in total three hours patient to include and collect all data, with 1.6% missing data showing the feasibility of the data acquisition. Six (12.5%) patients received red blood cells (RBCs), three patients (6.3%) received platelet concentrates, and two (4.2%) patients received plasma units. In total eight (16.7%) patients were transfused with one or more blood products. Median pre- and post-transfusion haemoglobin (Hb) levels were 7.6 (6.7-7.7) g/dL and 8.1 (7.6-8.7) g/dL, respectively. CONCLUSION: In this pilot-study we proved the feasibility of our protocol and observed in this small population a restrictive transfusion practice for all blood products.

Randomized clinical trial of an enhanced recovery after surgery programme versus conventional care in laparoscopic Roux-en-Y gastric bypass surgery.

Background: Enhanced recovery after surgery (ERAS) programmes have led to a decreased duration of hospital stay in several surgical fields, but have not been fully tested in patients undergoing laparoscopic Roux-en-Y gastric bypass (LRYGB) for obesity. This study aimed to investigate an ERAS programme versus standard care in these patients. Methods: Between January 2013 and July 2014, patients undergoing LRYGB were randomized to ERAS or conventional care. The primary outcome was functional hospital stay, defined as the time between end of surgery and when predefined discharge criteria (pain adequately controlled, fever and postoperative nausea and vomiting (PONV) absent, full liquid diet tolerated, mobilized and feeling fit for discharge) were met. Secondary outcomes were total length of hospital stay, 30-day complication and mortality rates, duration of surgery, time spent on the recovery ward and health-related quality of life. Results: A total 220 patients were randomized to ERAS (110 patients) or conventional (110) care. Patients in the ERAS group had shorter functional hospital stay (17·4 versus 20·5 h; P < 0·001), quicker pain control, tolerated liquid diet earlier, had earlier control of PONV, mobilized sooner and were comfortable with discharge sooner than those receiving conventional care. Total length of hospital stay, duration of surgery, time spent on the recovery ward, health-related quality of life, complication and readmission rates did not differ between the study groups. There were no deaths. Conclusion: Patients under ERAS care recovered faster after LRYGB surgery than those receiving conventional care, with no increase in readmission and postoperative morbidity rates. Registration number: NTR3853 (http://www.trialregister.nl/).

Sensitivity and Specificity of 50% Excess Weight Loss (50%EWL) and Twelve Other Bariatric Criteria for Weight Loss Success.

BACKGROUND: Criteria for bariatric weight loss success are numerous. Most of them are arbitrary. None of them is evidence-based. Our objective was to determine their sensitivity and specificity. METHODS: Thirteen common bariatric weight loss criteria were compared to a benchmark reflecting the gold standard in bariatric surgery. We used an elaborate baseline BMI-independent weight loss percentile chart, based on retrospective data after laparoscopic Roux-en-Y gastric bypass (LRYGB), performed between 2007 and 2017. Percentile curves p31.6 (patients' expectation), p25 (interquartile range), p15.9 (1 standard deviation (SD) below median), and p10.9 (surgeons' goal) were used as possible cutoff for success to determine true or false positive and negative results beyond 1 year. RESULTS: We operated 4497 primary LRYGB patients, with mean follow-up 22 (± 1 SD 19; range 0-109) months, 3031 patients with last result ≥ 1 year, 518 ≥ 5 years. For all four cutoff percentile curves for success, specificities were low (2-72%) for criteria < 35 body mass index (BMI), ≥ 25percentage excess BMI loss (%EBMIL), ≥ 50%EBMIL, ≥ 15 percentage total weight loss (%TWL), ≥ 20%TWL, ≥ 25 percentage excess weight loss (%EWL), and high (83-96%) for < 30 BMI. No criterion had > 80% specificity and sensitivity for a cutoff above p15.9. For p15.9, they were both > 80% for criteria ≥ 10 BMI reduction and ≥ 50%EWL, both > 90% for ≥ 25%TWL and ≥ 35 percentage alterable weight loss (%AWL). All criteria had high sensitivities for all cutoff percentile curves (87-100%), except < 30 BMI (65-78%). CONCLUSIONS: For the first time, common bariatric criteria for weight loss success were systematically validated. Most criteria recognized success very well (high sensitivities), but ≥ 15%TWL, ≥ 20%TWL, < 35BMI, ≥ 25%EWL, ≥ 25%EBMIL, and ≥ 50%EBMIL left too many poor responders unnoticed (low specificities). Bariatric weight loss success is best assessed by comparing results to percentile curve 1 SD below median (p15.9) in a bariatric baseline BMI-independent weight loss percentile chart. Criteria ≥ 35%AWL and ≥ 25%TWL came close to that curve, both with > 90% sensitivity and specificity. Among others, criterion ≥ 50%EBMIL did not.

[Cost variation in care groups?] / Verschillen zorgkosten tussen zorggroepen?

OBJECTIVE: Is the simple mean of the costs per diabetes patient a suitable tool with which to compare care groups? Do the total costs of care per diabetes patient really give the best insight into care group performance? DESIGN: Cross-sectional, multi-level study. METHOD: The 2009 insurance claims of 104,544 diabetes patients managed by care groups in the Netherlands were analysed. The data were obtained from Vektis care information centre. For each care group we determined the mean costs per patient of all the curative care and diabetes-specific hospital care using the simple mean method, then repeated it using the 'generalized linear mixed model'. We also calculated for which proportion the differences found could be attributed to the care groups themselves. RESULTS: The mean costs of the total curative care per patient were €3,092 - €6,546; there were no significant differences between care groups. The mixed model method resulted in less variation (€2,884 - €3,511), and there were a few significant differences. We found a similar result for diabetes-specific hospital care and the ranking position of the care groups proved to be dependent on the method used. The care group effect was limited, although it was greater in the diabetes-specific hospital costs than in the total costs of curative care (6.7% vs. 0.4%). CONCLUSION: The method used to benchmark care groups carries considerable weight. Simply stated, determining the mean costs of care (still often done) leads to an overestimation of the differences between care groups. The generalized linear mixed model is more accurate and yields better comparisons. However, the fact remains that 'total costs of care' is a faulty indicator since care groups have little impact on them. A more informative indicator is 'costs of diabetes-specific hospital care' as these costs are more influenced by care groups.

High-resolution proton magnetic resonance studies of the 3'-terminal colicin fragment of 16 S ribosomal RNA from Escherichia coli. Assignment of iminoproton resonances by nuclear Overhauser effect experiments and the influence of adenine dimethylation on the hairpin conformation.

The "colicin" fragments comprising the 49 3'-terminal nucleotides of 16 S ribosomal RNA have been isolated from wild-type Escherichia coli and from a kasugamycin-resistant mutant that lacks methylation of two geminal adenine residues. Proton nuclear magnetic resonance (n.m.r.) spectra (500 MHz) were recorded at various temperatures. The low-field resonances arising from the hydrogen-bonded iminoprotons of paired bases were assigned using the nuclear Overhauser effect (n.o.e.). Crucial to the interpretation of the spectra are the resonances that originate from the two hydrogen-bonded iminoprotons of a U X G basepair. Combined with temperature-jump relaxation kinetics experiments the n.o.e.s lead to the conclusion that a conserved A X U/U X G junction in the hairpin is a thermolabile dislocation in the helix. The n.m.r. spectra of the wild-type and mutant fragment are only different with respect to the iminoproton resonances of the two base-pairs adjoining the hairpin loop. The spectra recorded at various temperatures tend to indicate that dimethylation of the adenosines labilizes these base-pairs, but no definitive conclusions are drawn. The results confirm our previous views that dimethylation of the adenosine residues affects the conformation of the hairpin loop.

Hairpin formation in synthetic oligonucleotides.

The structure and dynamics of the homologous series of the (partly) self-complementary DNA fragments, d(ATCCTATnTAGGAT) n = 0-7, were investigated in a combined NMR, T-jump, and optical melting study. It is shown that all compounds in the series may adopt hairpin like conformations, even for n less than 3, although for these smaller n values this only occurs in significant amounts at relatively low concentrations (approximately 10 microM). The enthalpy change accompanying the hairpin-coil melting transition turns out to depend on the number of intervening thymidines, n. It is shown that this does not mean that the enthalpy of loop closure is significantly different from zero, but that loop formation stabilizes the base pair closing the loop. The results indicate that for DNA the optimal loop consists of four or five residues. The observation that hairpins are formed for n less than 3 and that the stability of DNA hairpins is at its maximum for loop lengths of four to five residues is at variance with earlier findings for RNA. In the latter case the optimal loop size consists of six to seven residues, whereas for less than three intervening residues only, dimer, and no hairpin formation, was observed [17, 20]. A direct comparison with RNA behaviour was made by studying r(AUCCUAUT4UAGGAU), T = ribothymidine. In contrast to its DNA analogue, d(ATCCTAT4TAGGAT), the ribo-fragment forms a dimer as well as a hairpin at low (10 microM) concentrations. With the thermodynamic melting parameters deduced from the present experiments the differences between DNA and RNA melting behaviour can be explained.

Cardiac and hemodynamic effects of hemodialysis and ultrafiltration.

Imbalance between cardiac oxygen supply and demand may trigger cardiac events in already vulnerable hemodialysis (HD) patients. We studied the effect of ultrafiltration (UF) and HD in nine chronic HD patients by continuously measuring blood volume (BV; by Critline), blood pressure (BP; by Portapres), and changes in hemodynamics (Modelflow) during isolated UF (iUF) of 500 mL in 30 minutes and subsequent HD combined with UF (HD + UF). Aortic pressure was reconstructed from finger pressure. Changes in cardiac oxygen supply were assessed by calculating the area under the aortic pressure curve during diastole (diastolic pressure time index [DPTI]). Changes in cardiac oxygen demand were assessed by calculating systolic pressure time index (SPTI). BV decreased 4.0% +/- 1.8% during UF and 7.3% +/- 3.3% during HD + UF (both P < 0.01). Systolic BP did not change; diastolic and mean BP increased 11 +/- 7.4 and 11 +/- 8.4 mm Hg during iUF, respectively (both P < 0.01), and stabilized during HD + UF. Overall pulse pressure decreased 19 +/- 11.1 mm Hg (P < 0.01). Heart rate increased 13 +/- 11 beats/min (P < 0.01) and systemic vascular resistance increased 59% +/- 51% (P < 0. 01), whereas stroke volume and cardiac output (CO) decreased by 40% +/- 17% and 30% +/- 13%, respectively (both P < 0.01). Both cardiac oxygen supply (DPTI) and demand (SPTI) increased during iUF, and both decreased during HD + UF. By the end of the procedure, DPTI/SPTI ratio had increased 9% +/- 8% (P < 0.05). Changes in CO correlated closely to changes in BV. Despite large changes in hemodynamics during uncomplicated UF and HD, the balance between cardiac oxygen supply and demand (DPTI/SPTI ratio) did not decrease, but improved slightly.

Differential hydrogen ion titrations of the histidine residues in Helix pomatia haemocyanin.

The properties of the histidine residues in Helix pomatia haemocyanin have been studied by differential hydrogen ion titrations. In oxy-and deoxyhaemocyanin 31 X 10(-5) histidine residues per g protein are titrated in contrast to 35 X 10(-5) residues in apohaemocyanin. The difference corresponds to a stoichiometry of one histidine residue per copper atom bound. Even in apohaemocyanin about 6 X 10(-5) histidine residues per g protein are not titrated in their normal pH region. In the presence of sufficient calcium to displace the dissociation completely out of the titration region, the titration curve of apohaemocyanin could be linarized according to the model of Linderstrom--Lang. In oxy-and deoxyhaemocyanin, however, a distinct deviation from linearity was found under the same conditions. In the absence of calcium the effect of the dissociation adds up to this deviation. The electrostatic interaction factors were determined for the protein at 0.1 M KC1 and for the dissociation products: halves and tenths at 1.0 M KC1. The electrostatic interaction factor for the wholes and the halves are much smaller than the values calculated from the Linderstrom--Lang equation, using the radius of the equivalent sphere either obtained from electron microscopy or from the partial specific volume. This probably due to solvent penetration. For the tenths at 1.0 M KC1, this effect is small.

The influence of organic phosphates on the Bohr effect of human hemoglobin valency hybrids.

The Bohr effect of hemoglobin and that of the aquomet and cyanomet valency hybrids was measured in the presence and the absence of IHP (inositol hexaphosphate) and DPG (2,3-diphosphoglycerate). In the absence of these organic phosphates the four hybrids show similar, but suppressed Bohr effects as compared to hemoglobin. Addition of IHP and DPG results in all cases in an increase of the Bohr effect. The additional phosphate induced Bohr effect of the hybrids with the alpha chain in the oxidized form is almost identical to that of hemoglobin, while this effect of the hybrids with oxidized beta chains is slighly lower than that of hemoglobin. The results suggest (a) that the Bohr effect is correlated to the ligation state of the hemoglobin molecule rather than to its quaternary structure (b) that the additional phosphate induced Bohr effect is related to the change in quaternary structure of the tetramer, and (c) that with respect to the Bohr effect of the hybrids there is no difference between high and low spin species.

The CO and NO Bohr effect of human hemoglobin with and without inositolhexaphosphate.

Using NO and CO as ligands the Bohr effect of human hemoglobin has been measured with and without inositolhexophosphate. It appears that in the absence and presence of inositolhexaphosphate hemoglobin shows a distinct ligand specificity with respect to the Bohr effect. Ligation with NO is accompanied by release of a larger number of Bohr effect. It is shown that this latter result is due to the fact that the number of protons taken up upon binding of inositolhexaphosphate to ligated hemoglobin is larger for HbNO than for HbCO. It is suggested that this additional proton uptake is partially due to a restoration of the saltbridge between His 146beta and Asp 94beta upon addition of IHP.