Occurrence of contaminants of emerging concern in the Eerste River, South Africa: Towards the optimisation of an urban water profiling approach for public- and ecological health risk characterisation.

The study evaluated the presence and fate of various contaminants of emerging concern (CECs) from a South African wastewater treatment works (WWTW) and surface waters located around an urban setting. A total of 45 CECs were quantified from nine sampling locations over an 11-month period. Daily loads (g/day) of the target analytes in the WWTW showed persistence of some CECs, along with population-normalised daily loads (mg/day/1000inh) of pharmaceuticals and drugs of abuse (DOA) that were estimated for the first time in the study area. Multiple chemical markers were recorded in river water located upstream of the WWTW discharge throughout the study period, suggesting a high degree of diffuse pollution from urban communities in the study area that are not connected to sewage networks or where sanitation services are limited. The potential of using defined surface water locations to perform community-wide substance use profiling for non-sewered communities was also explored. Environmental risk characterisation for the WWTW effluent and surface waters throughout the study period provided multiple risk quotients (RQ) for the target list of CECs spanning over various sentinel trophic levels. High risk profiles (RQ > 1.0) with a frequency of exceedance (FoE) larger than 75 % were recorded for several CECs in both WWTW effluent and surface water locations that suggest potential long-term ecological health risk impacts of pollution hotspot areas in the river catchment situated around the urban area. We present challenges in surface water quality within the study area that is relatable, or may even present more challenging, in other low- or middle-income country (LMICs) settings. The study also highlighted some challenges and limitations associated with the much-needed application of wastewater-based epidemiology (WBE) intervention in non-sewered communities that can inform on public health and communal substance use profiles of the entire urban setting.

Wastewater-based epidemiology in hazard forecasting and early-warning systems for global health risks.

With the advent of the SARS-CoV-2 pandemic, Wastewater-Based Epidemiology (WBE) has been applied to track community infection in cities worldwide and has proven succesful as an early warning system for identification of hotspots and changingprevalence of infections (both symptomatic and asymptomatic) at a city or sub-city level. Wastewater is only one of environmental compartments that requires consideration. In this manuscript, we have critically evaluated the knowledge-base and preparedness for building early warning systems in a rapidly urbanising world, with particular attention to Africa, which experiences rapid population growth and urbanisation. We have proposed a Digital Urban Environment Fingerprinting Platform (DUEF) - a new approach in hazard forecasting and early-warning systems for global health risks and an extension to the existing concept of smart cities. The urban environment (especially wastewater) contains a complex mixture of substances including toxic chemicals, infectious biological agents and human excretion products. DUEF assumes that these specific endo- and exogenous residues, anonymously pooled by communities' wastewater, are indicative of community-wide exposure and the resulting effects. DUEF postulates that the measurement of the substances continuously and anonymously pooled by the receiving environment (sewage, surface water, soils and air), can provide near real-time dynamic information about the quantity and type of physical, biological or chemical stressors to which the surveyed systems are exposed, and can create a risk profile on the potential effects of these exposures. Successful development and utilisation of a DUEF globally requires a tiered approach including: Stage I: network building, capacity building, stakeholder engagement as well as a conceptual model, followed by Stage II: DUEF development, Stage III: implementation, and Stage IV: management and utilization. We have identified four key pillars required for the establishment of a DUEF framework: (1) Environmental fingerprints, (2) Socioeconomic fingerprints, (3) Statistics and modelling and (4) Information systems. This manuscript critically evaluates the current knowledge base within each pillar and provides recommendations for further developments with an aim of laying grounds for successful development of global DUEF platforms.

An empirical investigation of climate and land-use effects on water quantity and quality in two urbanising catchments in the southern United Kingdom.

Using historical data of climate, land-use, hydrology and water quality from four catchments located in the south of England, this study identifies the impact of climate and land-use change on selected water quantity and water quality indicators. The study utilises a paired catchment approach, with two catchments that have experienced a high degree of urbanisation over the past five decades and two nearby, hydrologically similar, but undeveloped catchments. Multivariate regression models were used to assess the influence of rainfall and urbanisation on runoff (annual and seasonal), dissolved oxygen levels and temperature. Results indicate: (i) no trend in annual or seasonal rainfall totals, (ii) upward trend in runoff totals in the two urban catchments but not in the rural catchments, (iii) upward trend in dissolved oxygen and temperature in the urban catchments, but not in the rural catchments, and (iv) changes in temperature and dissolved oxygen in the urban catchments are not driven by climatic variables.

Detection and attribution of urbanization effect on flood extremes using nonstationary flood-frequency models.

Urbanization is found to have an impact on high flows in a urbanized catchmentThe use of point processes is advocated for trend detection and attributionThe use of process-related covariates gives a better representation of change.

Inducible nitric oxide synthase is responsible for nitric oxide release from murine pituicytes.

This study investigated whether pituicytes were able to produce and release nitric oxide (NO), and which type of nitric oxide synthase (NOS) would be responsible for this phenomenon. Lipopolysaccharide (LPS) 1 micro g/ml was used as inflammatory mediator. Because pituicytes are known to secrete interleukin (IL)-6 upon stimulation with LPS, this parameter was also investigated. Cultured pituicytes, from 4-week-old male mice, were stimulated with LPS for 6 h or 24 h. At 24 h, there was a significant increase in accumulated nitrite indicating NO formation. In contrast, IL-6 release was already significantly higher 6 h after stimulation and further increased at 24 h. The correlation between accumulated nitrite and secreted IL-6 was 0.84 after 24 h of incubation with LPS. The expression of inducible NOS (iNOS) mRNA in the pituicytes was significantly higher than the control level after 6 h and 24 h of exposure to LPS, with levels at 6 h being significantly higher than those at 24 h. There was no detected expression of endothelial NOS or neuronal NOS mRNA. Cultured pituicytes were also subjected to immunocytochemistry for iNOS protein at 6, 12, and 24 h after stimulation with LPS. Most cells were positive for iNOS, but there were no observable differences with the time points that we used. Collectively, these results show that pituicytes are able to produce NO, and that the inducible form of NOS is responsible for this production. Furthermore, there is a weak correlation between NO and IL-6 released from pituicytes after 24 h of stimulation with LPS.

Dopa decarboxylase genotypes may influence age at onset of schizophrenia.

Several lines of evidence implicate dopa decarboxylase (DDC) with schizophrenia. By analysis of two putative functional DDC variants in 173 schizophrenic patients and 204 controls we tested the hypotheses that DDC is involved in: (1) predisposition to schizophrenia; and (2) modulation of age at disease onset. No association was observed with schizophrenia as a whole, whereas an association between DDC genotypes and age at disease onset was suggested in males (P = 0.03). This association was most pronounced in relation to genotypes of haplotypes comprising both variants, suggesting an additive model where one variant mediates early and the other late onset. Accordingly, the haplotype-based genotypes could be assigned into three groups by their possible relative effect on age at onset: an "early", "neutral" and "late" group. Dividing the male schizophrenics into four groups with increasing age at onset, the "early" genotypes were seen to decrease in frequency from 51.5% to 16.7% while the "late" genotypes increased from 12.1% to 33.3% (P = 0.02). The difference in mean age at onset between male patients with "early" genotypes vs patients with "late" genotypes was close to 5 years (95% CI: 0.7-8.8). Thus, DDC may possibly act as a modulator of age at onset in male schizophrenics.

Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complex.

An insulin-containing fusion protein (ICFP, encoding the yeast prepro-alpha factor leader peptide fused via a lysine-arginine cleavage site to a single chain insulin) has been expressed in Saccharomyces cerevisiae where it is inefficiently secreted. Single gene disruptions have been identified that cause enhanced immunoreactive insulin secretion (eis). Five out of six eis mutants prove to be vacuolar protein sorting (vps)8, vps35, vps13, vps4, and vps36, which affect Golgi<-->endosome trafficking. Indeed, in wild-type yeast insulin is ultimately delivered to the vacuole, whereas vps mutants secrete primarily unprocessed ICFP. Disruption of KEX2, which blocks intracellular processing to insulin, quantitatively reroutes ICFP to the cell surface, whereas loss of the Vps10p sorting receptor is without effect. Secretion of unprocessed ICFP is not based on a dominant secretion signal in the alpha-leader peptide. Although insulin sorting mediated by Kex2p is saturable, Kex2p functions not as a sorting receptor but as a protease: replacement of Kex2p by truncated secretory Kex2p (which travels from Golgi to cell surface) still causes endoproteolytic processing and intracellular insulin retention. Endoproteolysis promotes a change in insulin's biophysical properties. B5His residues normally participate in multimeric insulin packing; a point mutation at this position permits ICFP processing but causes the majority of processed insulin to be secreted. The data argue that multimeric assembly consequent to endoproteolytic maturation regulates insulin sorting in the secretory pathway.

Yeast secretory expression of insulin precursors.

Since the 1980s, recombinant human insulin for the treatment of diabetes mellitus has been produced using either the yeast Saccharomyces cerevisiae or the prokaryote Escherichia coli. Here, development of the insulin secretory expression system in S. cerevisiae and its subsequent optimisation is described. Expression of proinsulin in S. cerevisiae does not result in efficient secretion of proinsulin or insulin. However, expression of a cDNA encoding a proinsulin-like molecule with deletion of threonine(B30) as a fusion protein with the S. cerevisiae alpha-factor prepro-peptide (leader), followed either by replacement of the human proinsulin C-peptide with a small C-peptide (e.g. AAK), or by direct fusion of lysine(B29) to glycine(A1), results in the efficient secretion of folded single-chain proinsulin-like molecules to the culture supernatant. The secreted single-chain insulin precursor can then be purified and subsequently converted to human insulin by tryptic transpeptidation in organic aqueous medium in the presence of a threonine ester. The leader confers secretory competence to the insulin precursor, and constructed (synthetic) leaders have been developed for efficient secretory expression of the insulin precursor in the yeasts S. cerevisiae and Pichia pastories. The Kex2 endoprotease, specific for dibasic sites, cleaves the leader-insulin precursor fusion protein in the late secretory pathway and the folded insulin precursor is secreted to the culture supernatant. However, the Kex2 endoprotease processing of the pro-peptide-insulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insulin precursor fusion protein is secreted to the culture supernatant in a hyperglycosylated form. A spacer peptide localised between the leader and the insulin precursor has been developed to optimise Kex2 endoprotease processing and insulin precursor fermentation yield.

The role of leaders in intracellular transport and secretion of the insulin precursor in the yeast Saccharomyces cerevisiae.

Pulse-chase analysis of folded and misfolded insulin precursor (IP) expressed in Saccharomyces cerevisiae was performed to establish the requirements for intracellular transport and the influence of the secretory pathway quality control mechanisms on secretion. Metabolic labelling of the IP expressed in S. cerevisiae showed that the effect of a leader was to stabilise the IP in the endoplasmic reticulum (ER), and facilitate intracellular transport of the fusion protein and rapid secretion. The first metabolically labelled IP appeared in the culture supernatant within 2-4 min of chase, and most of the secreted IP appeared within the first 15 min of chase. After enzymatic removal of the leader in a late Golgi apparatus compartment, the IP followed one of two routes: (1) to the plasma membrane and hence to the culture supernatant, or (2) to a Golgi or post-Golgi compartment from which secretion was restricted. Combined secretion and intracellular retention of the IP reflected either saturation of a Golgi or post-Golgi compartment and secretion as a consequence of overexpression, or competition between secretion and intracellular retention. IP which was misfolded, either due to amino acid substitution or because disulphide bond formation had been prevented with dithiothreitol (DTT), was transported from the ER to the Golgi apparatus but then retained in a Golgi or post-Golgi compartment and not exported to the culture supernatant.

Two novel variants in the DOPA decarboxylase gene: association with bipolar affective disorder.

DOPA decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase (AADC), is an enzyme involved in the synthesis of the important neurotransmitters dopamine, norepinephrine, and serotonin. In addition, it participates in the synthesis of trace amines; compounds suggested to act as endogenous modulators of central neurotransmission. Thus, DDC is regarded as a potential susceptibility gene for a variety of neuropsychiatric disorders. The aim of the present study was to examine the role of DDC in bipolar affective disorder (BPAD). By screening 10 individuals for sequence variations in the coding region of the DDC gene as well as in the neuron-specific promoter and 5' untranslated regions we were able to identify two fairly frequent variants: a 1-bp deletion in the promoter and a 4-bp deletion in the untranslated exon 1. Both deletions affect putative binding sites for known transcription factors, suggesting a possible functional impact at the level of expression. The two variants were applied in an association study including 80 Danish bipolar patients, 112 English bipolar patients, 223 Danish controls, and 349 English controls. Analyzing the combined material, a significant association was found between the 1-bp deletion and BPAD with P-values of 0.037 (allelic) and 0.021 (genotypic). The frequency of the 1-bp deletion was 13.3% in patients and 9.4% in controls with a corresponding odds ratio of 1. 48 (95% CI: 1.02-2.15). The results presented suggest that DDC may act as a minor susceptibility gene for bipolar affective disorder.

Secretory expression and characterization of insulin in Pichia pastoris.

The yeasts Pichia pastoris and Saccharomyces cerevisiae have similar overall features regarding the secretory expression of insulin. The S. cerevisiae mating factor alpha (alpha-factor) prepro-leader facilitated the secretion of an insulin precursor, but not proinsulin expressed in P. pastoris. Synthetic prepro-leaders developed for the secretory expression of the insulin precursor in S. cerevisiae also facilitated the secretion of the insulin precursor expressed in P. pastoris. In contrast with S. cerevisiae, only insulin precursor and no unprocessed hyperglycosylated alpha-factor pro-leader/insulin precursor fusion protein was secreted from P. pastoris. A spacer peptide in the fusion protein increased the fermentation yield of the insulin precursor in P. pastoris. A synthetic prepro-leader, but not an alpha-factor prepro-leader lacking N-glycosylation sites, facilitated the secretion of the insulin precursor in P. pastoris. P. pastoris has a capacity for secretory expression of the insulin precursor that is equal to or better than that of S. cerevisiae. Peptide mapping and MS indicated a structure of the insulin precursor expressed in P. pastoris identical with that of human insulin.

Secretory expression of human albumin domains in Saccharomyces cerevisiae and their binding of myristic acid and an acylated insulin analogue.

Albumin is organized in three homologous domains formed by double loops stabilized by disulfide bonds. Utilizing a secretory expression system based on a synthetic secretory prepro-leader, the three human serum albumin domains were expressed in the yeast Saccharomyces cerevisiae. Human serum albumin domains I and III were efficiently expressed and secreted, indicating that these domains can form independent structural units capable of folding into stable tertiary structures. In contrast, albumin domain II was not secreted and disappeared early in the secretory pathway. Human serum albumin has the ability to bind a large number of small molecule ligands, including fatty acids, presumably due to its structure and structural flexibility. Purified albumin domain III bound myristic acid, whereas purified albumin domain I did not bind myristic acid. A new soluble long-acting insulin an alogue acylated with myristic acid (Markussen J., et al., Diabetologia 39, 281-288, 1996) bound to domain III and bound markedly more weakly to domain I.

alpha-Factor pro-peptide N-linked oligosaccharides facilitate secretion of the insulin precursor in Saccharomyces cerevisiae.

To evaluate the possible relationship between N-linked glycosylation of the Saccharomyces cerevisiae alpha-factor pro-peptide and transport of the alpha-factor pro-peptide/insulin precursor fusion protein through the Saccharomyces cerevisiae secretory pathway, we analysed secretion of insulin precursor facilitated by alpha-factor pro-peptides with one or more of the three N-linked glycosylation sites removed. Mutation of the three alpha-factor pro-peptide N-linked glycosylation sites drastically decreased insulin precursor secretion. The three alpha-factor pro-peptide N-linked glycosylation sites differ in their ability to facilitate secretion of the insulin precursor. The two alpha-factor pro-peptide N-linked glycosylation sites localized closest to the insulin precursor contributed significantly to secretion, whereas the most N-terminally linked glycosylation site did not appear to facilitate secretion. Only correctly folded insulin precursor was found in the culture supernatant, regardless of the pro-peptide used for secretion, indicating that alpha-factor pro-peptide N-linked oligosaccharide chains are not necessary for correct folding of the insulin precursor. Thus, N-linked glycosylation facilitates intracellular transport of the alpha-factor propeptide/insulin precursor fusion protein through the Saccharomyces cerevisiae secretory pathway and secretion of the insulin precursor. N-linked glycosylation per se is not sufficient to facilitate secretion of the insulin precursor; the position of the N-linked oligosaccharide chain on the alpha-factor pro-peptide is important for facilitating efficient secretion.

Prepro-leaders lacking N-linked glycosylation for secretory expression in the yeast Saccharomyces cerevisiae.

Synthetic prepro-leaders lacking consensus N-linked glycosylation sites confers secretion competence of correctly folded insulin precursor expressed in the yeast species Saccharomyces cerevisiae with a yield comparable to, or better than the alpha-factor prepro-leader. In contrast, the S. cerevisiae alpha-factor prepro-leader's three N-linked oligosaccharide chains are necessary for the ability to facilitate secretion of the insulin precursor from S. cerevisiae (T. Kjeldsen et al., Biotechnol. Appl. Biochem. 27, 109-115, 1998). Synthetic prepro-leader lacking both N-glycosylation and the dibasic Kex2 endoprotease processing site also efficiently facilitated secretion of a pro-leader/insulin precursor fusion protein in which the insulin precursor was correctly folded. The unprocessed pro-leader/insulin-precursor fusion protein was purified from culture medium and matured in vitro to desB30 insulin by Achromobacter lyticus lysyl-specific protease providing an alternative yeast expression system not dependent on the Kex2 endoprotease. The synthetic prepro-leader lacking N-linked glycosylation provides the opportunity for secretory expression in yeast utilizing either in vivo Kex2 endoprotease maturation of the fusion protein during secretion or in vitro maturation of the purified fusion protein with a suitable enzyme.

Alanine scanning mutagenesis of insulin.

Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. A total of 21 new insulin analog constructs were made, and in addition 7 high pressure liquid chromatography-purified analogs were tested, covering alanine substitutions in positions B1, B2, B3, B4, B8, B9, B10, B11, B12, B13, B16, B17, B18, B20, B21, B22, B26, A4, A8, A9, A12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for binding were at positions TyrA19, GlyB8, LeuB11, and GluB13, resulting in decreases in affinity of 1,000-, 33-, 14-, and 8-fold, respectively, relative to wild-type insulin. In contrast, alanine substitutions at positions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala insulin retains high affinity binding to the receptor. GlyB20 is conserved in insulins from different species, and in the structure of the B-chain it appears to be essential for the shift from the alpha-helix B8-B19 to the beta-turn B20-B22. Thus, replacing GlyB20 with alanine most likely modifies the structure of the B-chain in this region, but this structural change appears to enhance binding to the insulin receptor.

Synthetic leaders with potential BiP binding mediate high-yield secretion of correctly folded insulin precursors from Saccharomyces cerevisiae.

Secretion leaders are essential for expression of many heterologous proteins including insulin in yeast. The function of secretion leaders and their interaction with the secretory pathway is not clear. To determine what constitutes functional pre-pro-leader sequences in Saccharomyces cerevisiae, synthetic leader sequences for secretion of the insulin precursor were developed by a combination of rational design and stepwise systematic optimization. The synthetic leaders efficiently facilitate secretion of the insulin precursor from S. cerevisiae when compared with the alpha-factor leader, leading to a high yield of correctly folded insulin precursor in the culture supernatant. The synthetic leaders feature two potential N-linked glycosylation sites which are efficiently glycosylated during secretion. Pulse-chase analysis indicates that the synthetic leaders/insulin precursor fusion protein have a prolonged residence in the endoplasmic reticulum compared to the alpha-factor leader/insulin precursor fusion protein. The longer transition time in the endoplasmic reticulum mediated by the synthetic leaders might provide additional time for correct folding of the insulin precursor and account for the increased fermentation yield.

A removable spacer peptide in an alpha-factor-leader/insulin precursor fusion protein improves processing and concomitant yield of the insulin precursor in Saccharomyces cerevisiae.

An alpha-factor leader/insulin precursor fusion protein was produced in Saccharomyces cerevisiae and metabolically labeled in order to analyse the efficiency of maturation and secretion. A substantial fraction of the secreted material was found in a hyperglycosylated unprocessed form, indicating incomplete Kex2p endopeptidase maturation. Introduction of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creating a N-terminal extension of the insulin precursor, greatly increased the Kex2p catalytic efficiency and the fermentation yield of insulin precursor. The N-terminal extension features a Lys to allow subsequent proteolytic removal by trypsin or the Achromobacter lyticus Lys-specific protease. Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu-Ala dipeptides from the extension was inhibited by adding a Glu N-terminally to the extension. Unexpectedly, this modified N-terminal extension (EEAEAEAK) was partially cleaved after the Lys during fermentation. This monobasic proteolytic activity was demonstrated to be associated with Yap3p. Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK).

A single-chain insulin-like growth factor I/insulin hybrid binds with high affinity to the insulin receptor.

1. To investigate the structure/function relationship of the interaction between ligand and receptor in the insulin-like growth factor I (IGF-I) and insulin receptor systems we have prepared and characterized a single-chain insulin/IGF-I hybrid. The single-chain hybrid consists of the insulin molecule combined with the C domain of IGF-I. The single-chain hybrid was found to bind with high affinity to both truncated soluble insulin receptors and membrane-bound holoreceptors. The affinity for interacting with the soluble truncated insulin receptors was 55-94% relative to insulin, and affinity for membrane-bound insulin receptors was 113% of that of insulin. Furthermore we found that the affinity of the single-chain hybrid molecule for IGF-I receptors was 19-28% relative to IGF-I. 2. The affinity of the single-chain hybrid for chimeric insulin/IGF-I receptors exceeded that of either natural ligand. This indicates that coordinately changing domains of the receptors and the ligands can induce higher affinity of ligand for receptor, supporting the idea that these receptors have a common ligand-binding site [Kjeldsen, Andersen, Wiberg, Rasmussen, Schäffer, Balschmidt, Møller and Møller (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4404-4408]. 3. In contrast with what was generally assumed about the ligand structure required for binding to the insulin receptor we demonstrate the first single-chain insulin analogue that can bind with high affinity to the insulin receptor.