Antibacterial, anti-invasive, and anti-inflammatory activity of bovine lactoferrin extracted from milk or colostrum versus whole colostrum.

Lactoferrin (Lf), a multifunctional cationic glycoprotein extracted from milk or colostrum, is able to chelate two ferric ions per molecule, inhibit the formation of reactive oxygen species, interact with the anionic components of bacteria or host cells, and enter inside host cell nucleus, thereby exerting antibacterial, anti-invasive, and anti-inflammatory activities. By virtue of Lf presence, bovine colostrum is expected to perform analogous functions to pure Lf, along with additional activities attributable to other bioactive constituents. The present research aims to compare the antibacterial, anti-invasive, and anti-inflammatory activities of bovine Lf purified from milk (mbLf) and colostrum (cbLf) in comparison to those exhibited by whole bovine colostrum (wbc). The results demonstrated a major efficacy of mbLf in inhibiting pathogenic bacteria and in exerting anti-invasive and anti-survival activities with respect to cbLf and wbc. Furthermore, mbLf lowered IL-6 levels to those of uninfected cells, while a less evident decrease was observed upon cbLf treatment. Conversely, wbc managed to slightly lower IL-6 levels compared to those synthesized by infected cells. These data demonstrate that, to obtain maximum effectiveness in such activities, Lf should be formulated/used without addition of other substances and should be sourced from bovine milk rather than colostrum.

LARP6C orchestrates posttranscriptional reprogramming of gene expression during hydration to promote pollen tube guidance.

Increasing evidence suggests that posttranscriptional regulation is a key player in the transition between mature pollen and the progamic phase (from pollination to fertilization). Nonetheless, the actors in this messenger RNA (mRNA)-based gene expression reprogramming are poorly understood. We demonstrate that the evolutionarily conserved RNA-binding protein LARP6C is necessary for the transition from dry pollen to pollen tubes and the guided growth of pollen tubes towards the ovule in Arabidopsis thaliana. In dry pollen, LARP6C binds to transcripts encoding proteins that function in lipid synthesis and homeostasis, vesicular trafficking, and polarized cell growth. LARP6C also forms cytoplasmic granules that contain the poly(A) binding protein and possibly represent storage sites for translationally silent mRNAs. In pollen tubes, the loss of LARP6C negatively affects the quantities and distribution of storage lipids, as well as vesicular trafficking. In Nicotiana benthamiana leaf cells and in planta, analysis of reporter mRNAs designed from the LARP6C target MGD2 provided evidence that LARP6C can shift from a repressor to an activator of translation when the pollen grain enters the progamic phase. We propose that LARP6C orchestrates the timely posttranscriptional regulation of a subset of mRNAs in pollen during the transition from the quiescent to active state and along the progamic phase to promote male fertilization in plants.

Structural basis of dimerization and nucleic acid binding of human DBHS proteins NONO and PSPC1.

The Drosophila behaviour/human splicing (DBHS) proteins are a family of RNA/DNA binding cofactors liable for a range of cellular processes. DBHS proteins include the non-POU domain-containing octamer-binding protein (NONO) and paraspeckle protein component 1 (PSPC1), proteins capable of forming combinatorial dimers. Here, we describe the crystal structures of the human NONO and PSPC1 homodimers, representing uncharacterized DBHS dimerization states. The structures reveal a set of conserved contacts and structural plasticity within the dimerization interface that provide a rationale for dimer selectivity between DBHS paralogues. In addition, solution X-ray scattering and accompanying biochemical experiments describe a mechanism of cooperative RNA recognition by the NONO homodimer. Nucleic acid binding is reliant on RRM1, and appears to be affected by the orientation of RRM1, influenced by a newly identified 'ß-clasp' structure. Our structures shed light on the molecular determinants for DBHS homo- and heterodimerization and provide a basis for understanding how DBHS proteins cooperatively recognize a broad spectrum of RNA targets.

Maternal Larp6 controls oocyte development, chorion formation and elevation.

La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes: larp6a and larp6b Viable and fertile single and double homozygous larp6a and larp6b zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However, larp6a mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from larp6b single mutant females showed minor chorion defects, but chorions from eggs laid by larp6a;larp6b double mutant females were more defective than those from larp6a single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation.

The risks associated with percutaneous native kidney biopsies: a prospective study.

BACKGROUND: The known risks and benefits of native kidney biopsies are mainly based on the findings of retrospective studies. The aim of this multicentre prospective study was to evaluate the safety of percutaneous renal biopsies and quantify biopsy-related complication rates in Italy. METHODS: The study examined the results of native kidney biopsies performed in 54 Italian nephrology centres between 2012 and 2020. The primary outcome was the rate of major complications 1 day after the procedure, or for longer if it was necessary to evaluate the evolution of a complication. Centre and patient risk predictors were analysed using multivariate logistic regression. RESULTS: Analysis of 5304 biopsies of patients with a median age of 53.2 years revealed 400 major complication events in 273 patients (5.1%): the most frequent was a ≥2 g/dL decrease in haemoglobin levels (2.2%), followed by macrohaematuria (1.2%), blood transfusion (1.1%), gross haematoma (0.9%), artero-venous fistula (0.7%), invasive intervention (0.5%), pain (0.5%), symptomatic hypotension (0.3%), a rapid increase in serum creatinine levels (0.1%) and death (0.02%). The risk factors for major complications were higher plasma creatinine levels [odds ratio (OR) 1.12 for each mg/dL increase, 95% confidence interval (95% CI) 1.08-1.17], liver disease (OR 2.27, 95% CI 1.21-4.25) and a higher number of needle passes (OR for each pass 1.22, 95% CI 1.07-1.39), whereas higher proteinuria levels (OR for each g/day increase 0.95, 95% CI 0.92-0.99) were protective. CONCLUSIONS: This is the first multicentre prospective study showing that percutaneous native kidney biopsies are associated with a 5% risk of a major post-biopsy complication. Predictors of increased risk include higher plasma creatinine levels, liver disease and a higher number of needle passes.

An overview on in vitro and in vivo antiviral activity of lactoferrin: its efficacy against SARS-CoV-2 infection.

Beyond the absolute and indisputable relevance and efficacy of anti-SARS-CoV-2 vaccines, the rapid transmission, the severity of infection, the absence of the protection on immunocompromised patients, the propagation of variants, the onset of infection and/or disease in vaccinated subjects and the lack of availability of worldwide vaccination require additional antiviral treatments. Since 1987, lactoferrin (Lf) is well-known to possess an antiviral activity related to its physico-chemical properties and to its ability to bind to both heparan sulfate proteoglycans (HSPGs) of host cells and/or surface components of viral particles. In the present review, we summarize in vitro and in vivo studies concerning the efficacy of Lf against DNA, RNA, enveloped and non-enveloped viruses. Recent studies have revealed that the in vitro antiviral activity of Lf is also extendable to SARS-CoV-2. In vivo, Lf oral administration in early stage of SARS-CoV-2 infection counteracts COVID-19 pathogenesis. In particular, the effect of Lf on SARS-CoV-2 entry, inflammatory homeostasis, iron dysregulation, iron-proteins synthesis, reactive oxygen formation, oxidative stress, gut-lung axis regulation as well as on RNA negativization, and coagulation/fibrinolysis balance will be critically reviewed. Moreover, the molecular mechanisms underneath, including the Lf binding to HSPGs and spike glycoprotein, will be disclosed and discussed. Taken together, present data not only support the application of the oral administration of Lf alone in asymptomatic COVID-19 patients or as adjuvant of standard of care practice in symptomatic ones but also constitute the basis for enriching the limited literature on Lf effectiveness for COVID-19 treatment.

Effect of bovine lactoferrin on recurrent urinary tract infections: in vitro and in vivo evidences.

Uropathogenic Escherichia coli (UPEC) strains are the primary cause of urinary tract infections (UTIs). UPEC strains are able to invade, multiply and persisting in host cells. Therefore, UPEC strains are associated to recurrent UTIs requiring long-term antibiotic therapy. However, this therapy is suboptimal due to the increase of multidrug-resistant UPEC. The use of non-antibiotic treatments for managing UTIs is required. Among these, bovine lactoferrin (bLf), a multifunctional cationic glycoprotein, could be a promising tool because inhibits the entry into the host cells of several intracellular bacteria. Here, we demonstrate that 100 µg/ml bLf hinders the invasion of 2.0 ± 0.5 × 104 CFU/ml E. coli CFT073, prototype of UPEC, infecting 2.0 ± 0.5 × 105 cells/ml urinary bladder T24 epithelial cells. The highest protection (100%) is due to the bLf binding with host surface components even if an additional binding to bacterial surface components cannot be excluded. Of note, in the absence of bLf, UPEC survives and multiplies, while bLf significantly decreases bacterial intracellular survival. After these encouraging results, an observational survey on thirty-three patients affected by recurrent cystitis was performed. The treatment consisted in the oral administration of bLf alone or in combination with antibiotics and/or probiotics. After the observation period, a marked reduction of cystitis episodes was observed (p < 0.001) in all patients compared to the episodes occurred during the 6 months preceding the bLf-treatment. Twenty-nine patients did not report cystitis episodes (87.9%) whereas the remaining four (12.1%) experienced only one episode, indicating that bLf could be a worthwhile and safe treatment in counteracting recurrent cystitis.

Moyamoya disease presenting with tubular dysfunction in a child: pitfalls in diagnosing an atypical hyponatremic-hypertensive syndrome.

BACKGROUND: Moyamoya disease, a cause of pediatric stroke, has been shown to affect furthermore extra-cranial districts, mostly the kidney arterial site, resulting in steno-occlusive changes. Unilateral renal artery stenosis accounts for 8%-10% out of cases of renovascular hypertension in childhood, however it rarely underlies a hyponatremic-hypertensive syndrome (HHS). CASE PRESENTATION: We describe an 18-month-old boy with a recent history of polyuria and polydipsia, who presented an acute febrile gastroenteritis with neurological impairment, severe dehydration, hyponatremia, hypokalemia, kidney tubular dysfunction, and elevated aldosterone and renin even with a normal blood pressure. Fluid and electrolytes correction was performed, with complete recovery. An abdominal ultrasound displayed a smaller right kidney. A brain magnetic resonance and an electroencephalogram did not show any relevant abnormalities. Five months later, the child experienced a left-side hemiparesis after a traumatic concussion, and a severe hypertension. A brain tomography documented a cerebral ischemia. Brain and kidney angiographic studies displayed puff of smoke findings of internal right carotid artery branches and a steno-occlusive pattern of right renal artery, respectively. Hence, moyamoya disease with HHS secondary to unilateral renal artery stenosis was diagnosed. After an unsuccessful antiplatelet and antihypertensive pharmacological treatment, the boy underwent a renal angioplasty and a cerebral STA-MCA bypass (direct superficial temporal artery-to-middle cerebral artery bypass), resulting in a significant improvement of both neurological and kidney disease. CONCLUSIONS: Although the association between unilateral renal artery stenosis and HHS has been previously shown, this is the first report of atypical HHS, with hypertension preceded by tubular dysfunction, recognized in the framework of moyamoya disease.

Expression pattern of perilipins in human brain during aging and in Alzheimer's disease.

AIMS: Perilipins are conserved proteins that decorate intracellular lipid droplets and are essential for lipid metabolism. To date, there is limited knowledge on their expression in human brain or their involvement in brain aging and neurodegeneration. The aim of this study was to characterise the expression levels of perilipins (Plin1-Plin5) in different cerebral areas from subjects of different age, with or without signs of neurodegeneration. METHODS: We performed real-time RT-PCR, western blotting, immunohistochemistry and confocal microscopy analyses in autoptic brain samples of frontal and temporal cortex, cerebellum and hippocampus from subjects ranging from 33 to 104 years of age, with or without histological signs of neurodegeneration. To test the possible relationship between Plins and inflammation, correlation analysis with IL-6 expression was also performed. RESULTS: Plin2, Plin3 and Plin5, but not Plin1 and Plin4, are expressed in the considered brain areas with different intensities. Plin2 appears to be expressed more in grey matter, particularly in neurons in all the areas analysed, whereas Plin3 and Plin5 appear to be expressed more in white matter. Plin3 seems to be expressed more in astrocytes. Only Plin2 expression is higher in old subjects and patients with early tauopathy or Alzheimer's disease and is associated with IL-6 expression. CONCLUSIONS: Perilipins are expressed in human brain but only Plin2 appears to be modulated with age and neurodegeneration and linked to an inflammatory state. We propose that the accumulation of lipid droplets decorated with Plin2 occurs during brain aging and that this accumulation may be an early marker and initial step of inflammation and neurodegeneration.

Allergic rhinitis, microbiota and passive smoke in children: A pilot study.

BACKGROUND: Allergic rhinitis (AR) is one of the most common allergic diseases affecting children. Objective assessment of nasal obstruction is possible through active anterior rhinomanometry (AAR). Several factors, such as passive smoke exposure (PSE), are triggers for worsening nasal obstruction and chronic inflammation. PSE affects bacterial eubiosis in the upper respiratory tract. This study evaluates the influence of PSE and cotinine levels on both nasal obstruction and local microbiome composition in children with AR. METHODS: Fifty patients (aged between 6 and 16 years) with AR monosensitized grass pollen were enrolled. They underwent skin prick tests, a nasal swab to evaluate the microbial composition of the anterior nostrils, a basal AAR, a post-decongestion AAR, and spirometry. Serum cotinine levels were assessed to evaluate PSE. RESULTS: A significantly lower percentage of mean nasal flow (mNF%) was observed before and after hydrazine administration in subjects exposed to passive smoke (Exp group) compared with the non-exposed group. In contrast, higher cotinine levels were observed in the Exp group than in the controls. PSE has been associated with a decrease in biodiversity and a change in the nasal microbiome composition; instead, although to a different extent, the abundance of specific taxa resulted in being correlated to cotinine levels and nasal flow. CONCLUSION: Children with AR exposed to passive smoke with positive serum cotinine could represent a risk factor for developing nasal obstruction and microbial dysbiosis, suggesting their possible role in pathophysiological processes.

Management of the congenital solitary kidney: consensus recommendations of the Italian Society of Pediatric Nephrology.

BACKGROUND: In recent years, several studies have been published on the prognosis of children with congenital solitary kidney (CSK), with controversial results, and a worldwide consensus on management and follow-up is lacking. In this consensus statement, the Italian Society of Pediatric Nephrology summarizes the current knowledge on CSK and presents recommendations for its management, including diagnostic approach, nutritional and lifestyle habits, and follow-up. We recommend that any antenatal suspicion/diagnosis of CSK be confirmed by neonatal ultrasound (US), avoiding the routine use of further imaging if no other anomalies of kidney/urinary tract are detected. A CSK without additional abnormalities is expected to undergo compensatory enlargement, which should be assessed by US. We recommend that urinalysis, but not blood tests or genetic analysis, be routinely performed at diagnosis in infants and children showing compensatory enlargement of the CSK. Extrarenal malformations should be searched for, particularly genital tract malformations in females. An excessive protein and salt intake should be avoided, while sport participation should not be restricted. We recommend a lifelong follow-up, which should be tailored on risk stratification, as follows: low risk: CSK with compensatory enlargement, medium risk: CSK without compensatory enlargement and/or additional CAKUT, and high risk: decreased GFR and/or proteinuria, and/or hypertension. We recommend that in children at low-risk periodic US, urinalysis and BP measurement be performed; in those at medium risk, we recommend that serum creatinine also be measured; in high-risk children, the schedule has to be tailored according to kidney function and clinical data.

Loss of Detection of sgN Precedes Viral Abridged Replication in COVID-19-Affected Patients-A Target for SARS-CoV-2 Propagation.

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.

Impact of high fat diet on the sterol regulatory element-binding protein 2 cholesterol pathway in the testicle.

Male fertility has been shown to be dependent on cholesterol homeostasis. This lipid is essential for testosterone synthesis and spermatogenesis, but its levels must be maintained in an optimal range for proper testicular function. In particular, sperm cells' development is very sensitive to high cholesterol levels, noticeably during acrosomal formation. The aim of this work was to study whether the molecular pathway that regulates intracellular cholesterol, the sterol regulatory element-binding protein (SREBP) pathway, is affected in the testicles of animals under a fat diet. To investigate this, we took advantage of the non-obese hypercholesterolemia (HC) model in New Zealand rabbits that displays poor sperm and seminal quality. The testicular expression of SREBP isoform 2 (SREBP2) and its target molecules 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) and low-density lipoprotein receptor (LDLR) were studied under acute (6 months) and chronic (more than 12 months) fat intake by RT-PCR, western blot and immunofluorescence. Our findings showed that fat consumption promoted down-regulation of the SREBP2 pathway in the testicle at 6 months, but upregulation after a chronic period. This was consistent with load of testicular cholesterol, assessed by filipin staining. In conclusion, the intracellular pathway that regulates cholesterol levels in the testicle is sensitive to dietary fats, and behaves differently depending on the duration of consumption: it has a short-term protective effect, but became deregulated in the long term, ultimately leading to a detrimental situation. These results will contribute to the understanding of the basic mechanisms of the effect of fat consumption in humans with idiopathic infertility.

The La-related proteins: structures and interactions of a versatile superfamily of RNA-binding proteins.

The La-related proteins (LaRPs) are an ancient superfamily of RNA-binding proteins orchestrating the major fates of RNA, from processing and maturation to regulation of mRNA translation. LaRPs are instrumental in modulating complex assemblies where the RNA is bound, folded, processed, escorted and presented to the functional effectors often through recruitment of protein partners. This intricate web of protein-RNA and protein-protein interactions is enabled by the modular nature of the LaRPs, comprising several structured domains connected by flexible linkers, and other sequences lacking recognizable folded motifs. Recent structures, together with biochemical and biophysical studies, have provided insights into how each LaRP family has evolved unique mechanisms of RNA recognition, not only through the conserved RNA-binding unit, the La-module, but also mediated by other family-specific motifs. Furthermore, in a series of unexpected twists and turns, they have revealed that the dynamic and conformational interplay of multi-structured domains and disordered regions operate in unison to achieve RNA substrate discrimination. This review proposes a perspective of our current knowledge of the structure-function relationship of the LaRP superfamily.

Structural dynamics in the La-module of La-related proteins.

The La-related proteins (LaRPs) are a superfamily of eukaryotic RNA-binding proteins with important and varied roles. To understand LaRP functions it is essential to unravel the divergent features responsible for their RNA target selectivity, which underlie their distinct identities and cellular roles. LaRPs are built on a common structural module called the 'La-module' that acts as a main locus for RNA recognition. The La-module is comprised of two tethered domains whose relative structural and dynamic interplay has been proposed to regulate RNA-target selection, albeit the mechanistic underpinning of this recognition remains to be elucidated. A main unsolved conundrum is how conserved La-modules across LaRPs are able to bind to extremely diverse RNA ligands.In this work, we employed Small Angle X-ray Scattering (SAXS) to investigate several human LaRP La-modules in the absence and, where applicable, in the presence of their RNA target, with the aim to explore the structural dynamics of their RNA recognition and provide information on the architectural landscape accessible to these proteins. Integration of these SAXS experiments with prior X-ray crystallography and NMR data suggests that RNA binding is generally accompanied by a compaction and loss of flexibility of the La-module. Nonetheless, the La-modules appear to experience a considerably different degree of inherent flexibility in their apo state. Furthermore, although they all exist in discrete subsets of accessible populations in equilibrium, these vary from LaRP to LaRP and can be either extended or compact. We propose that these divergent features may be critical for RNA substrate discrimination.

LARP4A recognizes polyA RNA via a novel binding mechanism mediated by disordered regions and involving the PAM2w motif, revealing interplay between PABP, LARP4A and mRNA.

LARP4A belongs to the ancient RNA-binding protein superfamily of La-related proteins (LARPs). In humans, it acts mainly by stabilizing mRNAs, enhancing translation and controlling polyA lengths of heterologous mRNAs. These activities are known to implicate its association with mRNA, protein partners and translating ribosomes, albeit molecular details are missing. Here, we characterize the direct interaction between LARP4A, oligoA RNA and the MLLE domain of the PolyA-binding protein (PABP). Our study shows that LARP4A-oligoA association entails novel RNA recognition features involving the N-terminal region of the protein that exists in a semi-disordered state and lacks any recognizable RNA-binding motif. Against expectations, we show that the La module, the conserved RNA-binding unit across LARPs, is not the principal determinant for oligoA interaction, only contributing to binding to a limited degree. Furthermore, the variant PABP-interacting motif 2 (PAM2w) featured in the N-terminal region of LARP4A was found to be important for both RNA and PABP recognition, revealing a new role for this protein-protein binding motif. Our analysis demonstrates the mutual exclusive nature of the PAM2w-mediated interactions, thereby unveiling a tantalizing interplay between LARP4A, polyA and PABP.

PDXK mutations cause polyneuropathy responsive to pyridoxal 5'-phosphate supplementation.

OBJECTIVE: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. METHODS: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. RESULTS: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP-binding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'-phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. INTERPRETATION: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels. ANN NEUROL 2019;86:225-240.

Recovery from 6-month spaceflight at the International Space Station: muscle-related stress into a proinflammatory setting.

The Sarcolab pilot study of 2 crewmembers, investigated before and after a 6-mo International Space Station mission, has demonstrated the substantial muscle wasting and weakness, along with disruption of muscle's oxidative metabolism. The present work aimed at evaluating the pro/anti-inflammatory status in the same 2 crewmembers (A, B). Blood circulating (c-)microRNAs (miRs), c-proteasome, c-mitochondrial DNA, and cytokines were assessed by real-time quantitative PCR or ELISA tests. Time series analysis was performed ( i.e., before flight and after landing) at 1 and 15 d of recovery (R+1 and R+15, respectively). C-biomarkers were compared with an age-matched control population and with 2-dimensional proteomic analysis of the 2 crewmembers' muscle biopsies. Striking differences were observed between the 2 crewmembers at R+1, in terms of inflamma-miRs (c-miRs-21-5p, -126-3p, and -146a-5p), muscle specific (myo)-miR-206, c-proteasome, and IL-6/leptin, thus making the 2 astronauts dissimilar to each other. Final recovery levels of c-proteasome, c-inflamma-miRs, and c-myo-miR-206 were not reverted to the baseline values in crewmember A. In both crewmembers, myo-miR-206 changed significantly after recovery. Muscle biopsy of astronaut A showed an impressive 80% increase of α-1-antitrypsin, a target of miR-126-3p. These results point to a strong stress response induced by spaceflight involving muscle tissue and the proinflammatory setting, where inflamma-miRs and myo-miR-206 mediate the systemic recovery phase after landing.-Capri, M., Morsiani, C., Santoro, A., Moriggi, M., Conte, M., Martucci, M., Bellavista, E., Fabbri, C., Giampieri, E., Albracht, K., Flück, M., Ruoss, S., Brocca, L., Canepari, M., Longa, E., Di Giulio, I., Bottinelli, R., Cerretelli, P., Salvioli, S., Gelfi, C., Franceschi, C., Narici, M., Rittweger, J. Recovery from 6-month spaceflight at the International Space Station: muscle-related stress into a proinflammatory setting.

Isothermal Titration Calorimetry Enables Rapid Characterization of Enzyme Kinetics and Inhibition for the Human Soluble Epoxide Hydrolase.

Isothermal titration calorimetry (ITC) is conventionally used to acquire thermodynamic data for biological interactions. In recent years, ITC has emerged as a powerful tool to characterize enzyme kinetics. In this study, we have adapted a single-injection method (SIM) to study the kinetics of human soluble epoxide hydrolase (hsEH), an enzyme involved in cardiovascular homeostasis, hypertension, nociception, and insulin sensitivity through the metabolism of epoxy-fatty acids (EpFAs). In the SIM method, the rate of reaction is determined by monitoring the thermal power, while the substrate is being depleted, overcoming the need for synthetic substrates and reducing postreaction processing. Our results show that ITC enables the detailed, rapid, and reproducible characterization of the hsEH-mediated hydrolysis of several natural EpFA substrates. Furthermore, we have applied a variant of the single-injection ITC method for the detailed description of enzyme inhibition, proving the power of this approach in the rapid screening and discovery of new hsEH inhibitors using the enzyme's physiological substrates. The methods described herein will enable further studies on EpFAs' metabolism and biology, as well as drug discovery investigations to identify and characterize hsEH inhibitors. This also promises to provide a general approach for the characterization of lipid catalysis, given the challenges that lipid metabolism studies pose to traditional spectroscopic techniques.

Satureja montana L. essential oil and its antimicrobial activity alone or in combination with gentamicin.

Many essential oils (EOs) are screened as potential sources of antimicrobial compounds. EOs from the genus Satureja have recognized biological properties, including analgesic, anti-inflammatory, immunomodulatory, anticancer, and antimicrobial activity. This study aimed to obtain a metabolite profile of commercial essential oil of S. montana L. (SEO) and to evaluate its antimicrobial properties, both alone and combined with gentamicin towards Gram-negative and Gram-positive bacterial strains. Untargeted analyses based on direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and on GC-MS have provided a high metabolome coverage, allowing to identify carvacrol, cymene and thymol as the major components of commercial SEO. SEO exerted an antimicrobial activity and induced a synergistic interaction with gentamicin against both reference and clinical bacterial strains. A significant reduction of Escherichia coli, Staphylococcus aureus and Listeria monocytogenes biofilm formation was induced by SEO. As a result of SEO treatment, clear morphological bacterial alterations were visualized by scanning electron microscopy: L. monocytogenes and S. aureus showed malformed cell surface or broken cells with pores formation, whereas E. coli displayed collapsed cell surface. These results encourage further studies about bactericidal and antibiotic synergistic effect of SEO for combined therapy in clinical setting as well as in agricultural systems.