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The decline of the estimated glomerular filtration rate (eGFR) and the presence of albuminuria are the typical hallmarks of kidney disease arising as one of the most frequent diabetic complications over a long period of time, generally known as diabetic nephropathy or diabetes kidney disease (DKD). However, a decline in the renal function may occur in diabetic patients for other reasons unrelated to glycemic control, and this condition is known as non-diabetic kidney disease (NDKD). In this opinion paper we will review these conditions, and we outline the importance of other investigations, such as kidney biopsy and the measurement of novel biomarkers, in order to identify the disease progression early, and to allow a timely intervention. We will also focus on the actual limits of the quantitative measurements of albumin in urine, especially with regards to potential interferences due to the treatment of patients with statins., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Broilers are an important reservoir of extended spectrum beta-lactamase and AmpC beta-lactamase (ESBL/pAmpC)-producing bacteria. In previous studies, a single supply of a competitive exclusion (CE) product before challenge with a high dose of ESBL/pAmpC-producing Escherichia coli led to reduced colonization, excretion, and transmission, but could not prevent colonization. The hypothesized mechanism is competition; therefore, in this study the effect of a prolonged supply of CE products on colonization, excretion, and transmission of ESBL-producing E. coli after challenge with a low dose at day 0 or day 5 was investigated. Day-old broilers (Ross 308) (n = 220) were housed in isolators. Two CE products, containing unselected fermented intestinal bacteria (CEP) or a selection of pre- and probiotics (SYN), were supplied in drinking water from day 0 to 14. At day 0 or 5, broilers were challenged with 0.5 mL with 10 1 or 10 2  cfu/mL E. coli encoding the beta-lactamase gene bla CTX-M-1 on an IncI plasmid (CTX-M-1-E. coli). Presence and concentration of CTX-M-1-E. coli were determined using cloacal swabs (days 0-14, 16, 19, and 21) and cecal content (day 21). Cox proportional hazard model and a mixed linear regression model were used to determine the effect of the intervention on colonization and excretion (log 10  cfu/g). When challenged on the day of hatch, no effect of CEP was observed. When challenged at day 5, both CEP and SYN led to a prevention of colonization with CTX-M-1-E. coli in some isolators. In the remaining isolators, we observed reduced time until colonization (hazard ratio between 3.71 × 10 -3 and 3.11), excretion (up to -1.60 log 10  cfu/g), and cecal content (up to -2.80 log 10  cfu/g), and a 1.5 to 3-fold reduction in transmission rate. Colonization after a low-dose challenge with ESBL-producing E. coli can be prevented by CE products. However, if at least 1 bird is colonized it spreads through the whole flock. Prolonged supply of CE products, provided shortly after hatch, may be applicable as an intervention to reduce the prevalence of ESBL/pAmpC-producing bacteria in the broiler production chain., (Copyright © 2020. Published by Elsevier Inc.)

The presence of SXT/R391-related integrating conjugative elements (ICEs) in Vibrio cholerae O1 and non-O1/non-O139 isolated from clinical and environmental samples in Haiti in 2010 was studied. The main finding of this work was the identification of the novel ICEVchHai2 among closely related V. cholerae non-O1/non-O139 clinical strains. The mosaic structure of this element confirms the role of ICEs as efficient recombination systems whereby new genetic material can be acquired and exchanged, according V. cholerae strains new accessory functions., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Staphylococcal enterotoxins (SEs) produced by Staphylococcus spp. are superantigens responsible for food-poisoning and are associated to mobile genetic elements such as Staphylococcus aureus pathogenicity islands (SaPI). The presence of 13 enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sel, sek, seq, and tst) was tested in 15 S. aureus and 24 coagulase-negative Staphylococcus (CNS) multi-resistant strains isolated from ovine milk in Sardinia. All CNS isolates were enterotoxin-negative, whereas co-presence of sec, sel and tst was observed in most of the S. aureus strains. One isolate of S. aureus was characterized by tst alone. A multiplex PCR assay aimed at discriminating between the integrase genes of pathogenicity islands SaPI2, SaPIbov1, and SaPIMW2 was developed. We demonstrated that strains harboring sec, sel and tst were associated with SaPIbov1, whereas the strain positive for tst was associated with SaPI2. Borderline oxacillin resistant S. aureus strains were also detected. RAPD analysis of the Staphylococcus strains showed that clonal relationships were correlated with pathogenic profiles., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The objective of the present study was to assess (I) the potential presence of a recently discovered thioether variant in commercially available recombinant human growth hormone (r-hGH) preparations, and (II) the impact of the thioether modification on the in-vivo bioactivity and the receptor binding kinetics. Samples were tested employing European (EP) and US Pharmacopeia (USP) Somatropin monograph and mass spectrometry methods. None of the international standards contained this variant. All products conformed to EP specifications but six out of eight lots contained the variant. An artificially enriched thioether sample exhibited a significantly reduced in vivo biopotency and altered receptor-binding properties compared with a control. The absence of the variant in the pituitary hGH standard, and the possibility to generate it artificially suggests that it is not naturally occurring and that it may arise from an uncontrolled manufacturing process. Controlled studies may be required to assess its clinical efficacy and safety. EP and USP methods may need to be adapted to reliably detect the presence of the variant., (2009 Wiley-Liss, Inc. and the American Pharmacists Association)

Advances in proteomic technology have enabled contaminant proteins to be identified from complex protein mixtures. The purity of two purified urinary gonadotrophin products, human menopausal gonadotrophin (u-HMG) and human FSH (u-hFSH), was compared with a preparation of recombinant human FSH (r-hFSH). After separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis showed that the recombinant preparation contained only FSH, whereas the urine-derived preparations exhibited several non-FSH or LH-related bands. These urinary components were further investigated by a proteomic approach using two-dimensional SDS-PAGE followed by mass spectrometric identification. The proteomic approach detected a total of 23 non-gonadotrophin-related proteins, at variable levels in different batches of the urine-derived preparations. Of these, 16 co-purified proteins have not been previously reported to be present in urine-derived gonadotrophins. These results indicate that the process used to purify urinary gonadotrophins may not remove all non-gonadotrophin proteins. By using a comprehensive proteomic approach, it has been shown that the recombinant FSH preparation has greater purity than either of the urine-derived preparations.

Aim: To determine which timing of therapy with formoterol (FOR) and/or tiotropium (TIO) shows the greater and more continuous functional improvement during 24 h in patients with moderate to severe COPD., Methods: In this randomised, blind, crossover study 80 patients with stable COPD (40 moderate and 40 severe) received 5 different bronchodilator 30-day treatments in a random order. Treatments (Tr) were: Tr1: TIO 18 microg once-daily (8 am); Tr2: TIO 18 microg (8 am) + FOR 12 microg (8 pm); Tr3: FOR 12 microg twice-daily (8 am and 8 pm); Tr4: TIO 18 microg (8 am) + FOR 12 microg twice-daily (8 am and 8 pm); Tr5: FOR 12 microg twice-daily (8 am and 8 pm) + TIO 18 microg (8 pm). Spirometries were performed during 24 h (13 steps) on Day1 and Day30. End-points were: gain of FEV(1) (DeltaFEV(1)) from baseline of the Day1 and Day30, AUC (Area Under Curve), Dyspnoea Index, and as-needed use of salbutamol., Results: Sixty-eight patients completed all treatments. The greater and continuous daily functional improvement was showed during Tr4 and Tr5 (Day1 +135.8 mL and +119.1 mL; Day30 +160.2 mL, and +160.5 mL, respectively). Daily means of DeltaFEV(1) were significantly different between single-drug treatments and combination therapy. Dyspnoea was greater in single-drug treatments. Less use of rescue salbutamol was reported in Tr4 (0.80 puffs/die) and Tr5 (0.71 puffs/die)., Conclusions: In patients with moderate to severe COPD, combination therapy with tiotropium administered in the morning (Tr4) was the most effective; in patients with prevailing night-symptoms, treatment with tiotropium in the evening (Tr5) reduced symptoms and use of salbutamol. Tr5 showed less variability of FEV(1) during the 24 h (CV=0.256). These results are relevant for opening new ways in clinical practice.

The revolutionary development of biotechnology-derived therapeutic proteins has provided the expected improvements in quality, purity and consistency, as demonstrated in recombinant human FSH (rhFSH). However, the development of urine-derived gonadotrophins has not always shown comparable improvements. More recently, highly purified urine-derived FSH (uFSH-HP) products have become widely available. The relative purity, level of urine-derived contaminants, and consistency of one such highly purified human uFSH (uhFSH) (urofollitropin) has been assessed and directly compared with rhFSH (follitropin alpha). It has been demonstrated that the highly purified urofollitropin contains variable levels of urine-derived contaminant proteins and demonstrates a variable level of FSH purity, FSH isoforms, and delivered dose. These variable factors may contribute to the control of ovarian stimulation. The relative purity, variable consistency and the presence of contaminants indicates that the urofollitropin is, at best, a partially purified uFSH that is not able to meet the quality attributes of follitropin alpha (rhFSH).

The fluorescent dye 10-N-nonyl acridine orange (NAO) is extensively used for location and quantitative assays of cardiolipin in living cells on the assumption of its high specificity for cardiolipin; however, the limits and the mechanism of this specificity are not clear. Moreover, whether factors such as the membrane potential in mitochondria may limit the consistency of the results obtained by this method is open to discussion. The aim of this research was to investigate the effects of some experimental factors on the selective fluorescence of NAO in the presence of cardiolipin in artificial and natural membranes (mitochondria). The results show that the fluorescence of NAO, due to interaction with cardiolipin, is significantly modified by factors that control the spatial arrangement of cardiolipin molecules within the space of the membrane under investigation. Moreover, the present observations suggest that the specific effect of cardiolipin is to facilitate the dimerization of this fluorescent dye, thus confirming that reliable measurements of cardiolipin concentration can be obtained only when the NAO/cardiolipin molar ratio is equal to 2. The finding is also reported that in isolated respiring mitochondria the interaction of NAO with cardiolipin is somewhat related to the respiratory state of mitochondria.

Myopathy often complicates Zidovudine (AZT) treatment in patients with acquired immunodeficiency syndrome (AIDS). The pathogenesis of the myopathy is controversial, since clinical phenomena intrinsic to AIDS may interfere per se with the onset of the myopathy. In the present work we investigated the in vivo effect of AZT in an animal model species (rat) not susceptible to HIV infection. Histochemical and electron microscopic analyses demonstrated that, under the experimental conditions used, the in vivo treatment with AZT does not cause in skeletal muscle true dystrophic lesions, but rather mitochondrial alterations confined to the fast fibers. In the same animal models, the biochemical analysis confirmed that mitochondria are the target of AZT toxicity in muscles. The effects of AZT on mitochondria energy transducing mechanisms were investigated in isolated mitochondria both in vivo and in vitro. Membrane potential abnormalities, due to a partial impairment of the respiratory chain capability observed in muscle mitochondria from AZT-treated rats, closely resemble those of control mitochondria in the presence of externally added AZT. mtDNA deletion analysis by PCR amplification and Southern blot analysis did not show any relevant deletion, while mtDNA depletion analysis demonstrated a significant decrease in mtDNA in AZT-treated rats. The present findings show that AZT causes damage to mitochondria by two mechanisms: a short-term mechanism that affects directly the respiratory chain, and a long-term mechanism that alters the mitochondrial DNA thus impairing the mitochondrial protein synthesis. In addition, the ultrastructural observations indicate that the fiber types are differently affected upon AZT treatment, which poses a number of questions as to the pathogenesis of this myopathy.

The concentration of total iron in the hepatic tissue and mitochondria from rats fed a 2.5% carbonyl iron supplemented diet progressively increased up to 40 days, then reached nearly a steady-state. By contrast the level of free iron (desferrioxamine-chelatable) exhibited a transient but significant increase at 40 days of treatment, only in this period of treatment the induction of lipid peroxidation and the resulting mitochondrial abnormalities in calcium transport was observed too. The enhancement of the energy dissipating mitochondrial calcium cycling was found to be associated with a significant decrease of endogenous mitochondrial ATP content. As to the pathophysiological mechanism for hepatocellular injury in iron overload, these results indicated that the transit pool of free iron may play a critical role in initiating organelle dysfunctions, at least in this experimental model of iron overload.

It has been found that acute ethanol (EtOH) intoxication to rats caused approximately 40% depletion of mitochondrial reduced glutathione (GSH). A GSH reduction of similar extent was also observed after the administration to rats of buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis. The combined treatment of EtOH plus BSO induced a further mitochondrial GSH decrease up to 70% with respect to control. The presence of lipid hydroperoxides in the mitochondrial membrane was observed whenever an additional oxidative stress was associated to a condition of GSH depletion as in the case of EtOH or EtOH plus BSO. Under these conditions a severe derangement in mitochondrial oxidative functions occurred.

31P-NMR spectroscopy of rat liver perchloric acid extracts was utilized to assess the hepatic energy state in an experimental model of chronic dietary iron overload. Oral administration of iron for a period of 65 days that induces a steady ten-fold increase in hepatic iron concentration causes a significant decrease in the hepatic ATP level not associated with appreciable modifications of ADP and Pi levels. The phosphorylation ratio appears on the average decreased. The values of the energy state parameters revert to the normal if the concentration of iron in the liver is reversed below the critical level upon withdrawal of iron treatment after 45 days for a period of 20 days. The implication of these energy modifications for the pathogenesis of cell damage in the siderosis is discussed.

A possible link between changes in iron and porphyrin content in liver mitochondria, from rats treated with either hexachlorobenzene, iron, or hexachlorobenzene plus iron, as a function of treatment time and their structural-functional properties, has been investigated. Normal oxidative phosphorylation in mitochondria from rats treated with iron has been shown. By contrast a significant and constant uncoupling of the phosphorylative process, fully reversed by albumin, in mitochondria from rats treated with hexachlorobenzene and hexachlorobenzene plus iron has been presented. A possible involvement of pentachlorophenol in causing these abnormalities has been proposed.

Liver mitochondria isolated from rats treated with hexachlorobenzene plus iron, present a lower content of total porphyrin in respect to that of mitochondria from rats fed hexachlorobenzene alone. The in vitro mitochondrial porphyrin accumulation processes have been studied in mitochondria from iron loaded rats. It has been found that under these conditions the active porphyrin uptake process, which is driven by the K+ transmembrane gradient, is maximally inhibited in the presence of pentachlorophenol at a concentration similar to that found in vivo in the hexachlorobenzene experimental porphyria. By contrast the same degree of inhibition is presented by control mitochondria only in the presence of pentachlorophenol plus valinomycin, a condition which collapses the transmembrane K+ gradient. A strict correlation between porphyrin uptake and K+ concentration has been found in control as well as in iron treated mitochondria. A possible involvement of peroxidative reactions in the mitochondrial membranes has been proposed as a cause of the changes in the permeability properties of the mitochondrial membranes in the experimental chronic hepatic porphyria under conditions of iron overload.

The effect of in vivo hepatic iron overload, induced by two different amounts of iron, on the energy-transducing efficiency of the mitochondrial membrane has been examined. It has been found that when the epatic iron concentration is up to a threshold value mitochondria present an anomalous membrane potential. Addition of oligomycin fully restitutes it. A low content of intramitochondrial K+ is connected with this pathological condition. A relative lack of antioxidant capability is parallely exhibited by these mitochondria. A possible involvement of lipid peroxidation process in vivo in causing the membrane potential drop and the net efflux of intramitochondrial K+ is suggested.

The in vitro effects of iron (III)-gluconate complex on the production of malondialdehyde and on the Ca2+ transport in isolated rat liver mitochondria were studied. A correlation between the concentration of iron added and the formation of malondialdehyde was found. The enhancement by iron of lipid peroxidative process in the mitochondrial membrane brought about the induction of Ca2+ release from mitochondria. Experimental evidence based on the membrane potential pattern of mitochondria pre-loaded with a low pulse of Ca2+ suggested that Ca2+ efflux was not due to a nonspecific increase in the inner membrane permeability, i.e. to a collapse of membrane potential, but rather to the activation of an apparently selective pathway for Ca2+ release.