Use of pure recombinant human enzymes to assess the disease-causing potential of missense mutations in urea cycle disorders, applied to N-acetylglutamate synthase deficiency.

N-acetylglutamate synthase (NAGS) makes acetylglutamate, the essential activator of the first, regulatory enzyme of the urea cycle, carbamoyl phosphate synthetase 1 (CPS1). NAGS deficiency (NAGSD) and CPS1 deficiency (CPS1D) present identical phenotypes. However, they must be distinguished, because NAGSD is cured by substitutive therapy with the N-acetyl-L-glutamate analogue N-carbamyl-L-glutamate, while curative therapy of CPS1D requires liver transplantation. Since their differentiation is done genetically, it is important to ascertain the disease-causing potential of CPS1 and NAGS genetic variants. With this goal, we previously carried out site-directed mutagenesis studies with pure recombinant human CPS1. We could not do the same with human NAGS (HuNAGS) because of enzyme instability, leading to our prior utilization of a bacterial NAGS as an imperfect surrogate of HuNAGS. We now use genuine HuNAGS, stabilized as a chimera of its conserved domain (cHuNAGS) with the maltose binding protein (MBP), and produced in Escherichia coli. MBP-cHuNAGS linker cleavage allowed assessment of the enzymatic properties and thermal stability of cHuNAGS, either wild-type or hosting each one of 23 nonsynonymous single-base changes found in NAGSD patients. For all but one change, disease causation was accounted by the enzymatic alterations identified, including, depending on the variant, loss of arginine activation, increased Km Glutamate, active site inactivation, decreased thermal stability, and protein misfolding. Our present approach outperforms experimental in vitro use of bacterial NAGS or in silico utilization of prediction servers (including AlphaMissense), illustrating with HuNAGS the value for UCDs of using recombinant enzymes for assessing disease-causation and molecular pathogenesis, and for therapeutic guidance.

A New Paradigm in the Relationship between Gut Microbiota and Breast Cancer: ß-glucuronidase Enzyme Identified as Potential Therapeutic Target.

Breast cancer (BC) is the most frequently occurring malignancy and the second cancer-specific cause of mortality in women in developed countries. Over 70% of the total number of BCs are hormone receptor-positive (HR+), and elevated levels of circulating estrogen (E) in the blood have been shown to be a major risk factor for the development of HR+ BC. This is attributable to estrogen's contribution to increased cancer cell proliferation, stimulation of angiogenesis and metastasis, and resistance to therapy. The E metabolism-gut microbiome axis is functional, with subjacent individual variations in the levels of E. It is conceivable that the estrobolome (bacterial genes whose products metabolize E) may contribute to the risk of malignant neoplasms of hormonal origin, including BC, and may serve as a potential biomarker and target. It has been suggested that ß-glucuronidase (GUS) enzymes of the intestinal microbiome participate in the strobolome. In addition, it has been proposed that bacterial GUS enzymes from the gastrointestinal tract participate in hormone BC. In this review, we discuss the latest knowledge about the role of the GUS enzyme in the pathogenesis of BC, focusing on (i) the microbiome and E metabolism; (ii) diet, estrobolome, and BC development; (iii) other activities of the bacterial GUS; and (iv) the new molecular targets for BC therapeutic application.

Impact of SARS-CoV-2 Infection on Patients with Cancer: Retrospective and Transversal Studies in Spanish Population.

BACKGROUND: Studies of patients with cancer affected by coronavirus disease 2019 (COVID-19) are needed to assess the impact of the disease in this sensitive population, and the influence of different cancer treatments on the COVID-19 infection and seroconversion. MATERIAL AND METHODS: We performed a retrospective analysis of all patients hospitalized with RT-PCR positive for COVID-19 in our region to assess the prevalence of cancer patients and describe their characteristics and evolution (Cohort 1). Concurrently, a transversal study was carried out in patients on active systemic cancer treatment for symptomatology and seroprevalence (IgG/IgM by ELISA-method) against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (Cohort 2). RESULTS: A total of 215 patients (Cohort 1) were admitted to hospital with a confirmed COVID-19 infection between February 28 and April 30, 2020, and 17 died (7.9%). A medical record of cancer was noted in 43 cases (20%), 6 of them required Intensive care unit ICU attention (14%), and 7 died (16%). There were thirty-six patients (83%) who tested IgG/IgM positive for SARS-CoV-2. Patients on immunosuppressive therapies presented a lower ratio of seroconversion (40% vs. 8%; p = 0.02). In Cohort 2, 166 patients were included in a symptoms-survey and tested for SARS-CoV-2. Any type of potential COVID-19-related symptom was referred up to 67.4% of patients (85.9% vs. 48.2% vs. 73.9%, for patients on chemotherapy, immunotherapy and targeted therapies respectively, p < 0.05). The seroprevalence ratio was 1.8% for the whole cohort with no significant differences by patient or treatment characteristics. CONCLUSION: Patients with cancer present higher risks for hospital needs for COVID-19 infection. The lack of SARS-CoV-2 seroconversion may be a concern for patients on immunosuppressive therapies. Patients receiving systematic therapies relayed a high rate of potentially COVID-19-related symptoms, particularly those receiving chemotherapy. However, the seroconversion rate remains low and in the range of general population.

Bifidobacterium pseudocatenulatum CECT 7765 reverses the adverse effects of diet-induced obesity through the gut-bone axis.

Obesity and the associated chronic metabolic diseases (e.g., type-2 diabetes) adversely affect bone metabolism and health. Gut microbiota is considered to be involved in the pathophysiology of obesity and also represents a therapeutic target. This study has investigated the contribution of diet-induced obesity to alterations in bone health and metabolism and whether these could be restored by oral administration of Bifidobacterium pseudocatenulatum CECT 7765. To do so, adult male wild-type C57BL-6 mice were fed either a standard or high-fat diet (HFD), supplemented or not with B. pseudocatenulatum CECT 7765 (109 CFU/day) for 14 weeks. Effects on bone mass density (BMD), bone mineral content, bone remodeling, bone structure and gene expression were assessed. In HFD-fed mice, bone microstructural properties at the distal femur showed deteriorated trabecular architecture in bone volumetric fraction, trabecular number and trabecular pattern factor. Besides, the HFD reduced the volumetric bone mineral density in the trabecular bone, but not in the cortical bone. All these bone microstructural alterations found in obese mice were reversed by B. pseudocatenulatum CECT 7765. Administration of the bacterium increased (p < .05) the Wnt/ß-catenin pathway gene expression, which could mediate effects on BMD. Bifidobacterium pseudocatenulatum CECT 7765 supplementation increased (p < .05) serum osteocalcin (OC, bone formation parameter), and decreased serum C-terminal telopeptide (CTX) (p < .01) and parathormone (PTH) (p < .05) (both bone resorption parameters). It also altered the microstructure of the femur. In summary, HFD interfered with the normal bone homeostasis leading to increased bone loss. In obese mice, B. pseudocatenulatum CECT 7765 lowered bone mass loss and enhanced BMD by decreasing bone resorption and increasing bone formation.

Advances in drug-induced cholestasis: Clinical perspectives, potential mechanisms and in vitro systems.

Despite growing research, drug-induced liver injury (DILI) remains a serious issue of increasing importance to the medical community that challenges health systems, pharmaceutical industries and drug regulatory agencies. Drug-induced cholestasis (DIC) represents a frequent manifestation of DILI in humans, which is characterised by an impaired canalicular bile flow resulting in a detrimental accumulation of bile constituents in blood and tissues. From a clinical point of view, cholestatic DILI generates a wide spectrum of presentations and can be a diagnostic challenge. The drug classes mostly associated with DIC are anti-infectious, anti-diabetic, anti-inflammatory, psychotropic and cardiovascular agents, steroids, and other miscellaneous drugs. The molecular mechanisms of DIC have been investigated since the 1980s but they remain debatable. It is recognised that altered expression and/or function of hepatobiliary membrane transporters underlies some forms of cholestasis, and this and other concomitant mechanisms are very likely in DIC. Deciphering these processes may pave the ways for diagnosis, prognosis and prevention, for which currently major gaps and caveats exist. In this review, we summarise recent advances in the field of DIC, including clinical aspects, the potential mechanisms postulated so far and the in vitro systems that can be useful to investigate and identify new cholestatic drugs.

Predicting drug-induced cholestasis: preclinical models.

INTRODUCTION: In almost 50% of patients with drug-induced liver injury (DILI), the bile flow from the liver to the duodenum is impaired, a condition known as cholestasis. However, this toxic response only appears in a small percentage of the treated patients (idiosyncrasy). Prediction of drug-induced cholestasis (DIC) is challenging and emerges as a safety issue that requires attention by professionals in clinical practice, regulatory authorities, pharmaceutical companies, and research institutions. Area covered: The current synopsis focuses on the state-of-the-art in preclinical models for cholestatic DILI prediction. These models differ in their goal, complexity, availability, and applicability, and can widely be classified in experimental animals and in vitro models. Expert opinion: Drugs are a growing cause of cholestasis, but the progress made in explaining mechanisms and differences in susceptibility is not growing at the same rate. We need reliable models able to recapitulate the features of DIC, particularly its idiosyncrasy. The homogeneity and the species-specific differences move animal models away from a fair predictability. However, in vitro human models are improving and getting closer to the real hepatocyte phenotype, and they will likely be the choice in the near future. Progress in this area will not only need reliable predictive models but also mechanistic insights.

Development of synchrotron X-ray micro-tomography under extreme conditions of pressure and temperature.

X-ray tomography is a non-destructive three-dimensional imaging/microanalysis technique selective to a wide range of properties such as density, chemical composition, chemical states and crystallographic structure with extremely high sensitivity and spatial resolution. Here the development of in situ high-pressure high-temperature micro-tomography using a rotating module for the Paris-Edinburgh cell combined with synchrotron radiation is described. By rotating the sample chamber by 360°, the limited angular aperture of ordinary high-pressure cells is surmounted. Such a non-destructive high-resolution probe provides three-dimensional insight on the morphological and structural evolution of crystalline as well as amorphous phases during high pressure and temperature treatment. To demonstrate the potentials of this new experimental technique the compression behavior of a basalt glass is investigated by X-ray absorption tomography, and diffraction/scattering tomography imaging of the structural changes during the polymerization of C60 molecules under pressure is performed. Small size and weight of the loading frame and rotating module means that this apparatus is portable, and can be readily installed on most synchrotron facilities to take advantage of the diversity of three-dimensional imaging techniques available at beamlines. This experimental breakthrough should open new ways for in situ imaging of materials under extreme pressure-temperature-stress conditions, impacting diverse areas in physics, chemistry, geology or materials sciences.

Safety Assessment of Bacteroides uniformis CECT 7771 Isolated from Stools of Healthy Breast-Fed Infants.

BACKGROUND: Bacteroides uniformis CECT 7771 is a potential probiotic strain, originally isolated from the stools of healthy breast-feed infants. The strain showed pre-clinical efficacy in a mouse obesity model. The objective of this study was to evaluate its potential toxicity and translocation ability after acute oral administration to mice. METHODS AND FINDINGS: A safety study was conducted in immunocompetent and immunosuppressed C57BL-6 mice. Both mouse groups (n = 10 per group) were fed orally 2 x 10(9) colony forming units (cfu)/day of B. uniformis CECT 7771 or placebo by gavage for 6 days. Throughout this time, feed and water intake and body weight were monitored. Afterwards, mice were sacrificed and biological samples were collected to analyze blood and urine biochemistry, inflammatory and immune markers; gut mucosal histology and bacterial translocation to peripheral tissues. The results demonstrated that acute ingestion of this Bacteroides strain had no adverse effects on the animals' general health status or food intake, nor did it affect biochemical indicators of liver, kidney and pancreatic function or gut mucosal histology. Findings also demonstrated that administration did not lead to bacterial translocation to blood, liver or mesenteric lymph nodes. B. uniformis CECT 7771 also downregulated gene and protein expression (iNOS and PPAR-γ) and inflammatory cytokines induced by immunosuppression. CONCLUSIONS: The findings indicate that the acute oral consumption of B. uniformis CECT 7771 does not raise safety concerns in mice. Further studies in humans should be conducted.

Use of Relief Contrast(®) objective to improve sperm morphometric analysis by Isas(®) casa system in the ram.

The aim of this study was to develop a new method for morphometric assessment of the sperm head and acrosome in the ram. Ejaculates from 10 adult males were collected using an artificial vagina. For each ejaculate, 10 semen smears were prepared, air-dried and divided (in pairs) into the following five treatment groups: (i) washed in distilled water and allowed to dry without further processing (DRY); (ii) fixed in 50% methanol (MET); (iii) fixed in 2% glutaraldehyde (GLUT); (iv) fixed and stained with Hemacolor(®) (HEM) and (v) fixed and stained with SpermBlue(®) (SB). The prepared slides were examined with a 40 × Relief Contrast(®) objective (RC) and processed with ISAS(®) commercial software. The use of RC optics increased the contrast between acrosome and sperm head, allowing capture and morphometric analysis by ISAS of sperm heads and the acrosome, even in non-stained samples. MET and GLUT groups resulted in a lower number of acceptable, that is, correctly delineated, sperm heads than those in the SB, and SB and HEM groups, respectively (p < 0.05). The higher proportion of sperm discarded in MET and GLUT groups may be explained by a higher presence of artefacts. For the majority of the primary morphometric parameters of the sperm head and the acrosomal area, the relationship between treatments was the following: GLUT> HEM≥ MET≥ SB> DRY. When studying the proportion of the sperm head covered by the acrosome, the relation between treatments was: MET> DRY = GLUT = SB> HEM. It was concluded that the new method for sperm morphometric assessment allows the simultaneous assessment of sperm head and acrosome in the ram by the first time, even in unprocessed semen smears.

"Compressed graphite" formed during C60 to diamond transformation as revealed by scattering computed tomography.

The collapsing of C60 into polycrystalline diamond has been studied after nonhydrostatic pressurization at ambient temperature using x-ray scattering computed tomography. Using this selective structural probe we provide evidence of concentric coexistence of "compressed graphite" (d(00l)∼3.09-3.11 Å), sp2-graphitelike phase (d(00l)∼3.35-3.42 Å), and sp3-like amorphous carbon surrounding polycrystalline diamond (a∼3.56-3.59 Å). The so-called "compressed graphite" exhibits a collapsed c axis and is textured with disordered layers. This latter phase is better described as a short interlayered carbon phase with buckled sp2-sp3 layers with possible interlayer bonding. Additionally, our 3D maps of phase distribution and of the residual stress retained in the polycrystalline diamond phase support the importance of stressed synthesis conditions for diamond formation.

High efficiency multichannel collimator for structural studies of liquids and low-Z materials at high pressures and temperatures.

A high efficiency multichannel collimator (MCC) device has been developed at the high pressure beamline ID27 of the European Synchrotron Radiation Facility to drastically reduce the x-ray background from the sample environment in the Paris-Edinburgh press. The main technical difficulty, which resides in the minimum slits size achievable using the classical mono-bloc design, has been resolved using an original concept based on a set of independent slits. Then, a very small slit size of 50 µm was manufactured resulting in a great improvement of the signal to background ratio. In addition, the transfer function of the MCC has been measured using the x-ray diffusion signal of a metal doped glass and efficiently applied to correct the raw data. The potential of this new device is illustrated in two challenging examples: iron-sulfur liquid structures and C(60) polymerization process at high pressure and high temperature.

Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor.

N-acetyl-L-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K(m) for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity.

Basis of arginine sensitivity of microbial N-acetyl-L-glutamate kinases: mutagenesis and protein engineering study with the Pseudomonas aeruginosa and Escherichia coli enzymes.

N-acetylglutamate kinase (NAGK) catalyzes the second step of arginine biosynthesis. In Pseudomonas aeruginosa, but not in Escherichia coli, this step is rate limiting and feedback and sigmoidally inhibited by arginine. Crystal structures revealed that arginine-insensitive E. coli NAGK (EcNAGK) is homodimeric, whereas arginine-inhibitable NAGKs, including P. aeruginosa NAGK (PaNAGK), are hexamers in which an extra N-terminal kinked helix (N-helix) interlinks three dimers. By introducing single amino acid replacements in PaNAGK, we prove the functionality of the structurally identified arginine site, as arginine site mutations selectively decreased the apparent affinity for arginine. N-helix mutations affecting R24 and E17 increased and decreased, respectively, the apparent affinity of PaNAGK for arginine, as predicted from enzyme structures that revealed the respective formation by these residues of bonds favoring inaccessible and accessible arginine site conformations. N-helix N-terminal deletions spanning > or = 16 residues dissociated PaNAGK to active dimers, those of < or = 20 residues decreased the apparent affinity for arginine, and complete N-helix deletion (26 residues) abolished arginine inhibition. Upon attachment of the PaNAGK N-terminal extension to the EcNAGK N terminus, EcNAGK remained dimeric and arginine insensitive. We concluded that the N-helix and its C-terminal portion after the kink are essential but not sufficient for hexamer formation and arginine inhibition, respectively; that the N-helix modulates NAGK affinity for arginine and mediates signal transmission between arginine sites, thus establishing sigmoidal arginine inhibition kinetics; that the mobile alphaH-beta16 loop of the arginine site is the modulatory signal receiver; and that the hexameric architecture is not essential for arginine inhibition but is functionally essential for physiologically relevant arginine control of NAGK.

Genomic instability: on the birth and death of cancer.

The presence of an abnormal chromosomal content is probably the most universally conserved hallmark of cancer cells. Predicted at the beginning of the 20th century as the origin of tumours, and extensively documented thereafter, genomic instability lies at the core of neoplastic development. Regardless of this classic model, the actual impact that deficient control of genomic integrity has on human health and particularly on cancer development only started to gain attention from the scientific community two decades ago. From a bird's eye view and with a cancer-oriented perspective, in this work we will try to cover some of the concepts obtained from recent research in genomic instability. The review will end up presenting suggestive evidence which proposes that genomic instability might turn out to be not just the driving force but also the Achilles' heel of cancer.