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In this paper, we investigated whether cholecalciferol supplementation may increase the risk of stone recurrence in patients with calcium nephrolithiasis and Vitamin D deficiency. Thirty-three stone formers (56 ± 17 years old, 12 males) with 25(OH)D 1 mean supersaturation. Cholecalciferol was prescribed as oral bolus of 100,000–200,000 IU, followed by weekly (5000–10,000 IU) or monthly (25,000–50,000 IU) doses. Calcium intake varied between 800 and 1000 mg/day. In urine, total nitrogen (TNE) was taken as an index of protein intake, sodium as a marker of dietary intake, and net acid excretion (NAE) as an index of acid–base balance. TNE, sodium, and NAE did not change during the study (p = ns). Compared to baseline values, after cholecalciferol, both serum calcium and phosphate did not vary (p = ns); 25(OH)D increased from 11.8 ± 5.5 to 40.2 ± 12.2 ng/mL (p

Background Interleukin-1 plays a pivotal role in the inflammatory response during cytokine storm syndromes. Objective To analyse the efficacy and safety of early anti-inflammatory treatment (AIT) with i.v anakinra with or without glucocorticoids (GC) in COVID19 pneumonia. Methods We performed a retrospective single-centre cohort study on patients admitted for COVID19 pneumonia from February 26th to April 29th, 2020 to assess the efficacy of early AIT with i.v. Anakinra (100mg every 8 hours for 3 days, with tapering) alone or in combination with GC (i.v. methylprednisolone, 1-2 mg/kg daily, with tapering). Standard of care (SOC) treatment was: hydroxychloroquine and/or azithromycin with or without antivirals and anticoagulants. Late rescue AIT with anakinra or tocilizumab was also evaluated. Treatment effect on Overall Survival (OS) was assessed by a Propensity Score adjusted Cox model. Results 128 patients were analyzed; 63 received early AIT (30 anakinra, 33 anakinra and CG) at admission; 65 patients (44 with SOC, 21 SOC plus late rescue AIT) did not and were used as controls. After adjusting for all the unbalanced baseline covariates, early AIT reduced the hazard of mortality by 74% (adjusted HR=0.26, p, Graphical abstract, Capsule summary: This study provide evidence that the early use of Anakinra alone or with glucocorticoids allows a prompt amelioration of the inflammatory and respiratory parameters, reducing the mortality rate in patients with severe COVID-19 pneumonia.

The article shows dataset of the proteolysis of a natural variant of apolipoprotein A-I (apoA-I) with a substitution of a leucine by and arginine in position 60 (L60R), in comparison with the protein with the native sequence (Wt). This information demonstrates the potential of in vitro partial proteolysis experiments as it may be applicable to different approaches in the biophysical field. We have analyzed by different electrophoresis techniques apoA-I variants, quantified the degree of proteolysis after staining and compared the proteolysis efficiency with the computed cleavage patterns. The data shown here clearly strengthen the usefulness of this approach to test protein flexibility, as it may be attained with enzymes which are not expected to modify in vivo this protein but have a well-known digestion pattern. In addition it is appropriate for evaluating protein catabolism, as it is exemplified here by the evidence with metalloproteinase 12 (MMP-12), which is a physiological protease that may elicit the pro-inflammatory processing of this variant within the lesions. We support the work “Structural analysis of a natural apolipoprotein A-I variant (L60R) associated with amyloidosis” (Gaddi, et al., 2020), gaining insights on protein folding from a characterization by proteolysis analysis., Instituto de Investigaciones Bioquímicas de La Plata

Background: The identification of dysfunctional human apolipoprotein A-I (apoA-I) in atherosclerotic plaques suggests that protein structure and function may be hampered under a chronic pro inflammatory scenario. Moreover, the fact that natural mutants of this protein elicit severe cardiovascular diseases (CVD) strongly indicates that the native folding could shift due to the mutation, yielding a structure more prone to misfold or misfunction. To understand the events that determine the failure of apoA-I structural flexibility to fulfill its protective role, we took advantage of the study of a natural variant with a deletion of the residue lysine 107 (K107del) associated with atherosclerosis. Methods: Biophysical approaches, such as electrophoresis, fluorescence and spectroscopy were used to characterize proteins structure and function, either in the native conformation or under oxidation or intramolecular crosslinking. Results: K107del structure was more flexible than the protein with the native sequence (Wt) but interactions with artificial membranes were preserved. Instead, structural restrictions by intramolecular crosslinking impaired the Wt and K107del lipid solubilization function. In addition, controlled oxidation decreased the yield of the native dimer conformation for both variants. Conclusions: We conclude that even though mutations may alter protein structure and spatial arrangement, the highly flexible conformation compensates the mild shift from the native folding. Instead, post translational apoA-I modifications (probably chronic and progressive) are required to raise a protein conformation with significant loss of function and increased aggregation tendency. General Significance: The results learnt from this variant strength a close association between amyloidosis and atherosclerosis., Instituto de Investigaciones Bioquímicas de La Plata, Facultad de Ciencias Médicas

Background and Aims Post transplantation anaemia (PTA) is a common finding in kidney transplant patients, with reported prevalence ranging from 25 to 40%, and it is associated with adverse outcomes for the kidney allograft and the recipient. The pathogenesis of PTA is multifactorial, including iron deficiency, reduced graft function, untoward effects of drugs (immunosuppressants, ACE-I and ARB), folic acid and vitamin B12 deficiency, inflammatory states. Ferric carboxymaltose (FCM) reportedly improved haemoglobin (Hb) levels and replenished depleted iron stores in various populations of patients with iron-deficiency anaemia, but there are few data about its use in PTA. In this study we evaluated the effectiveness and tolerability of high intravenous doses of FCM (500 mg) for the treatment of iron deficiency and anaemia in patients with kidney transplant. Method Absolute iron deficiency was considered as transferrin saturation (TSAT) less than 20% with ferritin levels below 100 µg/L. Anaemia was defined as Hb Out of 101 patients with kidney transplant, 56 presented with absolute iron deficiency (59±13 years old, M/F=0.95, 8±4.6 years of transplantation; serum creatinine 1.45±0.64 mg/dL): of these, 40 patients had anaemia (7 were also taking ESAs) and 16 had normal Hb levels. The 16 patients with absolute iron deficiency and normal Hb were not prescribed iron supplements, fearing of causing erythrocytosis, whereas 36 of 40 patients with anaemia and iron deficiency were given FCM 500 mg, as i.v. infusion for 30 minutes. Hb, ferritin and TSAT were assayed either before and 45-60 days after FCM supplementation, which was repeated if Hb levels were less than 0.5 g/dL below the normal value. Data were reported as mean±SD. Results Among the 56 patients with absolute iron deficiency, there were not any significant differences in serum levels of folic acid and vitamin B12 between those with and without anaemia (p=ns). After the first dose of FCM, in 36 patients with iron deficiency and anaemia, ferritin levels increased from 55±69 ng/mL to 111±107 ng/mL (p Out of the 17 patients still showing anaemia after the first FCM dose, 10 had Hb levels less than 0.5 g/dL below the normal value and were given a second dose of FCM. In this subset, Hb level increased on average from 11.4±0.5 g/dL to 12±0.9 g/dL (p After the FCM course, in 2 out of 7 patients ESAs were discontinued, whereas in 3 patients their doses were reduced and in 2 patients remained unchanged. Only one patient had an adverse effect during FCM infusion, namely, a transient skin flushing on her face during the second dose, which immediately remitted when the infusion was interrupted. Conclusion In patients with PTA and absolute iron deficiency, single or repeated doses of 500 mg of i.v. FCM were effective in improving Hb levels, well tolerated and reduced the need for ESAs. Of 56 patients with kidney transplant and absolute iron deficiency, 16 (28%) had normal Hb levels. We did not treat such subjects, fearing of causing erythrocytosis. However, given that iron deficiency may affect negatively both heart and muscle metabolism, what is the best management of iron deficiency in transplanted patients without anaemia is still an open question.

Apolipoprotein A-I (apoA-I) has a key function in the reverse cholesterol transport mediated by the high-density lipoprotein (HDL) particles. However, aggregation of apoA-I single point mutants can lead to hereditary amyloid pathology. Although several studies have tackled the biophysical and structural impacts introduced by these mutations, there is little information addressing the relationship between the evolutionary and structural features that contribute to the amyloid behavior of apoA-I. We combined evolutionary studies, in silico saturation mutagenesis and molecular dynamics (MD) simulations to provide a comprehensive analysis of the conservation and pathogenic role of the aggregation-prone regions (APRs) present in apoA-I. Sequence analysis demonstrated the pervasive conservation of an APR, designated here APR1, within the N-terminal ɑ-helix bundle. Moreover, stability analysis carried out with the FoldX engine showed that this motif contributes to the marginal stability of apoA-I. Structural properties of the full-length apoA-I model suggest that aggregation is avoided by placing APRs into highly packed and rigid portions of its structure. Compared to HDL-deficiency or natural silent variants extracted from the gnomAD database, the thermodynamic and pathogenic impact of apoA-I point mutations associated with amyloid pathologies were found to show a higher destabilizing effect. MD simulations of the amyloid variant G26R evidenced the partial unfolding of the ɑ-helix bundle and the occurrence of β-strand secondary elements at the C-terminus of apoA-I. Our findings highlight APR1 as a relevant component for apoA-I structural integrity and emphasize a destabilizing effect of amyloid variants that leads to the exposure of APRs. This information contributes to our understanding of how apoA-I, with its high degree of structural flexibility, maintains a delicate equilibrium between its native structure and intrinsic tendency to form amyloid aggregates. In addition, our stability measurements could be used as a proxy to interpret the structural impact of new mutations affecting apoA-I., Instituto de Investigaciones Bioquímicas de La Plata, Instituto de Biotecnologia y Biologia Molecular

The cascade of molecular events leading to Human apolipoprotein A–I (apoA–I) amyloidosis is not completely understood, not even the pathways that determine clinical manifestations associated to systemic protein deposition in organs such as liver, kidney and heart. About twenty natural variants of apoA–I were described as inducing amyloidosis, but the mechanisms driving their aggregation and deposition are still unclear. We previously identified that the mutant Gly26Arg but not Lys107-0 induced the release of cytokines and reactive oxygen species from cultured RAW 264.7 murine macrophages, suggesting that part of the pathogenic pathway could elicit of an inflammatory signal. In this work we gained deep insight into this mechanism and determined that Gly26Arg induced a specific pro-inflammatory cascade involving activation of NF-κB and its translocation into the nucleus. These findings suggest that some but not all apoA–I natural variants might promote a pro-oxidant microenvironment which could in turn result in oxidative processing of the variants into a misfolded conformation. Fil: Ramella, Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Andújar, Isabel. Fundación Hospital Universitario Dr. Peset; España. Universidad de Valencia; España Fil: Ríos, José Luis. Universidad de Valencia; España. Fundación Hospital Universitario Dr. Peset; España Fil: Rosu, Silvana Antonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Tricerri, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Schinella, Guillermo Raúl. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina

Nephrolithiasis is one of the most frequent causes of hospitalization in nephrology and urology units in our country and is an important source of discomfort in affected patients. Despite the availability of modern, minimally invasive endourological procedures to remove stones from the urinary tract, prevention of stone recurrences remains an essential strategy. Chemical analysis of stones, whether passed spontaneously or removed surgically, is a crucial step in etiological diagnostics aimed at devising adequate prevention strategies. Modern endoscopic lithotripsy techniques provide complete fragmentation of stones and, compared to extracorporeal shock wave lithotripsy, have the advantage of avoiding painful elimination of fragments through the urinary tract. This means, however, that stone fragments are often unavailable for analysis, thereby depriving the nephrologist of an important tool for diagnosis. As a consequence, metabolic evaluation tends to be the only means of establishing the etiology of sto...

The reason that determines the pathological deposition of human apolipoprotein A-I variants inducing organ failure has been under research since the early description of natural mutations in patients. To shed light into the events associated with protein aggregation, we studied the structural perturbations that may occur in the natural variant that shows a substitution of a Leucine by an Arginine in position 60 (L60R). Circular dichroism, intrinsic fluorescence measurements, and proteolysis analysis indicated that L60R was more unstable, more sensitive to cleavage and the N-terminus was more disorganized than the protein with the native sequence (Wt). A higher tendency to aggregate was also detected when L60R was incubated at physiological pH. In addition, the small structural rearrangement observed for the freshly folded variant led to the release of tumor necrosis factor-α and interleukin-1β from a model of macrophages. However, the mutant preserved both its dimeric conformation and its lipid-binding capacity. Our results strongly suggest that the chronic disease may be a consequence of the native conformation loss which elicits the release of protein conformations that could be either cytotoxic or precursors of amyloid conformations.

Background Different protein conformations may be involved in the development of clinical manifestations associated with human amyloidosis. Although a fibrillar conformation is usually the signature of damage in the tissues of patients, it is not clear whether this species is per se the cause or the consequence of the disease. Hereditary amyloidosis due to variants of apolipoprotein A-I (apoA-I) with a substitution of a single amino acid is characterized by the presence of fibrillar protein within the lesions. Thus mutations result in increased protein aggregation. Here we set up to characterize the folding of a natural variant with a mutation leading to a deletion at position 107 (apoA-I Lys107–0). Patients carrying this variant show amyloidosis and severe atherosclerosis. Methods We oxidized this variant under controlled concentrations of hydrogen peroxide and analyzed the structure obtained after 30-day incubation by fluorescence, circular dichroism and microscopy approaches. Neutrophils activation was characterized by confocal microscopy. Results We obtained a high yield of well-defined stable fibrillar structures of apoA-I Lys107–0. In an in vitro neutrophils system, we were able to detect the induction of Neutrophils Extracellular Traps (NETs) when we incubated with oxidized apoA-I variants. This effect was exacerbated by the fibrillar structure of oxidized Lys 107–0. Conclusions We conclude that a pro-inflammatory microenvironment could result in the formation of aggregation-prone species, which, in addition may induce a positive feed-back in the activation of an inflammatory response. General significance These events may explain a close association between amyloidosis due to apoA-I Lys107–0 and atherosclerosis.

Since the early description of different human apolipoprotein A-I variants associated to amyloidosis, the reason that determines its deposition inducing organ failure has been under research. To shed light into the events associated to protein aggregation, we studied the effect of the structural perturbations induced by the replacement of a Leucine in position 60 by an Arginine as it occurs in the natural amyloidogenic variant (L60R). Circular dichroism, intrinsic fluorescence measurements and assays of binding to ligands indicate that L60R is more unstable, more sensitive to proteolysis and interacts with sodium dodecyl sulfate (a model of negative lipids) more than the protein with the native sequence and other natural variant tested, involving a replacement of a Trytophan by and Arginine in the amino acid 50 (W50R). In addition, the small structural rearrangement observed under physiological pH leads to the release of tumor necrosis factor α and interleukin-lβ from a model of macrophages. Our results strongly suggest that the chronic disease may be a consequence of the loss in the native conformation which alters the equilibrium among native and cytotoxic proteins conformation., Instituto de Investigaciones Bioquímicas de La Plata, Facultad de Ciencias Médicas, Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas

Serotonin signaling influences satiety and motivation through known actions in the hindbrain and hypothalamus. Recently, we reported that some classes of serotonin receptors also modulate food intake through actions in the ventral tegmentum and the nucleus accumbens. In the current experiments, we examined whether activation or blockade of individual serotonin receptor subtypes in the ventral tegmentum might also affect appetitive motivation for sugar pellets as assessed in a progressive ratio (PR) task. Separate groups of rats were tested following stimulation or blockade of ventral tegmental serotonin 1A, 1B, 2A, 2B, 2C, or 3 receptors. Rats within each group received multiple doses of a single drug across days; each test was separated by 72 h. Progressive ratio break point was significantly affected by stimulation of ventral tegmental serotonin 1A receptors with 8-OH-DPAT (0, 2, 4, 8 μg/side) or stimulation of serotonin 3 receptors with mCPBG (0, 10, & 20 μg/side). High doses of both agents tended to decrease break point. Additionally, stimulation of serotonin 2C receptors with RO60-0175 (at 0, 2, and 5 μg/side) reduced total lever presses and demonstrated a trend towards reducing break point. There were no effects of stimulating ventral tegmental serotonin 1B, 2A, or 2B receptors on break point; neither did antagonism of any of the serotonin receptor subtypes significantly affect performance. These data provide additional evidence that serotonergic signaling in the mesolimbic pathway affects motivated behavior, and demonstrate that a subset of serotonin receptors impact not only food consumption, but appetitive food-seeking as well.