Profiling metabolites and lipoproteins in COMETA,an Italian cohort of COVID-19 patients

Metabolomics and lipidomics have been used in several studies to define the biochemical alterations induced by COVID-19 in comparison with healthy controls. Those studies highlighted the presence of a strong signature, attributable to both metabolites and lipoproteins/lipids. Here, ¹H NMR spectra were acquired on EDTA-plasma from three groups of subjects: i) hospitalized COVID-19 positive patients (≤21 days from the first positive nasopharyngeal swab); ii) hospitalized COVID-19 positive patients (>21 days from the first positive nasopharyngeal swab); iii) subjects after 2–6 months from SARS-CoV-2 eradication. A Random Forest model built using the EDTA-plasma spectra of COVID-19 patients ≤21 days and Post COVID-19 subjects, provided a high discrimination accuracy (93.6%), indicating both the presence of a strong fingerprint of the acute infection and the substantial metabolic healing of Post COVID-19 subjects. The differences originate from significant alterations in the concentrations of 16 metabolites and 74 lipoprotein components. The model was then used to predict the spectra of COVID-19>21 days subjects. In this group, the metabolite levels are closer to those of the Post COVID-19 subjects than to those of the COVID-19≤21 days; the opposite occurs for the lipoproteins. Within the acute phase patients, characteristic trends in metabolite levels are observed as a function of the disease severity. The metabolites found altered in COVID-19≤21 days patients with respect to Post COVID-19 individuals overlap with acute infection biomarkers identified previously in comparison with healthy subjects. Along the trajectory towards healing, the metabolome reverts back to the “healthy” state faster than the lipoproteome.     

The influence of dietary vitamin C and E supplementation on the physiological response of pirarucu, Arapaima gigas, in net culture

This study evaluated the efficacy of dietary vitamin C (ascorbic acid or AA), vitamin E (alpha-tocopherol or alpha-T), and C+E supplementation on the blood parameters of Arapaima gigas grown in net cages for 45 days. Four treatments were tested: control (commercial feed); C800; E500 and C+E (800+500) with supplementation of 800 mg AA kg(-1), 500 mg alpha-T kg(-1) and 800+500 mg AA+alpha-T kg(-1), respectively. Hematocrit (Ht), red blood cells (RBC), and hemoglobin concentration (Hb) (oxidative status indicators), thrombocytes and leukocytes (immunological indicators), plasma protein and glucose were evaluated. Fish fed vitamin C and C+E supplemented diets showed greater weight gain and survival. Dietary vitamin C and C+E diet supplementation resulted in increased Ht, Hb, RBC, MCHC, total leukocytes, total proteins, thrombocytes and eosinophils compared to the control and alpha-T. The alpha-tocopherol-supplemented diet reduced the number of total thrombocytes, lymphocytes and neutrophils and increased glucose and eosinophils relatively to the control. In general, leukocytes and thrombocytes were good indicators of the efficiency of vitamin on the defense mechanism of the A. gigas reared in cages. Results indicate that high alpha-T diet supplementation provides no benefit for the maintenance of the oxidative or the immunological status of A. gigas. However, it was demonstrated that high dietary AA improves A. gigas immunological status. Red blood cell indices and immune system indicators showed no synergistic effect between the vitamins after supplementing the A. gigas diet with alpha-T+AA.     

Kinetic properties of native and mutagenized isoforms of mitochondrial alcohol dehydrogenase III purified from Kluyveromyces lactis

By computer modelling and protein engineering we have investigated changes in two amino acid residues located in the coenzyme pocket of the yeast Kluyveromyces lactis mitochondrial alcohol dehydrogenase III. These two residues, Gly 225 and Ala 274, were hypothesized to be involved in the enzyme discrimination between NAD(H) and NADP(H). Upon changing Gly 225 to Ala we produced an enzyme (mutant G225A) showing very little difference from the wild-type. On the contrary, change at position 274 of Phe instead of Ala (mutant A274F) caused a significant increase of K(m) values for NAD(P) and for NADPH and even a more marked decrease in catalytic activity. The k(cat)/K(m) rates for NADP(H) were also decreased in this mutant. Enzymes with the double changes at 225 and 274 (mutant G225A-A274F) showed, apart the substantial low K(m) value for NADPH and its high catalytic efficiency, kinetic parameters relative to coenzymes which were not additive over the single substitutions. Surprisingly, enzymes with changes at the two positions reduced efficiently acetaldehyde, displaying a K(m) value 10-fold lower and a catalytic efficiency sevenfold higher with respect to parent or singularly mutated enzymes. None of the engineered enzymes would convert formaldehyde, glutaraldehyde or aromatic aldehydes but all enzymes reduced propionaldehyde and butyraldehyde at relative reaction rates approximately half of that exhibited by acetaldehyde. Interestingly only mutant A274F was able to oxidize methanol almost as well as ethanol. In addition, this mutant was capable to convert secondary and cyclic alcohols, at a rate not detected in the other isoforms. These results are in general agreement with the prediction that increasing the size of amino acids in the proximity of the coenzyme pocket would hamper the accommodation of NADP but discord the increased affinity for NADPH as well as for alcoholic or aldehydic substrates with high steric hindrance.     

Nuclear reformation after mitosis requires downregulation of the Ran GTPase effector RanBP1 in mammalian cells

The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.     

Proteolysin,a Novel Highly Thermostable and Cosolvent-Compatible Protease from the Thermophilic Bacterium Coprothermobacter proteolyticus

Through genome mining, we identified a gene encoding a putative serine protease of the thermitase subgroup of subtilases (EC 3.4.21.66) in the thermophilic bacterium Coprothermobacter proteolyticus. The gene was functionally expressed in Escherichia coli, and the enzyme, which we called proteolysin, was purified to near homogeneity from crude cell lysate by a single heat treatment step. Proteolysin has a broad pH tolerance and is active at temperatures of up to 80°C. In addition, the enzyme shows good activity and stability in the presence of organic solvents, detergents, and dithiothreitol, and it remains active in 6 M guanidinium hydrochloride. Based on its stability and activity profile, proteolysin can be an excellent candidate for applications where resistance to harsh process conditions is required.     

Kidins220/ARMS is transported by a kinesin-1-based mechanism likely to be involved in neuronal differentiation

Kinase D-interacting substrate of 220 kDa/ankyrin repeat-rich membrane spanning (Kidins220/ARMS) is a conserved membrane protein mainly expressed in brain and neuroendocrine cells, which is a downstream target of the signaling cascades initiated by neurotrophins and ephrins. We identified kinesin light chain 1 (KLC1) as a binding partner for Kidins220/ARMS by a yeast two-hybrid screen. The interaction between Kidins220/ARMS and the kinesin-1 motor complex was confirmed by glutathione S-transferase-pull-down and coimmunoprecipitation experiments. In addition, Kidins220/ARMS and kinesin-1 were shown to colocalize in nerve growth factor (NGF)-differentiated PC12 cells. Using Kidins220/ARMS and KLC1 mutants, we mapped the regions responsible for the binding to a short sequence of Kidins220/ARMS, termed KLC-interacting motif (KIM), which is sufficient for the interaction with KLC1. Optimal binding of KIM requires a region of KLC1 spanning both the tetratricopeptide repeats and the heptad repeats, previously not involved in cargo recognition. Overexpression of KIM in differentiating PC12 cells impairs the formation and transport of EGFP-Kidins220/ARMS carriers to the tips of growing neurites, leaving other kinesin-1 dependent processes unaffected. Furthermore, KIM overexpression interferes with the activation of the mitogen-activated protein kinase signaling and neurite outgrowth in NGF-treated PC12 cells. Our results suggest that Kidins220/ARMS-positive carriers undergo a kinesin-1-dependent transport linked to neurotrophin action.     

Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.     

A Soap Technique for Cell Separation to Study the Seed Coat of Sesbania punicea

A technique is described for separating plant cells used for morphological studies. The plant material is placed in a concentrated solution of olive oil castile soap for 1-2 days or more. The material is then thoroughly washed and placed between two glass slides. The upper glass slide is lifted from the lower one, then gently pressed down several times. Through this procedure Malpighian cells of the seed coat of Sesbania punicea. mesophyll cells of Euphorbia peplus and of Trifolium pratense and cortical cells of the aerial roots of Monstera deliciosa have been separated. Various shapes of the Malpighian cells of the Sesbania punicea seed coat can be observed along with intermediates.     

Effect of the leader sequence on the expression of recombinant C. rugosa lipase by S. cerevisiae cells

Summary The Candida rugosa lipase I gene has been expressed in Saccharomyces cerevisiae. The recombinant lipase was efficiently synthesized only following the replacement of the enzyme endogenous leader sequence with the signal peptide of the Kluyveromyces lactis killer toxin. Amount of accumulated lipase was about 10–20 mg/l in batch culture and over 1g/l in a computer-controlled fed-batch fermentation system.