A Perspective on the Experimental Techniques for Studying Lamins

3Lamins are type V intermediate filaments that collectively form a meshwork underneath the inner nuclear membrane, called nuclear lamina. Furthermore, they are also present in the nucleoplasm. Lamins are experiencing a growing interest, since a wide range of diseases are induced by mutations in the gene coding for A-type lamins, globally known as laminopathies. Moreover, it has been demonstrated that lamins are involved in other pathological conditions, like cancer. The role of lamins has been studied from several perspectives, exploiting different techniques and procedures. This multidisciplinary approach has contributed to resolving the unique features of lamins and has provided a thorough insight in their role in living organisms. Yet, there are still many unanswered questions, which constantly generate research in the field. The present work is aimed to review some interesting experimental techniques performed so far to study lamins. Scientists can take advantage of this collection for their novel investigations, being aware of the already pursued and consolidated methodologies. Hopefully, advances in these research directions will provide insights to achieve better diagnostic procedures and effective therapeutic options.openopenPecorari, Ilaria; Borin, Daniele; Sbaizero, OrfeoPecorari, Ilaria; Borin, Daniele; Sbaizero, Orfe

An impaired metabolic response to hydrostatic pressure explains Alcanivorax borkumensis recorded distribution in the deep marine water column

Alcanivorax borkumensis is an ubiquitous model organism for hydrocarbonoclastic bacteria, which dominates polluted surface waters. Its negligible presence in oil-contaminated deep waters (as observed during the Deepwater Horizon accident) raises the hypothesis that it may lack adaptive mechanisms to hydrostatic pressure (HP). The type strain SK2 was tested under 0.1, 5 and 10 MPa (corresponding to surface water, 500 and 1000 m depth, respectively). While 5 MPa essentially inactivated SK2, further increase to 10 MPa triggered some resistance mechanism, as indicated by higher total and intact cell numbers. Under 10 MPa, SK2 upregulated the synthetic pathway of the osmolyte ectoine, whose concentration increased from 0.45 to 4.71 fmoles cell(-1). Central biosynthetic pathways such as cell replication, glyoxylate and Krebs cycles, amino acids metabolism and fatty acids biosynthesis, but not beta-oxidation, were upregulated or unaffected at 10 MPa, although total cell number was remarkably lower with respect to 0.1 MPa. Concomitantly, expression of more than 50% of SK2 genes was downregulated, including genes related to ATP generation, respiration and protein translation. Thus, A. borkumensis lacks proper adaptation to HP but activates resistance mechanisms. These consist in poorly efficient biosynthetic rather than energy-yielding degradation-related pathways, and suggest that HP does represent a major driver for its distribution at deep-sea

An engineering insight into the relationship of selective cytoskeletal impairment and biomechanics of HeLa cells

It is widely accepted that the pathological state of cells is characterized by a modification of mechanical properties, affecting cellular shape and viscoelasticity as well as adhesion behaviour and motility. Thus, assessing these parameters could represent an interesting tool to monitor disease development and progression, but also the effects of drug treatments. Since biomechanical properties of cells are strongly related to cytoskeletal architecture, in this work we extensively studied the effects of selective impairments of actin microfilaments and microtubules on HeLa cells through force-deformation curves and stress relaxation tests with atomic force microscopy. Confocal microscopy was also used to display the effects of the used drugs on the cytoskeletal structure. In synergy with the aforementioned methods, stress relaxation data were used to assess the storage and loss moduli, as a complementary way to describe the influence of cytoskeletal components on cellular viscoelasticity. Our results indicate that F-actin and microtubules play a complementary role in the cell stiffness and viscoelasticity, and both are fundamental for the adhesion properties. Our data support also the application of biomechanics as a tool to study diseases and their treatments

Bacterial community structure and diversity along the halocline of Tyro deep-sea hypersaline anoxic basin

Purpose: Tyro is a deep hypersaline anoxic basin (DHAB) located at the seafloor of the Eastern Mediterranean sea. Tyro hosts a stratified eukaryotic microbiome moving from seawater to the brine, but no reports are available on its prokaryotic community. We provide the first snapshot of the bacterial community structure in Tyro brine, seawaterbrine interface, and the overlaying deep seawater. Methods: In this study, we combined the use of molecular analyses, i.e., DNA fingerprinting and 16S rRNA pyrosequencing for the description of the bacterial community structure and taxonomy. PiCRUST2 was used to infer information on the prokaryotes functional diversity. A culture-dependent approach was applied to enrich bacteria of interest for marine biotechnology. Results: Bacterial communities sharply clustered moving from the seawater to the Tyro brine, in agreement with the abrupt increase of salinity values. Moreover, specific taxonomic groups inhabited the seawater-brine interface compared to the overlaying seawater and their identification revealed converging taxonomy with other DHABs in the Eastern Mediterranean sea. Functional traits inferred from the prokaryote taxonomy in the upper interface and the overlaying seawater indicated metabolic pathways for the synthesis of osmoprotectants, likely involved in bacterial adaptation to the steep increasing salinity. Metabolic traits related to methane and methylated compounds and to hydrocarbon degradation were also revealed in the upper interface of Tyro. The overall capability of the Tyro microbiome for hydrocarbon metabolism was confirmed by the isolation of hydrocarbonoclastic bacteria in the sediments. Conclusions: Our results suggest that Tyro seawater-brine interface hosts a specific microbiome adapted to the polyextreme condition typical of DHABs with potential metabolic features that could be further explored for the characterization of the metabolic network connecting the brine with the deep seawater through the chemocline. Moreover, Tyro could be a reservoir of culturable microbes endowed with functionalities of interest for biotechnological applications like hydrocarbon bioremediation

Fluorescent-BOX-PCR for resolving bacterial genetic diversity, endemism and biogeography

<p>Abstract</p> <p>Background</p> <p>BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) is one of the most used techniques in biogeography studies of microbial isolates. However the traditional separation of BOX-PCR patterns by agarose gel electrophoresis suffers many limitations. The aim of this research was to set up a fluorescent BOX-PCR (F-BOX-PCR) assay in which separation of PCR products is automated in a capillary electrophoresis system. F-BOX-PCR was compared with the traditional BOX-PCR using bacterial strains with different G+C content (<it>Bacillus cereus</it>; <it>Escherichia coli</it>; isolates of the family <it>Geodermatophilaceae</it>). Resolution, discriminatory power and reproducibility were evaluated by assaying different electrophoretic runs, PCR reactions and independent DNA extractions. BOX-PCR and F-BOX-PCR were compared for the analysis of 29 strains of <it>Modestobacter multiseptatus </it>isolated from three different microsites in an altered carbonatic wall from Cagliari, Italy, and 45 strains of <it>Streptococcus thermophilus </it>isolated from 34 samples of the hand-made, yogurt-like product Matsoni, collected in different locations in Georgia.</p> <p>Results</p> <p>Fluorophore 6-FAM proved more informative than HEX and BOX-PCR both in agarose gel electrophoresis (<it>p </it>< 0.004 and <it>p </it>< 0.00003) and in capillary electrophoresis (compared only with HEX, <it>p </it>< 2 × 10^-7). 6-FAM- and HEX-based F-BOX-PCR respectively detected up to 12.0 and 11.3 times more fragments than BOX-PCR. Replicate separations of F-BOX-PCR showed an accuracy of the size calling of ± 0.5 bp until 500 bp, constantly decreasing to ± 10 bp at 2000 bp. Cluster analysis of F-BOX-PCR profiles grouped <it>M. multiseptatus </it>strains according to the microsite of isolation and <it>S. thermophilus </it>strains according to the geographical origin of Matsoni, but resulted intermixed when a BOX-PCR dataset was used.</p> <p>Conclusion</p> <p>F-BOX-PCR represents an improved method for addressing bacterial biogeography studies both in term of sensitivity, reproducibility and data analysis.</p

Cellular Biomechanic Impairment in Cardiomyocytes Carrying the Progeria Mutation: An Atomic Force Microscopy Investigation

Given the clinical effect of progeria syndrome, understanding the cell mechanical behavior of this pathology could benefit the patient's treatment. Progeria patients show a point mutation in the lamin A/C gene (LMNA), which could change the cell's biomechanical properties. This paper reports a mechano-dynamic analysis of a progeria mutation (c.1824 C > T, p.Gly608Gly) in neonatal rat ventricular myocytes (NRVMs) using cell indentation by atomic force microscopy to measure alterations in beating force, frequency, and contractile amplitude of selected cells within cell clusters. Furthermore, we examined the beating rate variability using a time-domain method that produces a Poincaré plot because beat-to-beat changes can shed light on the causes of arrhythmias. Our data have been further related to our cell phenotype findings, using immunofluorescence and calcium transient analysis, showing that mutant NRVMs display changes in both beating force and frequency. These changes were associated with a decreased gap junction localization (Connexin 43) in the mutant NRVMs even in the presence of a stable cytoskeletal structure (microtubules and actin filaments) when compared with controls (wild type and non-treated cells). These data emphasize the kindred between nucleoskeleton (LMNA), cytoskeleton, and the sarcolemmal structures in NRVM with the progeria Gly608Gly mutation, prompting future mechanistic and therapeutic investigations

Biomechanical defects and rescue of cardiomyocytes expressing pathologic nuclear lamins

Given the clinical impact of LMNA cardiomyopathies, understanding lamin function will fulfill a clinical need and will lead to advancement in the treatment of heart failure. A multidisciplinary approach combining cell biology, atomic force microscopy (AFM) and molecular modeling was used to analyze the biomechanical properties of human lamin A/C gene (LMNA) mutations (E161K, D192G, N195K) using an in vitro neonatal rat ventricular myocyte (NRVM) model

Hydrolytic Profile of the Culturable Gut Bacterial Community Associated With Hermetia illucens

Larvae of the black soldier fly (BSF) Hermetia illucens (L.) convert organic waste into high valuable insect biomass that can be used as alternative protein source for animal nutrition or as feedstock for biodiesel production. Since insect biology and physiology are influenced by the gut microbiome, knowledge about the functional role of BSF-associated microorganisms could be exploited to enhance the insect performance and growth. Although an increasing number of culture-independent studies are unveiling the microbiota structure and composition of the BSF gut microbiota, a knowledge gap remains on the experimental validation of the contribution of the microorganisms to the insect growth and development. We aimed at assessing if BSF gut-associated bacteria potentially involved in the breakdown of diet components are able to improve host nutrition. A total of 193 bacterial strains were obtained from guts of BSF larvae reared on a nutritious diet using selective and enrichment media. Most of the bacterial isolates are typically found in the insect gut, with major representatives belonging to the Gammaproteobacteria and Bacilli classes. The hydrolytic profile of the bacterial collection was assessed on compounds typically present in the diet. Finally, we tested the hypothesis that the addition to a nutritionally poor diet of the two isolates Bacillus licheniformis HI169 and Stenotrophomonas maltophilia HI121, selected for their complementary metabolic activities, could enhance BSF growth. B. licheniformis HI169 positively influenced the larval final weight and growth rate when compared to the control. Conversely, the addition of S. maltophilia HI121 to the nutritionally poor diet did not result in a growth enhancement in terms of larval weight and pupal weight and length in comparison to the control, whereas the combination of the two strains positively affected the larval final weight and the pupal weight and length. In conclusion, we isolated BSF-associated bacterial strains with potential positive properties for the host nutrition and we showed that selected isolates may enhance BSF growth, suggesting the importance to evaluate the effect of the bacterial administration on the insect performance

Cell phenotype changes and oxidative stress response in Vibrio spp. induced into viable but non-culturable (VBNC) state

Purpose: Aquatic bacteria of the genus Vibrio include animal and human pathogens. The occurrence of Vibrio-related diseases has been associated with the current climate change-driven increase of sea surface temperature. Vibrio spp. can enter into the viable but non-culturable (VBNC) state, as a consequence of starvation in seawater at low temperatures. In such physiological state, Vibrio cells are no longer culturable on standard media agar plates but can resuscitate if incubated at 30 °C prior to plating, retaining virulence. Since limited information is available on regards to this topic, in this work, we characterized the phenotypic changes of four Vibrio spp. strains (one laboratory strain and three environmental isolates) in cold seawater microcosms, investigating the relationship between resuscitation and a hydrogen peroxide-induced oxidative stress. Methods: Cell phenotypic changes and the effect of hydrogen peroxide and/or catalase addition to the medium were studied on VBNC and resuscitated cells by flow cytometry in microcosm experiments, paralleled by culturability experiments by plating. Results: The cells of all the Vibrio strains changed their phenotype upon the induction of the VBNC state resulting in cell dwarfing and decrease in DNA quantity, losing the ability to grow on solid media. These features were partially or totally reverted when the cells were treated for resuscitation. Hydrogen peroxide at concentrations as low as 0.007 mM prevented resuscitation and a prolonged exposure to hydrogen peroxide at concentrations far under those inhibiting the growth of log-phase cells permanently damaged VBNC cells, which could not be resuscitated. However, the potential of culturability of VBNC cells could be preserved, at least for a part of the population, by plating the cells in the presence of catalase. The study also showed that during the resuscitation process, the cells gradually increased their resistance to hydrogen peroxide. Conclusions: The timing and mode of induction of the VBNC state, as well as cell resuscitation and response to hydrogen peroxide, differed among Vibrio strains, indicating that induction and resuscitation from dormancy could vary in the context of species belonging to a single genus