Antiproliferative Effect and Cell Cycle Alterations Induced by Salvia officinalis Essential Oil and Its Three Main Components in Human Colon Cancer Cell Lines

Colon cancer is one of the most common human malignancies, and chemotherapy cannot yet prevent recurrence in all patients. Essential oils are phytocomplexes with antiproliferative properties. In this study, we elucidated the antiproliferative properties and the effect on cell cycle progression of Sicilian Salvia officinalis essential oil and its three main compounds, α‐thujone, 1,8‐cineole (eucalyptol) and camphor, on three human colon cancer cell lines. The essential oil was obtained by hydrodistillation and analyzed by gas chromatography. Cell proliferation was evaluated by MTT assay, and the cell cycle distribution was determined by flow cytometry. Thirty‐four compounds were identified in the tested essential oil. Growth inhibition was observed after 72 h, with an impact on cell cycle progression and no effect on the viability of normal colonic epithelial cells. The study shows that S. officinalis essential oil and its three main components have an in vitro antiproliferative effect on colon cancer cells.     

Direct amplification of new cellulase genes from woodland soil purified DNA

Eight genes encoding cellulolytic enzymes were obtained by direct PCR amplification of genomic DNA recovered from woodland soil samples. The direct amplifications were carried out by using primers designed from available online cellulase nucleotide sequences. The isolated genes were all different from each other and homologous to endo-β-1,4-glucanases of Bacillus subtilis. The cellulases were functionally expressed in Escherichia coli and tested on soluble substrate at 37 and 60 °C, showing different cellulolytic activities. Among these, the enzyme renamed CelWS6 exhibited good activity at higher temperatures. Further analysis of CelWS6 showed a high performance in acid environments (between pH 4.0 and 6.0) and at elevated temperatures with its maximum activity at pH 5.0 and 50 °C. At the optimum pH, it was very stable since more than 80 % of its original activity was maintained after an incubation of 120 min at 60 °C. Because the cellulases had different cellulolytic activities, but similar amino acid sequences, it was possible to assess the relationship between sequence and protein function.     

Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.     

The N-BAR domain protein,Bin3, regulates Rac1- and Cdc42-dependent processes in myogenesis

Actin dynamics are necessary at multiple steps in the formation of multinucleated muscle cells. BAR domain proteins can regulate actin dynamics in several cell types, but have been little studied in skeletal muscle. Here, we identify novel functions for the N-BAR domain protein, Bridging integrator 3 (Bin3), during myogenesis in mice. Bin3 plays an important role in regulating myofiber size in vitro and in vivo. During early myogenesis, Bin3 promotes migration of differentiated muscle cells, where it colocalizes with F-actin in lamellipodia. In addition, Bin3 forms a complex with Rac1 and Cdc42, Rho GTPases involved in actin polymerization, which are known to be essential for myotube formation. Importantly, a Bin3-dependent pathway is a major regulator of Rac1 and Cdc42 activity in differentiated muscle cells. Overall, these data classify N-BAR domain proteins as novel regulators of actin-dependent processes in myogenesis, and further implicate BAR domain proteins in muscle growth and repair.     

Clerocidin-mediated DNA footprinting discriminates among different G-quadruplex conformations and detects tetraplex folding in a duplex environment

Background

G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide.

Methods

Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer.

Results

The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlights stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context.

Conclusions

CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions.

General significance

Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions.     

Inhibition of bromodomain and extra‐terminal proteins increases sensitivity to venetoclax in chronic lymphocytic leukaemia

The development of drugs able to target BTK, PI3k‐delta and BCL2 has dramatically improved chronic lymphocytic leukaemia (CLL) therapies. However, drug resistance to these therapies has already been reported due to non‐recurrent changes in oncogenic pathways and genes expression signatures. In this study, we investigated the cooperative role of the BCL2 inhibitor venetoclax and the BRD4 inhibitor JQ1. In particular, we found that JQ1 shows additional activity with venetoclax, in CLL cell lines and in ex vivo isolated primary CD19⁺ lymphocytes, arguing in favour of combination strategies. Lastly, JQ1 is also effective in venetoclax‐resistant CLL cell lines. Together, our findings indicated that the BET inhibitor JQ1 could be a promising therapy in CLL, both as first‐line therapy in combination with venetoclax and as second‐line therapy, after the emergence of venetoclax‐resistant clones.     

Altered modulation of lamin A/C‐HDAC2 interaction and p21 expression during oxidative stress response in HGPS

Defects in stress response are main determinants of cellular senescence and organism aging. In fibroblasts from patients affected by Hutchinson–Gilford progeria, a severe LMNA‐linked syndrome associated with bone resorption, cardiovascular disorders, and premature aging, we found altered modulation of CDKN1A, encoding p21, upon oxidative stress induction, and accumulation of senescence markers during stress recovery. In this context, we unraveled a dynamic interaction of lamin A/C with HDAC2, an histone deacetylase that regulates CDKN1A expression. In control skin fibroblasts, lamin A/C is part of a protein complex including HDAC2 and its histone substrates; protein interaction is reduced at the onset of DNA damage response and recovered after completion of DNA repair. This interplay parallels modulation of p21 expression and global histone acetylation, and it is disrupted by LMNAmutations leading to progeroid phenotypes. In fact, HGPS cells show impaired lamin A/C‐HDAC2 interplay and accumulation of p21 upon stress recovery. Collectively, these results link altered physical interaction between lamin A/C and HDAC2 to cellular and organism aging. The lamin A/C‐HDAC2 complex may be a novel therapeutic target to slow down progression of progeria symptoms.     

Targeting Synaptic Pathology with a Novel Affinity Mass Spectrometry Approach

We report a novel strategy for studying synaptic pathology by concurrently measuring levels of four SNARE complex proteins from individual brain tissue samples. This method combines affinity purification and mass spectrometry and can be applied directly for studies of SNARE complex proteins in multiple species or modified to target other key elements in neuronal function. We use the technique to demonstrate altered levels of presynaptic proteins in Alzheimer disease patients and prion-infected mice.One prominent pathological feature of neuropsychiatric disorders such as Alzheimer disease (AD)¹ is severe synaptic loss (1–3). Previous reports of AD patients have shown that presynaptic dysfunction might occur early in the disease process (1, 4). Cortical synapse pathology has also been shown to correlate to the severity of dementia more closely than other pathological hallmarks of AD such as plaques and neurofibrillary tangles (5, 6). The SNARE proteins are essential components for the regulation of neurotransmitter exocytosis at the presynaptic site (7). Animal models suggest that changed expression or modification of SNARE complex proteins (synaptosomal-associated protein 25 (SNAP-25), syntaxin-1, and vesicle-associated membrane protein (VAMP)) alters synaptic function and is an interesting target for the development of therapeutics for neuropsychiatric illness (8, 9). The constituents of the SNARE complex are either localized in synaptic vesicles (VAMPs) or anchored at the presynaptic plasma membrane (SNAP-25 and syntaxin). The SNARE proteins are tightly assembled, and subsequent neurotransmitter release of the complex is quickly dissociated by N-ethylmaleimide-sensitive factor (7, 10–12). Because they are both strongly associated into complexes and membrane associated, the SNARE proteins are difficult to analyze via mass spectrometry, which is incompatible with most detergents necessary for the solubilization of proteins. Each SNARE complex protein exists in several isoforms that are differently distributed within the central nervous system (13–18). Post-translational modifications and truncated variants of the SNARE proteins make investigation of the protein expression even more complicated.In this study we developed an approach for the characterization and concurrent quantification of SNARE complex proteins that combines affinity purification by immunoprecipitation and mass spectrometry (IP-MS). We used precipitation with monoclonal antibodies against SNAP-25 to target the SNARE complex proteins and nanoflow LC–tandem mass spectrometry (LC-MS/MS) to characterize the co-immunoprecipitated interaction partners. Selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer coupled to a microflow LC system was used for quantification of the SNARE proteins. To demonstrate the usability of the IP-MS method, we performed a comparison of SNARE complex protein levels in brain tissue from AD patients and age-matched controls, as well as a study of SNARE complex protein levels in brain tissue from prion-infected mice.