Inferring hospital admissions from SARS-CoV-2 virus loads in wastewater in The Netherlands, August 2020 - February 2022.

Wastewater-based surveillance enables tracking of SARS-CoV-2 circulation at a local scale in near-real time. Here we investigate the relation between virus loads and the number of hospital admissions in the Netherlands. Inferred virus loads from August 2020 until February 2022 in each of the 344 Dutch municipalities are analysed in a Bayesian multilevel Poisson regression to relate virus loads to daily age-stratified (in groups of 20 years) hospital admissions. Covariates include municipal vaccination coverages stratified by age and dose (first, second, and booster) and prevalence of the circulating coronavirus variants (wildtype, Alpha, Delta, and Omicron (BA.1 and BA.2)). Our model captures the relation between hospital admissions and virus loads well. Estimated hospitalisation rates per 1,000,000 persons per day at a virus load of 1013 particles range from 0.18 (95 % Prediction Interval (PI): 0.046-0.48) in children (0-19 years) to 20.1 (95 % PI: 9.46-36.8) in the oldest age group (80 years and older) in an unvaccinated population with only wildtype SARS-CoV-2 circulation. The analyses indicate a nearly twofold (1.92 (95 % PI: 1.78-2.05)) decrease in the expected number of hospitalisations at a given virus load between the Alpha and the Omicron variant. Our analyses show that virus load estimates in wastewater are closely related to the expected number of hospitalisations and provide an attractive tool to detect increased SARS-CoV-2 circulation at a local scale, even when there are few hospital admissions. Our analyses enable integration of data at the municipality level into meaningful conversion rates to translate virus loads at a local level into expected numbers of hospital admissions, which would allow for a better interpretation of virus loads detected in wastewater.

Non-cystic fibrosis bronchiectasis: A single-centre retrospective study in Johannesburg, South Africa.

Background: Bronchiectasis, once rarely encountered, appears to be increasing in prevalence in South Africa (SA) and globally. There is a lack of published data on non-cystic fibrosis (CF) bronchiectasis, specifically in low- to middle-income countries, despite the high rates of risk factors such as HIV, pulmonary tuberculosis, and other infections. Objectives: Given this lack of data, to review the characteristics of adult patients with non-CF bronchiectasis at a tertiary academic hospital in Johannesburg, SA. Methods: This was a single-centre, retrospective record review, including all cases of non-CF bronchiectasis that were in the records of the adult pulmonology clinic at Charlotte Maxeke Johannesburg Academic Hospital as of April 2017. Results: There were 197 patients, with a slight predominance of males, and the patients were generally young. The HIV rate was higher than the national average (34.8% v. 13.7%), and the HIV-positive patients had a high TB prevalence (86.9%). Pseudomonas spp. were cultured from sputum in 15.3% of cases. Fewer than half of the cohort had the diagnosis of bronchiectasis confirmed by high-resolution chest tomography. Airway obstruction (forced expiratory volume in 1 second/forced vital capacity ratio <70%) was observed in 47.0% of patients. Treatment with a short-acting beta-2-agonist was prescribed in 62.9%, a long-acting beta-2-agonist in 43.6% and inhaled corticosteroids in 51.3%. Antibiotic therapy during exacerbations was used in 44.2%, mainly amoxycillin-clavulanate (66.7%). Conclusion: While single centre and retrospective, this study adds to the data on non-CF bronchiectasis in sub-Saharan Africa and should encourage further research to increase our understanding of adult non-CF bronchiectasis in SA. Study synopsis: What the study adds. This study adds to published data detailing the clinical characteristics of adult non-cystic fibrosis (CF) bronchiectasis in low- and middle-income countries (LMICs).Implications of the findings. As a retrospective descriptive study, the findings summarise the characteristics of adults with non-CF bronchiectasis in a cohort from Johannesburg, South Africa. The findings suggest that the characteristics of bronchiectasis in this region appear to be similar in several ways to those in other LMICs, but quite different from those in the developed world.

Structural and functional analysis of mutations in alkaptonuria.

Alkaptonuria (AKU), the prototypic inborn error of metabolism, was the first human disease to be interpreted as a Mendelian trait by Garrod and Bateson at the beginning of last century. AKU results from impaired function of homogentisate dioxygenase (HGO), an enzyme required for the catabolism of phenylalanine and tyrosine. With the novel 7 AKU and 22 fungal mutations reported here, a total of 84 mutations impairing this enzyme have been found in the HGO gene from humans and model organisms. Forty-three of these mutations result in single amino acid substitutions. This mutational information is analysed here in the context of the HGO structure and function using kinetic assays performed using purified AKU mutant enzymes and the crystal structure of human HGO. HGO is a topologically complex structure which assembles as a functional hexamer arranged as a dimer of trimers. We show how the intricate pattern of intra- and inter-subunit interactions and the extensive surfaces required for subunit folding and association of this oligomeric enzyme can be inactivated at multiple levels by single-residue substitutions. This explains, in part, the predominance of missense mutations (67%) in AKU.

Two-year mortality and its determinants following acute myocardial infarction in Trinidad and Tobago.

The purpose of this study was to determine the occurrence of coronary artery disease risk factors in patients presenting with acute myocardial infarction (AMI) to a tertiary care institution in Trinidad and to determine the factors associated with increased mortality following AMI. All patients admitted to the Eric Williams Medical Sciences Complex (EWMSC) between January 1 and December 31, 1996, with a diagnosis of AMI were identified using the hospital admissions and discharge diagnosis databases. Demographic, clinical and laboratory variables were extracted from the hospital case records of patients with confirmed AMI. Sixty-one AMI patients (38 men) were admitted during the study period. Mean age at admission was 60 +/- 11 years with an ethnic case mix of thirty-nine (62%) of East Indian descent, eight (13%) of African descent, twelve (20%) mixed ethnicity and three (5%) of Caucasian descent. Thirty patients (49%) were hypertensive. Thirty-two patients (53%) were diabetic and eighteen patients (30%) gave a history of cigarette smoking. The mean left ventricular ejection fraction was 53 +/- 14%. The mean serum cholesterol from 29 patients was 228.2 +/- 49.0 mg/dl. Increasing age, female gender, an ejection fraction less than 40%, non treatment with streptokinase and in-hospital ventricular fibrillation were associated with poor survival. Multiple regression analyses identified three independent predictors of mortality. These were gender (p = 0.04), in-hospital ventricular fibrillation (p = 0.001) and an ejection fraction less than 40% (p = 0.02). Diabetes mellitus, hypertension, hyperlipidaemia and cigarette smoking were prevalent amongst patients presenting with AMI. Ventricular function was a major determinant of two-year mortality following AMI. Aggressive risk factor modification is recommended to prevent both first and recurrent coronary events.

Crystal structure of human homogentisate dioxygenase.

Homogentisate dioxygenase (HGO) cleaves the aromatic ring during the metabolic degradation of Phe and Tyr. HGO deficiency causes alkaptonuria (AKU), the first human disease shown to be inherited as a recessive Mendelian trait. Crystal structures of apo-HGO and HGO containing an iron ion have been determined at 1.9 and 2.3 A resolution, respectively. The HGO protomer, which contains a 280-residue N-terminal domain and a 140-residue C-terminal domain, associates as a hexamer arranged as a dimer of trimers. The active site iron ion is coordinated near the interface between subunits in the HGO trimer by a Glu and two His side chains. HGO represents a new structural class of dioxygenases. The largest group of AKU associated missense mutations affect residues located in regions of contact between subunits.

Two-Year Mortality and its determinants following acute myocardial infarction in Trinidad and Tobago

The purpose of this study was to determine the occurrence of coronary artery disease risk factors in patients presenting with acute myocardial infarction(AMI) to a tertiary care institution in Trinidad and to determine the factors associated with increased mortality following AMI. All patients admitted to the Eric Williams Medical Sciences Complex (EWMSC) between January 1 and December 31, 1996, with a diagnosis of AMI were identified using the hospital admissions and discharge diagnosis databases. Demographic, clinical and laboratory variables were extracted from the hospital case records of patients with confirmed AMI. Sixty-one AMI patients (38 men) were admitted during the study period. Mean age of admittance was 60 ñ 11 years with an ethnic case mix of thirty-nine (62 percent) of East Indian descent, eight (13 percent) of African descent, twelve (20 percent) mixed ethnicity and three of Caucasian descent. Thirty patients (49 percent) were hypertensive. Thirty-two patients (53 percent) were diabetic and eighteen patients (30 percent) gave a history of cigarette smoking. The mean left venticular ejection fraction was 53 ñ 14 percent. The mean serum cholesterol from 29 patients was 228.2 ñ 49.0 mg/dl. Increasing age, female gender, an ejection fraction less than 40 percent, non treatment with streptokinase and in-hospital ventricular fibrillation were associated with poor survival. Multiple regression analyses identified three independent predictors of mortality. These were gender (p = 0.04), in-hospital ventricular fibrillation (p = 0.001) and an ejection fraction less than 40 percent (p = 0.02). Diabetes mellitus, hypertension, hyperlipidaemia and cigarette smoking were prevalent amongst patients presenting with AMI. Ventricular function was a major determinant of two-year mortality following AMI. Aggressive risk factor modification is recommended to prevent both first and recurrent coronary events.(AU)

Two-year mortality and its determinants following acute myocardial infarction in Trinidad and Tobago

The purpose of this study was to determine the occurrence of coronary artery disease risk factors in patients presenting with acute myocardial infarction(AMI) to a tertiary care institution in Trinidad and to determine the factors associated with increased mortality following AMI. All patients admitted to the Eric Williams Medical Sciences Complex (EWMSC) between January 1 and December 31, 1996, with a diagnosis of AMI were identified using the hospital admissions and discharge diagnosis databases. Demographic, clinical and laboratory variables were extracted from the hospital case records of patients with confirmed AMI. Sixty-one AMI patients (38 men) were admitted during the study period. Mean age of admittance was 60 ñ 11 years with an ethnic case mix of thirty-nine (62 percent) of East Indian descent, eight (13 percent) of African descent, twelve (20 percent) mixed ethnicity and three of Caucasian descent. Thirty patients (49 percent) were hypertensive. Thirty-two patients (53 percent) were diabetic and eighteen patients (30 percent) gave a history of cigarette smoking. The mean left venticular ejection fraction was 53 ñ 14 percent. The mean serum cholesterol from 29 patients was 228.2 ñ 49.0 mg/dl. Increasing age, female gender, an ejection fraction less than 40 percent, non treatment with streptokinase and in-hospital ventricular fibrillation were associated with poor survival. Multiple regression analyses identified three independent predictors of mortality. These were gender (p = 0.04), in-hospital ventricular fibrillation (p = 0.001) and an ejection fraction less than 40 percent (p = 0.02). Diabetes mellitus, hypertension, hyperlipidaemia and cigarette smoking were prevalent amongst patients presenting with AMI. Ventricular function was a major determinant of two-year mortality following AMI. Aggressive risk factor modification is recommended to prevent both first and recurrent coronary events.

Providing alternative health care: an ancient system for a modern age.

Ayurveda, the oldest health care system in the world, has unique potential waiting to be exploited by the advanced practice nurse (APN) practicing in family health and primary care settings. The background, paradigm, interventions, scientific research, and strategies to implement Ayurveda in APN practice are explored. Although little is known about Ayurveda in Western cultures, it offers many health promotive interventions that can help the APN fulfill the needs of families who seek a level of wellness not offered by conventional medicine.

Binding and autophosphorylating activity of human insulin analogs.

Insulin receptor binding and autophosphorylating activities of a number of synthetic analogs of human insulin have been examined using highly purified insulin receptor from human placenta. In general, autophosphorylation correlates well with the ability of the analogs to stimulate glucose oxidation and to inhibit lipolysis in adipocytes although their biological activities varied over a wide range. These findings support the hypothesis that autophosphorylation is an obligatory step in the pathways leading to glucose oxidation and inhibition of lipolysis. The relative biological potencies of the analogs in the autophosphorylation assay also correlated well with their receptor-binding affinities except for the peptides [endo-TyrB16a]insulin, in which an additional Tyr has been inserted between TyrB16 and LeuB17 and [ProA2]insulin. The relative receptor binding affinity of [endo-TyrB16a]insulin is significantly greater than its biological activity in the adipocyte or receptor autophosphorylation assays. The converse is true for [ProA2]insulin. These results demonstrate that the amino-acid residues involved in binding and receptor activation may not be identical.

The rat liver insulin receptor.

Using insulin affinity chromatography, we have isolated highly purified insulin receptor from rat liver. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the rat liver receptor contained the Mr 125,000 alpha-subunit, the Mr 90,000 beta-subunit, and varying proportions of the Mr 45,000 beta'-subunit. The specific insulin binding of the purified receptor was 25-30 micrograms of 125I-insulin/mg of protein, and the receptor underwent insulin-dependent autophosphorylation. Rat liver and human placental receptors differ from each other in several functional aspects: (1) the adsorption-desorption behavior from four insulin affinity columns indicated that the rat liver receptor binds less firmly to immobilized ligands; (2) the 125I-insulin binding affinity of the rat liver receptor is lower than that of the placental receptor; (3) partial reduction of the rat liver receptor with dithiothreitol increases its insulin binding affinity whereas the binding affinity of the placental receptor is unchanged; (4) at optimal insulin concentration, rat liver receptor autophosphorylation is stimulated 25-50-fold whereas the placental receptor is stimulated only 4-6-fold. Conversion of the beta-subunit to beta' by proteolysis is a major problem that occurs during exposure of the receptor to the pH 5.0 buffer used to elute the insulin affinity column. The rat receptor is particularly subject to destruction. Frequently, we have obtained receptor preparations that did not contain intact beta-subunit. These preparations failed to undergo autophosphorylation, but their insulin binding capacity and binding isotherms were identical with those of receptor containing beta-subunit. Proteolytic destruction and the accompanying loss of insulin-dependent autophosphorylation can be substantially reduced by proteolysis inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Avidin-biotin affinity chromatography: application to the isolation of human placental insulin receptor.

The ligand N alpha, B1-(6-biotinylamido)hexanoyl-insulin was attached noncovalently to Sepharose 4B immobilized succinoylavidin to form an insulin-affinity resin. This resin was used to isolate highly purified insulin receptor from human placental tissue by a four step process involving (i) preparation of a crude membrane fraction, (ii) solubilization with Triton X-100, (iii) wheat germ agglutinin purification, and (iv) insulin-affinity chromatography. NaDodSO4/PAGE of the purified 125I-labeled receptor under nonreducing conditions showed the presence of a major component with an approximate molecular weight of 350,000 and a minor component with a molecular weight of approximately equal to 166,000. Based on the assumption that the degree of labeling is comparable in both components, the material corresponding to the Mr 350,000 peak represents approximately equal to 94% of the receptor preparation as determined by scanning the autoradiograms. The specific insulin binding capacity of the preparation is 18 +/- 6 micrograms of 125I-labeled insulin per mg of protein as determined by the polyethylene glycol assay and analyzed by Scatchard plot. Insulin binding activity was stable at 4 degrees C and pH 7.6 for at least 12 weeks but was destroyed by freezing and thawing. The availability of highly purified receptor afforded the opportunity to explore its precipitability by polyethylene glycol under assay conditions. Whereas trichloroacetic acid precipitated 95% of the 125I-labeled receptor, polyethylene glycol precipitated only 30%. If the specific activity of the receptor is corrected for incomplete precipitability by polyethylene glycol, the apparent specific binding would be 3.5 +/- 1.2 mol of insulin per mol of receptor. These results are in disagreement with the current receptor model, which postulates that 1 mol of receptor (Mr, 350,000) binds 2 mol of insulin. Clearly, the problems associated with the method available for determining insulin binding are sufficiently serious to preclude their use in determining receptor valence.

Ligands for insulin receptor isolation.

Biotinylated insulins are bivalent molecules having the ability to bind to insulin receptors on the one hand and to "avidins" on the other. In order to be useful as ligands for insulin receptor isolation, biotinylated insulins must be developed that have the capacity to bind simultaneously to both and insulin receptor. The present investigation addresses this problem. A series of biotinylated and dethiobiotinylated insulins has been prepared in which the distance between the biotin carboxyl group and the insulin varies from 7 to 20 atoms. These compounds form complexes with succinoylavidin. The dissociation rates (K-1) of these complexes have been determined from the [14C]biotin exchange assay. The dissociation kinetics of most of these complexes are biphasic, and the kinetic constants reported are those corresponding to the slow rate. Ligands containing dethiobiotin dissociate more rapidly than the corresponding biotin derivatives. The interposition of a spacer arm substantially decreases the rate of dissociation. The [14C]biotin exchange assay could not be used with streptavidin complexes of the above ligand since biotin dissociates more rapidly from streptavidin than from succinoylavidin. However, the relative dissociation rates of a series of ligands could be determined and were as follows: 6-(dethiobiotinylamido)-hexanoic acid greater than dethiobiotinyl-A1-insulin greater than biotinylinsulin greater than biotinyl-A1-insulin greater than biotinyl-A2-insulin. Dethiobiotin and its amide failed to form complexes with streptavidin. The affinity of the ligands for insulin receptors was determined by measuring their ability to stimulate 14CO2 formation from [1-14C]glucose in rat epididymal adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin receptor isolation studies.

The observation that N alpha,B1-biotinylinsulin binds firmly to resins in which succinoylavidin is covalently attached to AH Sepharose 4B and can be retrieved by exposure of the resins to 20 mM biotin provided the basis for the present investigations. Solubilized, partially purified insulin receptor from human placenta binds to affinity resins in which N alpha,B1-biotinylinsulin is noncovalently attached to AH Sepharose 4B-immobilized-succinolylavidin. Exposure of the receptor loaded resin to 20 mM biotin results in liberation of a high molecular weight material containing bound 125I-biotinylinsulin, which precipitates with polyethyleneglycol and cross reacts with human insulin receptor antibodies. The technique is biospecific and appears to be applicable to the purification of insulin receptors on a preparative scale. Crude solubilized insulin receptor from human placenta is contaminated with "insulinase" which is inhibited by N-ethylmaleimide. HPLC provides a tool to assess "insulinase" activity that is more sensitive than the TCA precipitation method.

Recovery from thyroid-stimulating hormone-induced refractoriness in thyroid slices: effect of removal of hormone and new protein synthesis.

An initial incubation of bovine thyroid slices with TSH causes decreased responsiveness to the subsequent addition of the hormone when the adenylate cyclase -cAMP system and other metabolic parameters are measured. After the initial incubation with TSH, refractoriness persists despite incubation of thyroid slices for 24 h in the absence of added TSH. Removal of persistently bound TSH by trypsin or antibody to TSH did not reverse the refractoriness during a subsequent 2 h incubation without added TSH. However, normal TSH responsivity was restored by the removal of TSH bound during the first incubation by the addition of either trypsin or antibody to TSH at the beginning of a 24-h second incubation. Restitution of TSH responsiveness after treatment with trypsin or antibody to TSH requires new protein synthesis. While TSH-induced refractoriness does not modify stimulation of cAMP by cholera toxin, its effect on glucose oxidation is significantly diminished. Menadiol stimulation of glucose oxidation is not inhibited in thyroid slices refractory to TSH. Thus, the effect of menadiol is subsequent to the block induced by TSH, whereas that of cholera toxin is proximal to it.

Hormonal properties of avidin-biotinylinsulin and avidin-biotinylcorticotropin complexes.

In connection with the development of affinity columns (based on the avidin-biotin interaction) for retrieval of peptide and protein hormone receptors, the hormonal properties of a number of avidin-biotinylinsulin and avidin-bioinylcorticotropin complexes were examined. Of particular interest was an evaluation of streptavidin as a ligand for the attachment of biotinylated hormones to solid supports and its possible advantage over SpHPP-avidin (S = succinoylated; pHPP = 3-(p-hydroxyphenyl)propionyl). As concerns binding kinetics using rat liver plasma membranes, streptavidin was found superior to avidin since it does not display apparently nonsaturable binding. Scatchard analyses of the binding of 125I-streptavidin, 125I-S-streptavidin and 125I-SpHPP-avidin to rat liver plasma membranes gave KD value of 6.7, 13.2, and 10.6 nM respectively. The binding was saturable and the unlabeled proteins competed with their labeled counterparts for the membrane binding sites. Biotinylinsulin, attached to either streptavidin or SpHPP-avidin was able to compete for 125I-insulin-binding sites on rat liver plasma membranes though somewhat larger concentrations of the complexes than of insulin were required to achieve comparable inhibition. The ID50 values for insulin and the biotinylinsulin complexes were 5 and 80 nM respectively. Biotinylcorticotropin was found to be a more effective activator of particulate rat adrenal adenylate cyclase when complexed with unmodified avidin than with streptavidin, S-streptavidin or SpHPP-avidin.

Effects of thyrotropin and cholera toxin on the thyroidal adenylate cyclase-adenosine 3',5'-monophosphate system.

The present experiments examined the relationship between cholera toxin and TSH stimulation of the adenylate cyclase system in bovine thyroid tissue. Preincubation of thyroid slices for 20 min at 4 C with a maximal concentration of cholera toxin (100 microgram/ml) did not impair the subsequent stimulation of cAMP by submaximal amounts of TSH (1 mU/ml) during a 5-min incubation at 37 C. Incubation of cholera toxin or TSH with mixed gangliosides, followed by the addition of thyroid slices resulted in inhibition of the cholera toxin but not the TSH stimulation of cAMP formation. Previous exposure of thyroid slices to TSH induced refractoriness to subsequent stimulation of cAMP formation by TSH, but the response to cholera toxin was unchanged. NAD is necessary for cholera toxin, but not TSH, stimulation of adenylate cyclase. In the absence of NAD, cholera toxin inhibited the effect of maximal concentrations of TSH and prostaglandin E1 on adenylate cyclase activity but had no effect on NaF stimulation. In the presence of NAD, the stimulation of adenylate cyclase activity of bovine thyroid plasma membranes by a maximal amount of TSH was not influeced by maximal amounts of cholera toxin. Cholera toxin had a biphasic action on the binding of [125I]iodo-TSH, with low concentrations enhancing and high concentrations inhibiting binding. TSH augmented the binding of [125I]iodo-cholera toxin over the range of 1-100 mU/tube. Cholera toxin at 10 microgram/ml maximally inhibited binding. In addition to the requirement for ribosylation of adenylate cyclase, the present results indicate that the mechanisms of action of TSH and cholera toxin on cAMP formation are different.

Epidermal growth factor stimulation of human breast cancer cells in culture.

Epidermal growth factor (EGF), a polypeptide found in human and animal blood and secretions, has been found to stimulate a variety of tissues in vitro including normal and malignant rodent mammary epithelium and human breast epithelial cells and fibroadenoma. We have studied the influence of EGF on malignant human breast tissue with a model system comprising human breast carcinoma cells growing in tissue culture. EGF stimulated growth of MCF-7 cells in serum-free medium. After 7 days in culture, a 2-fold increase in cell number and DNA content and a 3-fold increase in total protein were observed in cells incubated with EGF (10 ng/ml). As little as 0.01 ng/ml of EGF stimulated growth; 10 ng/ml was maximal. EGF effects on growth were noted for cells plated at a high as well as sparse (cloning) density. EGF also stimulated the rates of thymidine, uridine, and leucine incorporation into macromolecules in a dose- and time-dependent fashion. Stimulation of uridine and leucine incorporation was evident by 3 hr, whereas EGF stimulation of thymidine incorporation was delayed until 12 to 18 hr. EGF increased the proportion of cells active in DNA synthesis by nearly 2-fold. The combination of optimal concentrations of insulin (also a growth factor for these cells) and EGF did not stimulate growth above that seen with either hormone alone, suggesting a common step in their mechanism of action. The EGF effect was not dependent on the presence of serum and was not enhanced by dexamethasone as reported for other types of cells. EGF had no effect on another human breast cancer cell line, the MDA-231. These studies suggest that growth of some human breast cancers may be influenced by EGF.