Detection of DZIP1L mutations by whole-exome sequencing in consanguineous families with polycystic kidney disease.

BACKGROUND: Autosomal recessive polycystic kidney disease is a cystic kidney disease with early onset and clinically characterized by enlarged echogenic kidneys, hypertension, varying degrees of kidney dysfunction, and liver fibrosis. It is most frequently caused by sequence variants in the PKHD1 gene, encoding fibrocystin. In more rare cases, sequence variants in DZIP1L are seen, encoding the basal body protein DAZ interacting protein 1-like protein (DZIP1L). So far, only four different DZIP1L variants have been reported. METHODS: Four children from three consanguineous families presenting with polycystic kidney disease were selected for targeted or untargeted exome sequencing. RESULTS: We identified two different, previously not reported homozygous DZIP1L sequence variants: c.193 T > C; p.(Cys65Arg), and c.216C > G; p.(Cys72Trp). Functional analyses of the c.216C > G; p.(Cys72Trp) variant indicated mislocalization of mutant DZIP1L. CONCLUSIONS: In line with published data, our results suggest a critical role of the N-terminal domain for proper protein function. Although patients with PKHD1-associated autosomal recessive polycystic kidney disease often have liver abnormalities, none of the present four patients showed any clinically relevant liver involvement. Our data demonstrate the power and efficiency of next-generation sequencing-based approaches. While DZIP1L-related polycystic kidney disease certainly represents a rare form of the disease, our results emphasize the importance of including DZIP1L in multigene panels and in the data analysis of whole-exome sequencing for cystic kidney diseases. A higher resolution version of the Graphical abstract is available as Supplementary information.

Children with acute pyelonephritis need medical re-evaluation when home-treated with oral antibiotics.

AIM: To investigate the efficacy and safety of home-treatment with oral piv-mecillinam or amoxicillin-clavulanate in children with acute pyelonephritis. METHODS: Children aged over 6 months diagnosed with culture confirmed pyelonephritis at Danish Paediatric Departments were home-treated with piv-mecillinam (tablets) or amoxicillin-clavulanate (liquid or tablets). Follow-up was performed by phone (second treatment day) and clinical review of the patients in the hospital (day three). RESULTS: Four hundred eighteen children were included. In total, 333/418 (80%) responded well to the initial oral antibiotic treatment. 85/418 (20%) were changed to another treatment of these 47/418 (11%) to a second-line oral antibiotic and 38/418 (9%) to intravenous antibiotics due to insufficient clinical improvement or bacterial resistance. Bacterial resistance was similar for piv-mecillinam and amoxicillin-clavulanate: 4/74 (5%) versus 33/333 (10%) (p = 0.22). Insufficient clinical improvement, despite no resistance, primarily occurred in children treated with piv-mecillinam: 16/74 (22%) versus 28/344 (8%) (p < 0.001), and predominantly occurred in piv-mecillinam treated children <5 years: 7/20 (35%) versus 9/54 (17%) (p < 0.05), potentially because of problems with piv-mecillinam tablets. In the study population no cases of death or septicemia developed after start of initial oral treatment. CONCLUSION: A home-treatment regime for pyelonephritis in children >6 months is safe; however, during treatment, clinical re-evaluation is required as in 20% of cases a change in treatment was necessary.

Planar scan vs. SPECT/low-dose CT for estimating split renal function by 99mTc-DMSA scintigraphy in children.

In the present study, we compared estimates of split renal function (SRF) in paediatric patients of various diagnostic subgroups by 99mTc-dimercaptosuccinic acid (DMSA) scintigraphy using either geometric mean (GM) based on planar scans or a volume of interest (VOI)-based analysis on single photon emission tomography combined with low-dose CT (SPECT/ldCT). Two experienced physicians blinded to patient diagnosis retrospectively analysed all paediatric 99mTc-DMSA scintigraphies that were conducted in our department between 2011 and 2016 and which included both a planar scan and SPECT/ldCT. All scintigraphies were performed on either a Phillips Precedence 16 slice CT or a Siemens Symbia 16 slice CT. SRF was estimated from planar scintigraphy using the geometric mean (GM), while the VOI-based analysis (VBA) was used for kidney segmentation on SPECT/ldCT. RESULTS: A total of 68 scintigraphies were included. A Bland-Altman plot-based analysis showed a bias for SRF of 2.1% with limits of agreement from - 7.5 to + 11.7% for the whole data set but showed larger differences between the two methods outside the normal range of 45-55%. In the GM-based SRF analyses, 29 cases were found to be outside the normal range, and in seven of these, VBA showed normal SRF. In the remaining 39 cases, VBA showed an abnormal SRF in only one case. CONCLUSION: Approximately a quarter of planar DMSA scintigraphies that show an abnormal SRF in paediatric patients may be normal when assessed by SPECT/ldCT, which likely reflects underestimation of the kidney with the poorest function when assessed by GM due to the lack of attenuation correction. Planar scans that show an abnormal SRF in paediatric patients should thus preferably be supplemented by SPECT/ldCT.

Overexpression of bifunctional fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase leads to enhanced photosynthesis and global reprogramming of carbon metabolism in Synechococcus sp. PCC 7002.

Cyanobacteria fix atmospheric CO2 to biomass and through metabolic engineering can also act as photosynthetic factories for sustainable productions of fuels and chemicals. The Calvin Benson cycle is the primary pathway for CO2 fixation in cyanobacteria, algae and C3 plants. Previous studies have overexpressed the Calvin Benson cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (hereafter BiBPase), in both plants and algae, although their impacts on cyanobacteria have not yet been rigorously studied. Here, we show that overexpression of BiBPase and RuBisCO have distinct impacts on carbon metabolism in the cyanobacterium Synechococcus sp. PCC 7002 through physiological, biochemical, and proteomic analyses. The former enhanced growth, cell size, and photosynthetic O2 evolution, and coordinately upregulated enzymes in the Calvin Benson cycle including RuBisCO and fructose-1,6-bisphosphate aldolase. At the same time it downregulated enzymes in respiratory carbon metabolism (glycolysis and the oxidative pentose phosphate pathway) including glucose-6-phosphate dehydrogenase (G6PDH). The content of glycogen was also significantly reduced while the soluble carbohydrate content increased. These results indicate that overexpression of BiBPase leads to global reprogramming of carbon metabolism in Synechococcus sp. PCC 7002, promoting photosynthetic carbon fixation and carbon partitioning towards non-storage carbohydrates. In contrast, whilst overexpression of RuBisCO had no measurable impact on growth and photosynthetic O2 evolution, it led to coordinated increase in the abundance of proteins involved in pyruvate metabolism and fatty acid biosynthesis. Our results underpin that singular genetic modifications in the Calvin Benson cycle can have far broader cellular impact than previously expected. These features could be exploited to more efficiently direct carbons towards desired bioproducts.

Rapid Reduction of the Diferric-Peroxyhemiacetal Intermediate in Aldehyde-Deformylating Oxygenase by a Cyanobacterial Ferredoxin: Evidence for a Free-Radical Mechanism.

Aldehyde-deformylating oxygenase (ADO) is a ferritin-like nonheme-diiron enzyme that catalyzes the last step in a pathway through which fatty acids are converted into hydrocarbons in cyanobacteria. ADO catalyzes conversion of a fatty aldehyde to the corresponding alk(a/e)ne and formate, consuming four electrons and one molecule of O2 per turnover and incorporating one atom from O2 into the formate coproduct. The source of the reducing equivalents in vivo has not been definitively established, but a cyanobacterial [2Fe-2S] ferredoxin (PetF), reduced by ferredoxin-NADP(+) reductase (FNR) using NADPH, has been implicated. We show that both the diferric form of Nostoc punctiforme ADO and its (putative) diferric-peroxyhemiacetal intermediate are reduced much more rapidly by Synechocystis sp. PCC6803 PetF than by the previously employed chemical reductant, 1-methoxy-5-methylphenazinium methyl sulfate. The yield of formate and alkane per reduced PetF approaches its theoretical upper limit when reduction of the intermediate is carried out in the presence of FNR. Reduction of the intermediate by either system leads to accumulation of a substrate-derived peroxyl radical as a result of off-pathway trapping of the C2-alkyl radical intermediate by excess O2, which consequently diminishes the yield of the hydrocarbon product. A sulfinyl radical located on residue Cys71 also accumulates with short-chain aldehydes. The detection of these radicals under turnover conditions provides the most direct evidence to date for a free-radical mechanism. Additionally, our results expose an inefficiency of the enzyme in processing its radical intermediate, presenting a target for optimization of bioprocesses exploiting this hydrocarbon-production pathway.

Effect of Continuous Glucose Monitoring Accuracy on Clinicians' Retrospective Decision Making in Diabetes: A Pilot Study.

BACKGROUND: The use of continuous glucose monitoring (CGM) in clinical decision making in diabetes could be limited by the inaccuracy of CGM data when compared to plasma glucose measurements. The aim of the present study is to investigate the impact of CGM numerical accuracy on the precision of diabetes treatment adjustments. METHOD: CGM profiles with maximum 5-day duration from 12 patients with type 1 diabetes treated with a basal-bolus insulin regimen were processed by 2 CGM algorithms, with the accuracy of algorithm 2 being higher than the accuracy of algorithm 1, using the median absolute relative difference (MARD) as the measure of accuracy. During 2 separate and similar occasions over a 1-month interval, 3 clinicians reviewed the processed CGM profiles, and adjusted the dose level of basal and prandial insulin. The precision of the dosage adjustments were defined in terms of the interclinician agreement and the intraclinician reproducibility of the decisions. The Cohen's kappa coefficient was used to assess the precision of the decisions. The study was based on retrospective and blind CGM data. RESULTS: For the interclinician agreement, in the first occasion, the kappa of algorithm 1 was .32, and that of algorithm 2 was .36. For the interclinician agreement, in the second occasion, the kappas of algorithms 1 and 2 were .17 and .22, respectively. For the intraclinician reproducibility of the decisions, the kappas of algorithm 1 were .35, .22, and .80 and the kappas of algorithm 2 were .44, .52, and .32, for the 3 clinicians, respectively. For the interclinician agreement, the relative kappa change from algorithm 1 to algorithm 2 was 86.06%, and for the intraclinician reproducibility, the relative kappa change from algorithm 1 to algorithm 2 was 53.99%. CONCLUSIONS: Results indicated that the accuracy of CGM algorithms might potentially affect the precision of the CGM-based insulin adjustments for type 1 diabetes patients. However, a larger study with several clinical centers, with higher number of clinicians and patients is required to validate the impact of CGM accuracy on decisions precision.

Substrate-triggered addition of dioxygen to the diferrous cofactor of aldehyde-deformylating oxygenase to form a diferric-peroxide intermediate.

Cyanobacterial aldehyde-deformylating oxygenases (ADOs) belong to the ferritin-like diiron-carboxylate superfamily of dioxygen-activating proteins. They catalyze conversion of saturated or monounsaturated C(n) fatty aldehydes to formate and the corresponding C(n-1) alkanes or alkenes, respectively. This unusual, apparently redox-neutral transformation actually requires four electrons per turnover to reduce the O2 cosubstrate to the oxidation state of water and incorporates one O-atom from O2 into the formate coproduct. We show here that the complex of the diiron(II/II) form of ADO from Nostoc punctiforme (Np) with an aldehyde substrate reacts with O2 to form a colored intermediate with spectroscopic properties suggestive of a Fe2(III/III) complex with a bound peroxide. Its Mössbauer spectra reveal that the intermediate possesses an antiferromagnetically (AF) coupled Fe2(III/III) center with resolved subsites. The intermediate is long-lived in the absence of a reducing system, decaying slowly (t(1/2) ~ 400 s at 5 °C) to produce a very modest yield of formate (<0.15 enzyme equivalents), but reacts rapidly with the fully reduced form of 1-methoxy-5-methylphenazinium methylsulfate ((MeO)PMS) to yield product, albeit at only ~50% of the maximum theoretical yield (owing to competition from one or more unproductive pathway). The results represent the most definitive evidence to date that ADO can use a diiron cofactor (rather than a homo- or heterodinuclear cluster involving another transition metal) and provide support for a mechanism involving attack on the carbonyl of the bound substrate by the reduced O2 moiety to form a Fe2(III/III)-peroxyhemiacetal complex, which undergoes reductive O-O-bond cleavage, leading to C1-C2 radical fragmentation and formation of the alk(a/e)ne and formate products.

Does nuclear tissue infected with bacteria following disc herniations lead to Modic changes in the adjacent vertebrae?

PURPOSE: To investigate the prevalence of infected herniated nucleus material in lumbar disc herniations and to determine if patients with an anaerobic infected disc are more likely to develop Modic change (MC) (bone oedema) in the adjacent vertebrae after the disc herniation. MCs (bone oedema) in vertebrae are observed in 6 % of the general population and in 35-40 % of people with low back pain. These changes are strongly associated with low back pain. There are probably a mechanical cause and an infective cause that causes MC. Several studies on nuclear tissue from herniated discs have demonstrated the presence of low virulent anaerobic microorganisms, predominantly Propionibacterium acnes, in 7-53 % of patients. At the time of a herniation these low virulent anaerobic bacteria may enter the disc and give rise to an insidious infection. Local inflammation in the adjacent bone may be a secondary effect due to cytokine and propionic acid production. METHODS: Patients undergoing primary surgery at a single spinal level for lumbar disc herniation with an MRI-confirmed lumbar disc herniation, where the annular fibres were penetrated by visible nuclear tissue, had the nucleus material removed. Stringent antiseptic sterile protocols were followed. RESULTS: Sixty-one patients were included, mean age 46.4 years (SD 9.7), 27 % female. All patients were immunocompetent. No patient had received a previous epidural steroid injection or undergone previous back surgery. In total, microbiological cultures were positive in 28 (46 %) patients. Anaerobic cultures were positive in 26 (43 %) patients, and of these 4 (7 %) had dual microbial infections, containing both one aerobic and one anaerobic culture. No tissue specimens had more than two types of bacteria identified. Two (3 %) cultures only had aerobic bacteria isolated. In the discs with a nucleus with anaerobic bacteria, 80 % developed new MC in the vertebrae adjacent to the previous disc herniation. In contrast, none of those with aerobic bacteria and only 44 % of patients with negative cultures developed new MC. The association between an anaerobic culture and new MCs is highly statistically significant (P = 0.0038), with an odds ratio of 5.60 (95 % CI 1.51-21.95). CONCLUSION: These findings support the theory that the occurrence of MCs Type 1 in the vertebrae adjacent to a previously herniated disc may be due to oedema surrounding an infected disc. The discs infected with anaerobic bacteria were more likely (P < 0.0038) to develop MCs in the adjacent vertebrae than those in which no bacteria were found or those in which aerobic bacteria were found.

High plasma aldosterone is associated with a risk of reversible decreased eGFR in childhood idiopathic nephrotic syndrome.

BACKGROUND: Oedema formation in nephrotic syndrome (NS) may be associated with volume overload or volume contraction. The present study investigates if plasma aldosterone was related to a clinical course symptomatic of either volume expansion or hypovolaemia. METHODS: Twenty patients with NS were included. Blood and urine samples were collected before treatment of NS and at stable remission. Aldosterone and other vasoactive hormones were measured in plasma and the patients were classified based on the aldosterone concentrations. RESULTS: Five patients were classified with stimulated aldosterone, mean 611 pg/mL [95% confidence interval (CI): 365-993], 12 with suppressed aldosterone, mean 13 pg/mL (95% CI: 6-26), and 3 with unchanged aldosterone, mean 117 pg/mL (95% CI: 101-135). Patients with high aldosterone were characterized by lower estimated glomerular filtration rate (eGFR) (87 ± 30 versus 142 ± 30, P < 0.01), and increased albuminuria (14 ± 11 versus 6 ± 4 g/L, P = 0.03) compared with the remaining patients. eGFR was normalized rapidly by volume expansion in four of these five patients. CONCLUSIONS: Elevated plasma aldosterone during NS may be associated with a risk of temporary reduced eGFR. The normalization of eGFR by volume expansion supports the hypothesis of functional hypovolaemia in some patients. Our data suggest that acute measurement of aldosterone may have implications for the management of oedema.

Conversion of fatty aldehydes to alka(e)nes and formate by a cyanobacterial aldehyde decarbonylase: cryptic redox by an unusual dimetal oxygenase.

Cyanobacterial aldehyde decarbonylase (AD) catalyzes conversion of fatty aldehydes (R-CHO) to alka(e)nes (R-H) and formate. Curiously, although this reaction appears to be redox-neutral and formally hydrolytic, AD has a ferritin-like protein architecture and a carboxylate-bridged dimetal cofactor that are both structurally similar to those found in di-iron oxidases and oxygenases. In addition, the in vitro activity of the AD from Nostoc punctiforme (Np) was shown to require a reducing system similar to the systems employed by these O(2)-utilizing di-iron enzymes. Here, we resolve this conundrum by showing that aldehyde cleavage by the Np AD also requires dioxygen and results in incorporation of (18)O from (18)O(2) into the formate product. AD thus oxygenates, without oxidizing, its substrate. We posit that (i) O(2) adds to the reduced cofactor to generate a metal-bound peroxide nucleophile that attacks the substrate carbonyl and initiates a radical scission of the C1-C2 bond, and (ii) the reducing system delivers two electrons during aldehyde cleavage, ensuring a redox-neutral outcome, and two additional electrons to return an oxidized form of the cofactor back to the reduced, O(2)-reactive form.

Detection of formate, rather than carbon monoxide, as the stoichiometric coproduct in conversion of fatty aldehydes to alkanes by a cyanobacterial aldehyde decarbonylase.

The second of two reactions in a recently discovered pathway through which saturated fatty acids are converted to alkanes (and unsaturated fatty acids to alkenes) in cyanobacteria entails scission of the C1-C2 bond of a fatty aldehyde intermediate by the enzyme aldehyde decarbonylase (AD), a ferritin-like protein with a dinuclear metal cofactor of unknown composition. We tested for and failed to detect carbon monoxide (CO), the proposed C1-derived coproduct of alkane synthesis, following the in vitro conversion of octadecanal (R-CHO, where R = n-C(17)H(35)) to heptadecane (R-H) by the Nostoc punctiforme AD isolated following its overproduction in Escherichia coli. Instead, we identified formate (HCO(2)(-)) as the stoichiometric coproduct of the reaction. Results of isotope-tracer experiments indicate that the aldehyde hydrogen is retained in the HCO(2)(-) and the hydrogen in the nascent methyl group of the alkane originates, at least in part, from solvent. With these characteristics, the reaction appears to be formally hydrolytic, but the improbability of a hydrolytic mechanism having the primary carbanion as the leaving group, the structural similarity of the ADs to other O(2)-activating nonheme di-iron proteins, and the dependence of in vitro AD activity on the presence of a reducing system implicate some type of redox mechanism. Two possible resolutions to this conundrum are suggested.

Rapid and quantitative activation of Chlamydia trachomatis ribonucleotide reductase by hydrogen peroxide.

We recently showed that the class Ic ribonucleotide reductase (RNR) from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor in its R2 subunit to initiate catalysis [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. The Mn (IV) site of the novel cofactor functionally replaces the tyrosyl radical used by conventional class I RNRs to initiate substrate radical production. As a first step in evaluating the hypothesis that the use of the alternative cofactor could make the RNR more robust to reactive oxygen and nitrogen species [RO(N)S] produced by the host's immune system [Högbom, M., Stenmark, P., Voevodskaya, N., McClarty, G., Gräslund, A., and Nordlund, P. (2004) Science 305, 245-248], we have examined the reactivities of three stable redox states of the Mn/Fe cluster (Mn (II)/Fe (II), Mn (III)/Fe (III), and Mn (IV)/Fe (III)) toward hydrogen peroxide. Not only is the activity of the Mn (IV)/Fe (III)-R2 intermediate stable to prolonged (>1 h) incubations with as much as 5 mM H 2O 2, but both the fully reduced (Mn (II)/Fe (II)) and one-electron-reduced (Mn (III)/Fe (III)) forms of the protein are also efficiently activated by H 2O 2. The Mn (III)/Fe (III)-R2 species reacts with a second-order rate constant of 8 +/- 1 M (-1) s (-1) to yield the Mn (IV)/Fe (IV)-R2 intermediate previously observed in the reaction of Mn (II)/Fe (II)-R2 with O 2 [Jiang, W., Hoffart, L. M., Krebs, C., and Bollinger, J. M., Jr. (2007) Biochemistry 46, 8709-8716]. As previously observed, the intermediate decays by reduction of the Fe site to the active Mn (IV)/Fe (III)-R2 complex. The reaction of the Mn (II)/Fe (II)-R2 species with H 2O 2 proceeds in three resolved steps: sequential oxidation to Mn (III)/Fe (III)-R2 ( k = 1.7 +/- 0.3 mM (-1) s (-1)) and Mn (IV)/Fe (IV)-R2, followed by decay of the intermediate to the active Mn (IV)/Fe (III)-R2 product. The efficient reaction of both reduced forms with H 2O 2 contrasts with previous observations on the conventional class I RNR from Escherichia coli, which is efficiently converted from the fully reduced (Fe 2 (II/II)) to the "met" (Fe 2 (III/III)) form [Gerez, C., and Fontecave, M. (1992) Biochemistry 31, 780-786] but is then only very inefficiently converted from the met to the active (Fe 2 (III/III)-Y (*)) form [Sahlin, M., Sjöberg, B.-M., Backes, G., Loehr, T., and Sanders-Loehr, J. (1990) Biochem. Biophys. Res. Commun. 167, 813-818].