Diets for inflammatory bowel disease: What do we know so far?

In recent years, the role of diet in the pathogenesis of inflammatory bowel disease (IBD) has gained great interest within the scientific community. Eating habits from industrialised countries (the so-called western diet or WD) have been associated with a higher incidence of IBD in observational studies, although the dietary factors responsible for the development of the disease are still to be elucidated. Some components of the diet with proinflammatory potential may cause changes in immunity and intestinal microbiota, leading to the inflammatory reaction that causes IBD-related lesions. The quality of available evidence is low, due to methodological issues, such as the lack of intervention studies, small sample size and heterogeneity of studies. For this reason, scientific societies have offered their recommendations using clinical practice guidelines and consensus documents, in order to establish a common criterion in the nutritional treatment of patients with IBD. The objective of this review was to summarise the data published regarding diet in IBD and review the recommendations given by scientific societies.

Nutritional management and clinical outcome of critically ill patients with COVID-19: A retrospective study in a tertiary hospital.

BACKGROUND & AIMS: Severe COVID-19 infection is characterized by an inflammatory response and lung injury that can evolve into an acute respiratory distress syndrome that needs support treatment in intensive care unit. Nutritional treatment is an important component of the management of critically ill patients and should be started in the first 48 h of ICU admission to avoid malnutrition. This study describes the characteristics of the patients treated in a tertiary hospital in Madrid during the months of March-May 2020 (first wave), the medical nutrition treatment employed and its influence in the clinical outcome of these patients. METHODS: This is a retrospective study including COVID-19 patients admitted in ICU that needed medical nutrition treatment (MNT). Collected variables included sex, age, BMI, underlying diseases, time from hospitalisation to ICU admission, type of respiratory support (invasive mechanical ventilation (IMV) or high flow nasal cannula (HFNC) or non-invasive ventilation (non-IMV)), caloric and protein requirements (25 kcal/kg adjusted body weight (ABW), 1.3 g/kg ABW/day), MNT type (enteral nutrition (EN), parenteral nutrition (PN), mixed EN + PN), total calories (including propofol) and proteins administered, percentage of caloric and protein goal in ICU day 4th and 7th, metabolic complications, acute kidney failure (AKF), length of stay (LOS) and mortality. Data are expressed as mean ± SD, median (IQR) or frequencies. Statistical analysis was performed with the IBM SPSS Statistics for Windows, Version 25.0. p < 0.05 were considered statistically significant. RESULTS: A total of 176 patients were included (72.7% male), 60.1 ± 13.5 years, BMI 29.9 ± 5.4 kg/m2. Underlying diseases included 47.4% overweight, 39.8% obesity, 49.1% hypertension, 41.4% dyslipidaemia. 88.6% of patients needed IMV, 89.1% prone position, 2.9% ECMO. Time to ICU admission: 2 (4.75) days. Estimated caloric and protein requirements were 1775 ± 202 kcal and 92.4 ± 10.3 g. Calories and proteins administered at days 4th and 7th were 1425 ± 577 kcal and 66 ± 26 g and 1574 ± 555 and 74 ± 37, respectively. Most of the patients received PN (alone or complementary to EN) to cover nutritional requirements (82.4% at day 4th and 77.9% at day 7th). IVM patients received more calories and proteins during the first week of ICU admission. Complications included 77.8% hyperglycaemia, 13.2% hypoglycaemia, 83.8% hypertriglyceridemia, and 35.1% AKF. ICU LOS was 20.5 (26) days. The mortality rate was 36.4%. CONCLUSIONS: In our series, the majority of patients reached energy and protein requirements in the first week of ICU admission due to the use of PN (total or complementary to EN). Patients with HFNC or non-IMV may be at risk of malnutrition if total or complementary PN to oral diet/ONS/tube feeding is not used to cover nutritional requirements. Therefore, if EN is not possible or insufficient, PN can be safely used in critically ill patients with COVID-19 with a close monitoring of metabolic complications.

Social and economic costs of home parenteral nutrition.

BACKGROUND AND AIMS: Home parenteral nutrition (HPN) is a lifesaving treatment for people with chronic intestinal failure. Although HPN has been studied from an economic point of view, the categories of costs usually included direct costs, frequently excluding personal costs and productivity costs. The purpose of the present paper was to study the total costs of HPN from a societal perspective. METHODS: Observational, retrospective, transverse study of all adult patients who were on HPN for more than 3 months and were treated at Gregorio Marañón University Hospital (Madrid, Spain), from June 2018-2019. Data on personal costs and productivity costs were collected from questionnaires completed by patients receiving HPN. We also updated the direct healthcare and non-healthcare costs studied by our group previously to Euros (€) for the year 2019. RESULTS: Twenty-two patients were included. Personal costs were €729.49 per patient (€3.45 per patient per day) and productivity costs were €256.39 per patient (€1.21 per patient per day). Total HPN costs amounted to €14,460.87 per patient (€131.58 per patient per day). The direct healthcare and non-healthcare costs accounted for 96.46% of overall costs, the personal costs for the patients receiving HPN accounted for 2.62% and productivity costs for 0.92%. CONCLUSIONS: From a societal perspective, the direct healthcare and non-healthcare costs accounted for the majority of HPN expenditure, followed by personal costs and productivity costs.

Discovery of large molecules as new biomarkers in wastewater using environmental proteomics and suitable polymer probes.

The capability of monitoring large molecules as possible biomarkers in wastewater will be an important contribution to the new field of sewage epidemiology. Here, we explore the use of polymer probes together with untargeted proteomics for large scale protein analysis in sewage and treated water. Polymeric probes were immersed in the influent, anoxic reactor and effluent waters of a Spanish WWTP during 11 days. Proteins sorbed were extracted and identified by mass spectrometry. A total of 690 proteins from bacteria, plants and animals, including human, were identified showing different proteome profiles in the different sites. Bacterial proteins (510) pointed at 175 genera distributed in 22 bacterial classes. The most abundant were EF-Tu, GroEL and ATP synthase which were contributed by a high number of species. Human was the species contributing the greatest number of identified proteins (57), some in high abundance like keratins. Human proteins dominated in the influent water and were efficiently removed at the effluent. Several of the proteins identified (S100A8, uromodulin, defensins) are known disease biomarkers. This study provides the first insight into the proteome profiles present in real wastewater.

Acute pancreatitis promotes the generation of two different exosome populations.

Exosomes are small extracellular vesicles that act as intercellular messengers. Previous studies revealed that, during acute pancreatitis, circulating exosomes could reach the alveolar compartment and activate macrophages. However, proteomic analysis suggested that the most likely origin of these exosomes could be the liver instead of the pancreas. The present study aimed to characterize the exosomes released by pancreas to pancreatitis-associated ascitic fluid (PAAF) as well as those circulating in plasma in an experimental model of taurocholate-induced acute pancreatitis in rats. We provide evidence that during acute pancreatitis two different populations of exosomes are generated with relevant differences in cell distribution, protein and microRNA content as well as different implications in their physiological effects. During pancreatitis plasma exosomes, but not PAAF exosomes, are enriched in the inflammatory miR-155 and show low levels of miR-21 and miR-122. Mass spectrometry-based proteomic analysis showed that PAAF exosomes contains 10-30 fold higher loading of histones and ribosomal proteins compared to plasma exosomes. Finally, plasma exosomes have higher pro-inflammatory activity on macrophages than PAAF exosomes. These results confirm the generation of two different populations of exosomes during acute pancreatitis. Deep understanding of their specific functions will be necessary to use them as therapeutic targets at different stages of the disease.

Direct costs of a home parenteral nutrition programme.

BACKGROUND & AIMS: Home parenteral nutrition (HPN) is a lifesaving treatment for people with chronic intestinal failure and its cost has been reported to be very high. The purpose of the present paper was to study the direct healthcare and non-healthcare costs associated with the HPN programme managed by a tertiary hospital. METHODS: Observational, retrospective study of all adult patients on HPN from 11.1.2014 to 10.31.2015 treated at Gregorio Marañón University Hospital (Madrid, Spain). An economic evaluation was undertaken to calculate the direct healthcare (HPN provision, outpatient monitoring and management of complications) and non-healthcare costs (transportation process) of the HPN programme. The variables were collected from medical records, the dispensary and the hospital's financial services. The unit costs were taken from official price lists. RESULTS: Thirty-two patients met the inclusion criteria. Total direct healthcare and non-healthcare costs amounted to €13,363.53 per patient (€124.02 per patient per day). The direct healthcare costs accounted for 98.32% of overall costs, while the non-healthcare costs accounted for the remaining 1.68%. HPN provision accounted for the majority of the costs (74.25%), followed by management of complications (21.85%) and outpatient monitoring (2.23%). CONCLUSIONS: The direct healthcare costs accounted for the majority of HPN expenditure, specifically HPN provision was the category with the highest percentage.

A functional glycoproteomics approach identifies CD13 as a novel E-selectin ligand in breast cancer.

BACKGROUND: The glycan moieties sialyl-Lewis-X and/or -A (sLeX/A) are the primary ligands for E-selectin, regulating subsequent tumor cell extravasation into distant organs. However, the nature of the glycoprotein scaffolds displaying these glycans in breast cancer remains unclear and constitutes the focus of the present investigation. METHODS: We isolated glycoproteins that bind E-selectin from the CF1_T breast cancer cell line, derived from a patient with ductal carcinoma. Proteins were identified using bottom-up proteomics approach by nanoLC-orbitrap LTQ-MS/MS. Data were curated using bioinformatics tools to highlight clinically relevant glycoproteins, which were validated by flow cytometry, Western blot, immunohistochemistry and in-situ proximity ligation assays in clinical samples. RESULTS: We observed that the CF1_T cell line expressed sLeX, but not sLeA and the E-selectin reactivity was mainly on N-glycans. MS and bioinformatics analysis of the targeted glycoproteins, when narrowed down to the most clinically relevant species in breast cancer, identified CD44 glycoprotein (HCELL) and CD13 as key E-selectin ligands. Additionally, the co-expression of sLeX-CD44 and sLeX-CD13 was confirmed in clinical breast cancer tissue samples. CONCLUSIONS: Both CD44 and CD13 glycoforms display sLeX in breast cancer and bind E-selectin, suggesting a key role in metastasis development. Such observations provide a novel molecular rationale for developing targeted therapeutics. GENERAL SIGNIFICANCE: While HCELL expression in breast cancer has been previously reported, this is the first study indicating that CD13 functions as an E-selectin ligand in breast cancer. This observation supports previous associations of CD13 with metastasis and draws attention to this glycoprotein as an anti-cancer target.

Recurrent attacks of acute hepatic porphyria: major role of the chronic inflammatory response in the liver.

BACKGROUND: Acute intermittent porphyria (AIP) is an inherited disorder of haem metabolism characterized by life-threatening acute neurovisceral attacks due to the induction of hepatic δ-aminolevulinic acid synthase 1 (ALAS1) associated with hydroxymethylbilane synthase (HMBS) deficiency. So far, the treatment of choice is hemin which represses ALAS1. The main issue in the medical care of AIP patients is the occurrence of debilitating recurrent attacks. OBJECTIVE: The aim of this study was to determine whether chronic hemin administration contributes to the recurrence of acute attacks. METHODS: A follow-up study was conducted between 1974 and 2015 and included 602 French AIP patients, of whom 46 had recurrent AIP. Moreover, we studied the hepatic transcriptome, serum proteome, liver macrophage polarization and oxidative and inflammatory profiles of Hmbs-/- mice chronically treated by hemin and extended the investigations to five explanted livers from recurrent AIP patients. RESULTS: The introduction of hemin into the pharmacopeia has coincided with a 4.4-fold increase in the prevalence of chronic patients. Moreover, we showed that both in animal model and in human liver, frequent hemin infusions generate a chronic inflammatory hepatic disease which induces HO1 remotely to hemin treatment and maintains a high ALAS1 level responsible for recurrence. CONCLUSION: Altogether, this study has important impacts on AIP care underlying that hemin needs to be restricted to severe neurovisceral crisis and suggests that alternative treatment targeting the liver such as ALAS1 and HO1 inhibitors, and anti-inflammatory therapies should be considered in patients with recurrent AIP.

Uso de células troncales derivadas de lipoaspirado en un modelo experimental animal de rotura de manguito rotador / Use of adipose-derived stem cells in an experimental rotator cuff fracture animal model

Objetivo. La reparación del manguito rotador tiene una alta tasa de fracaso. Se investiga si la aplicación de células troncales derivadas de lipoaspirado mejorará la resistencia de la reparación y recreará la entesis original. Material y métodos. Estudio experimental en 44 ratas BDIX con sección y reparación con sutura del tendón supraespinoso y asignación aleatoria a uno de 3 grupos: grupo A, nada (control); grupo B, aplicación local de vehículo de fibrina; y grupo C, aplicación de 2 x 106 células troncales derivadas de lipoaspirado. Se realiza estudio mecánico en célula de carga y estudio histológico en hematoxilina-eosina. Resultados. En el estudio mecánico no hubo diferencias entre grupos. La carga hasta el fracaso aumentó de los grupos de 4-8 semanas. En el estudio histológico se observó la unión hueso-tendón mediante un tejido fibrovascular desorganizado. En el grupo C se observó un aumento de células plasmáticas a las 4 y 8 semanas. Conclusión. La utilización de células troncales derivadas de lipoaspirado no recrea la organización celular de la entesis ni mejoran las propiedades biomecánicas de la misma. Son necesarios más estudios para investigar técnicas que mejoren la cicatrización del tendón (AU)

Aim. Rotator cuff repairs have shown a high level of re-ruptures. We hypothesized that the use of adipose-derived stem cells (ASC) could improve the biomechanical and histological properties of the repair. Material and methods. Controlled experimental study conducted on 44 BDIX rats with section and repair of the supraspinatus tendon and randomization to one of three groups: group A, no intervention (control); group B, local applications of a fibrin sealant; and group C, application of the fibrin sealant with 2 x 106 ASC. At 4 and 8 weeks a biomechanical and histological analysis was performed. Results. There were no differences in load-to-failure at 4 and 8 weeks between groups. The load-to-failure did increase between week 4 and week 8. Histologically the tendon-to bone union showed a disorganized fibrovascular tissue. Group C showed a different inflammatory pattern, with less presence of neutrophils and more presence of plasma cells. Conclusion. The use of ASC does not improve the biomechanical or histological properties of the repair site. More studies are needed to improve techniques that enhance the healing site of the repair (AU)

[Use of adipose-derived stem cells in an experimental rotator cuff fracture animal model]. / Uso de células troncales derivadas de lipoaspirado en un modelo experimental animal de rotura de manguito rotador.

AIM: Rotator cuff repairs have shown a high level of re-ruptures. We hypothesized that the use of adipose-derived stem cells (ASC) could improve the biomechanical and histological properties of the repair. MATERIAL AND METHODS: Controlled experimental study conducted on 44 BDIX rats with section and repair of the supraspinatus tendon and randomization to one of three groups: group A, no intervention (control); group B, local applications of a fibrin sealant; and group C, application of the fibrin sealant with 2 x 10(6) ASC. At 4 and 8 weeks a biomechanical and histological analysis was performed. RESULTS: There were no differences in load-to-failure at 4 and 8 weeks between groups. The load-to-failure did increase between week 4 and week 8. Histologically the tendon-to bone union showed a disorganized fibrovascular tissue. Group C showed a different inflammatory pattern, with less presence of neutrophils and more presence of plasma cells. CONCLUSION: The use of ASC does not improve the biomechanical or histological properties of the repair site. More studies are needed to improve techniques that enhance the healing site of the repair.

Evaluation of HLA Matchmaker compatibility as predictor of graft survival and presence of Anti-HLA antibodies.

BACKGROUND: HLA Matchmaker is a computer algorithm developed to evaluate donor/receptor compatibility comparing sequences of polymorphic aminoacids (eplets) present in human leukocyte antigen (HLA) molecules. The aim of this study was to evaluate the predictive value of HLA Matchmaker for patient and graft survival, graft survival free of rejection, and the presence of anti HLA antibodies. METHODS: Using this program, 62 of 173 kidney transplant patients, were retrospectively analyzed. HLA-I loci eplet mismatch value (EMM) was determined and correlated with graft survival, graft survival free of rejection, and the presence of anti HLA-I antibodies. EMM was compared with the traditional HLA antigen mismatch value (MM) in terms of the presence of anti HLA-I antibodies. RESULTS: Graft survival and graft survival free of rejection showed no statistical differences (P-value .975 and .365, respectively) while comparing patients with less or more than 10 HLA-I EMM. Patients with > or =6 HLA-B EMM had an odds ratio (OR) of 5.6 (95% confidence interval [CI], 0.47-66.45) of presenting anti HLA-I antibodies, with a sensitivity of 80% and specificity of 58.3%. For > or =2 HLA-B MM, the OR was 2.58 (95% CI, 0.46-14.5), with a sensitivity of 40% and specificity of 75%. CONCLUSION: Even though in our study population compatibility by HLA Matchmaker did not correlate with graft survival or rejection-free graft survival, it showed a better sensitivity than traditional HLA antigen matching for the presence of anti HLA-I antibodies. HLA Matchmaker is a promising tool in predicting the appearance of anti-HLA antibodies.

The effectiveness of intermittent rat parathyroid hormone (1-34) treatment on low bone mass due to oestrogen or androgen depletion in skeletally mature rats.

Rat parathyroid hormone (PTH) 1-34 (4 microg/kg/day) was applied for 2.5 months to 9 month-old rats immediately after ovariectomy or orchidectomy or to 15 month-old rats with low bone mass which had been castrated 6 months before in order to know the effects on serum biochemistry parameters, lumbar and femoral bone mineral density, histology, cancellous and cortical bone histomorphometry, mineralisation content profile in cortical bone by backscattered-electron microscopy, and femoral torsion biomechanical testing. In ovariectomised rats, preventive PTH treatment avoided cancellous bone loss in tibial metaphysis and partially in lumbar vertebra, while in cortical bone, PTH increased endosteal resorption and periosteal formation. In intervention study, PTH did not restore cancellous bone but a strong endosteal and periosteal new bone formation was detected. In orchidectomised rats, PTH, in preventive study, avoided cancellous bone loss in metaphysis and lumbar vertebra, and a mild new bone formation in cortical bone was found. In intervention study, PTH maintained baseline cancellous bone mass, but in cortical bone a strong endosteal and periosteal new bone formation was detected. The PTH-induced new bone formation was confirmed by histology and by mineral content profiles. After castration, biomechanical properties were affected in females but not in male rats and PTH reverted this effect.

Bone disease induced by phenytoin therapy: clinical and experimental study.

Previous studies have made references to prolonged treatment with phenytoin as a possible risk factor in the development of osteoporosis and/or osteomalacia. We studied a group of 30 epileptic patients who were under long-term treatment with phenytoin (DPH) in an ambulatory regimen. We found the prevalence of osteoporosis to be 3.3% and of osteopenia to be 56.6%, affecting predominantly the femur, without any significant decrease in bone mineral density of the lumbar spine. These patients were showing signs of bone turnover uncoupling with increases in bone resorption markers. At this time, they also exhibited slight alterations in their phosphocalcium metabolism with trends to hypocalcemia and secondary hyperparathyroidism that was found not to be caused by a vitamin D deficiency as the serum levels of 25(OH)D and 1,25(OH)(2)D were normal. With the aims of corroborating these results and to investigate the physiopathological effects on the bone induced by anticonvulsant drugs we developed a further experimental study in which we administered DPH over a 6-week period with a dose of 5 g/kg/day to male Wistar rats that were in the growth phase. This treatment produced a decrease in overall BMD and bone mineral content in the femur. We did not find osteomalacia in the vertebral biopsy, but the administration of DPH to these animals decreased trabecular volume as well as lessened the thickness of osteoid edges together with an uncoupling in bone turnover. There was also a marked decrease in bone formation and a tendency towards increased bone resorption. We have also found a decrease in resistance to fracture by torsion in the biomechanical assay, which translates into an increase in bone fragility. In these male Wistar rats, the administration of DPH produced a tendency towards increasing the markers of resorption and, though changes in serum levels of calcium and phosphorus were not observed, to provoke an increase in the parathyroid hormone levels; with normal levels of 1,25(OH)(2)D which has produced the same inclination in rats as in humans.

Effect of risedronate on bone mass, remodelling and biomechanical strength in orchidectomized rats.

BACKGROUND: The ability of risedronate to prevent and/or treat orchidectomy-induced osteoporosis in male rats was studied. METHODS: Ninety-five 10-week-old male Wistar rats were sham-operated or orchidectomized. Prevention study: Sham: sham-operated rats; ORX: orchidectomized rats; ORX + RSD: orchidectomized rats, treated for 6 weeks with risedronate. Animals were sacrificed 6 weeks after surgery. Treatment study: Sham(1) and ORX(1): sham and orchidectomized rats sacrificed 3 months after orchidectomy; Sham(2), ORX(2) and ORX(2) + RSD: sham-operated, and orchidectomized rats treated with placebo or risedronate for 6 weeks starting 3 months after orchidectomy, and then sacrificed. Risedronate (0.5 mg/kg/day) and placebo (saline) were administered via oral gavage. After sacrifice, bone mineral density by DEXA, bone volume (BV/TV), osteocalcin (BGP), and serum carboxyterminal telopeptide of collagen type I (CTX) were measured. Femur low-rate torsion testing was performed. RESULTS: Orchidectomy produced an increase in bone remodelling with loss of BV/TV, without effects on torsional strength. Risedronate treatment partially prevented these effects. In the treatment study, risedronate reduced bone remodelling and restored BV/TV to levels higher than those of the sham group, improving biomechanical parameters. CONCLUSIONS: These results suggest that risedronate could be used as a prevention or treatment of male osteoporosis due to hypogonadism.

Effect of vitamin C on fracture healing in elderly Osteogenic Disorder Shionogi rats.

We studied the effect of vitamin C on fracture healing in the elderly. A total of 80 elderly Osteogenic Disorder Shionogi rats were divided into four groups with different rates of vitamin C intake. A closed bilateral fracture was made in the middle third of the femur of each rat. Five weeks after fracture the femora were analysed by mechanical and histological testing. The groups with the lower vitamin C intake demonstrated a lower mechanical resistance of the healing callus and a lower histological grade. The vitamin C levels in blood during healing correlated with the torque resistance of the callus formed (r = 0.525). Therefore, the supplementary vitamin C improved the mechanical resistance of the fracture callus in elderly rats. If these results are similar in humans, vitamin C supplementation should be recommended during fracture healing in the elderly.

Biochemical characterization of recombinant and circulating human Spalpha.

Human Spalpha is a soluble protein expressed by macrophages present in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), for which little functional and structural information is available. It belongs to the group B of the scavenger receptor cysteine-rich superfamily (SRCR-SF) that includes the lymphocyte surface receptors CD5 and CD6 among others. Spalpha is able to bind to different cells of the immune system (monocytes and lymphocytes), which suggests that it may play an important role in the regulation of this system. To study Spalpha, an episomal mammalian expression system (pCEP-Pu/HEK 293-EBNA) was used to produce a recombinant form (rSpalpha) that was utilized for biochemical studies and for the generation of specific hybridomas. Four monoclonal antibodies were selected for their reactivity against rSpalpha by Western blot, immunoprecipitation, and enzyme-linked immunosorbent assays. The monoclonal antibodies recognized three different epitopes on Spalpha. The monoclonal antibodies revealed the existence of two Spalpha isoforms of 38 and 40 kDa, resulting from different sialic acid content. They also showed that Spalpha is a relatively abundant serum protein (60 micro g/ml) that mostly circulates in association with other serum proteins. Accordingly, rSpalpha allowed affinity chromatography isolation of polyclonal and monoclonal immunoglobulin M (IgM). These data indicate that Spalpha is a circulating protein that may play a role in the homeostasis of IgM antibodies.

Paraplejia secundaria a hematoma subdural espinal agudo con compresión medular / Paraplegia secondary to acute subdural spinal haematoma with spinal cord compression

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Automated strong cation exchange extraction of fatty acid esters of 3-(N-phenylamino)-1,2-propanediol from oil samples for routine quantification by HPLC-APCI/MS/MS.

Fatty acid esters of 3-(N-phenylamino)-1,2-propanediol are currently considered the best chemical markers of toxic oils related to the Spanish toxic oil syndrome. Recent research in this area has undertaken the exhaustive and quantitative characterization of these compounds in oils collected during the epidemic outbreak. Current methods developed in this laboratory are based on solid phase extraction (SPE) using SCX cartridges followed by HPLC-APCI/MS/MS quantification. To circumvent the long and tedious extraction procedure, the SPE protocol was adapted for automatic extraction and the problems derived from the use of the immiscible solvents required for the SCX extraction were solved. Linearity of the analytical method was found in the same range as for the manual method. Extraction recoveries were 87 and 75% for 2-hydroxy-3-(N-phenylamino)propyl linoleate and 2-(linoleyloxy)-3-(N-phenylamino)propyl linoleate, respectively, and the corresponding coefficients of variation were approximately 1%, greatly improving reproducibility over manual procedures.

Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS.

The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli.