Modeling the endosomal escape of cell-penetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers

Cell-penetrating peptides (CPPs) are able to mediate the efficient cellular uptake of a wide range of cargoes. Internalization of a number of CPPs requires uptake by endocytosis, initiated by binding to anionic cell surface heparan sulfate (HS), followed by escape from endosomes. To elucidate the endosomal escape mechanism, we have modeled the process for two CPPs: penetratin (pAntp) and the N-terminal signal peptide of the unprocessed bovine prion protein (bPrPp). Large unilamellar phospholipid vesicles (LUVs) were produced encapsulating either peptide, and an ionophore, nigericin, was used to create a transmembrane pH gradient (DeltapH(mem), inside acidic) similar to the one arising in endosomes in vivo. In the absence of DeltapH(mem), no pAntp escape from the LUVs is observed, while a fraction of bPrPp escapes. In the presence of DeltapH(mem), a significant amount of pAntp escapes and an even higher degree of bPrPp escape takes place. These results, together with the differences in kinetics of escape, indicate different escape mechanisms for the two peptides. A minimum threshold peptide concentration exists for the escape of both peptides. Coupling of the peptides to a cargo reduces the fraction escaping, while complexation with HS significantly hinders the escape. Fluorescence correlation spectroscopy results show that during the escape process the LUVs are intact. Taken together, these results suggest a model for endosomal escape of CPPs: DeltapH(mem)-mediated mechanism, following dissociation from HS of the peptides, above a minimum threshold peptide concentration, in a process that does not involve lysis of the vesicles.     

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Loss or inactivation of BLM, a helicase of the RecQ family, causes Bloom syndrome, a genetic disorder with a strong predisposition to cancer. Although the precise function of BLM remains unknown, genetic data has implicated BLM in the process of genetic recombination and DNA repair. Previously, we demonstrated that BLM can disrupt the RAD51-single-stranded DNA filament that promotes the initial steps of homologous recombination. However, this disruption occurs only if RAD51 is present in an inactive ADP-bound form. Here, we investigate interactions of BLM with the active ATP-bound form of the RAD51-single-stranded DNA filament. Surprisingly, we found that BLM stimulates DNA strand exchange activity of RAD51. In contrast to the helicase activity of BLM, this stimulation does not require ATP hydrolysis. These data suggest a novel BLM function that is stimulation of the RAD51 DNA pairing. Our results demonstrate the important role of the RAD51 nucleoprotein filament conformation in stimulation of DNA pairing by BLM.Mutations of BLM helicase cause Bloom syndrome (BS),² a rare autosomal disorder, which is associated with stunted growth, facial sun sensitivity, immunodeficiency, fertility defects, and a greatly elevated incidence of many types of cancer occurring at an early age (1). BLM belongs to the highly conserved family of RecQ helicases that are required for the maintenance of genome integrity in all organisms (2, 3). There are five RecQ helicases in humans; mutations in three of them, WRN, RECQ4, and BLM, have been associated with the genetic abnormalities known as Werner, Rothmund-Thomson, and Bloom syndrome, respectively (4, 5). The cells from BS patients display genomic instability; the hallmark of BS is an increase in the frequency of sister chromatid and interhomolog exchanges (1, 6). Because homologous recombination (HR) is responsible for chromosomal exchanges, it is thought that BLM helicase functions in regulating HR (7–9). Also, BLM helicase is required for faithful chromosome segregation (10) and repair of stalled replication forks (11, 12), the processes that are linked to HR (13–15). BLM was found to interact physically with RAD51, a key protein of HR (16) that catalyzes the central steps in HR including the search for homology and the exchange of strands between homologous ssDNA and dsDNA sequences (17). In cells, BLM forms nuclear foci, a subset of which co-localize with RAD51. Interestingly, the extent of RAD51 and BLM co-localization increases in response to ionizing radiation, indicating a possible role of BLM in the repair of DNA double-strand breaks (16).Biochemical studies suggest that BLM may perform several different functions in HR. BLM was shown to promote the dissociation of HR intermediates (D-loops) (18–20), branch migration of Holliday junctions (21), and dissolution of double Holliday junctions acting in a complex with TopoIIIα and BLAP75 (22–24). BLM may also facilitate DNA synthesis during the repair process by unwinding the DNA template in front of the replication fork (25). In addition, BLM and its yeast homolog Sgs1 may play a role at the initial steps of DNA double-strand break repair by participating in exonucleolitic resection of the DNA ends to generate DNA molecules with the 3′-ssDNA tails, a substrate for RAD51 binding (26–29).In vivo, the process of HR is tightly regulated by various mechanisms (30). Whereas some proteins promote HR (14, 31), others inhibit this process, thereby preventing its untimely initiation (32, 33). Disruption of the Rad51-ssDNA nucleoprotein filament appears to be an especially important mechanism of controlling HR. This filament disruption activity was demonstrated for the yeast Srs2 helicase (34, 35) and human RECQ5 helicase (36). Recently, we found that BLM can also catalyze disruption of the RAD51-ssDNA filament (25). This disruption only occurs if the filament is present in an inactive ADP-bound form, e.g. in the presence of Mg²⁺. Conversion of RAD51 into an active ATP-bound form, e.g. in the presence of Ca²⁺ (37), renders the filament resistant to BLM disruption (25). In this study, we analyze the interactions of BLM with an active ATP-bound RAD51-ssDNA filament. Surprisingly, we found that BLM stimulates the DNA strand exchange activity of RAD51. Thus, depending on the conformational state of the RAD51 nucleoprotein filament, BLM may either inhibit or stimulate the DNA strand exchange activity of RAD51. Our analysis demonstrated that, in contrast to several known stimulatory proteins that act by promoting formation of the RAD51-ssDNA filament, BLM stimulates the DNA strand exchange activity of RAD51 at a later stage, during synapsis. Stimulation appears to be independent of the ATPase activity of BLM. We suggest that this stimulation of RAD51 may represent a novel function of BLM in homologous recombination.     

Analysis of the structure of DNA cistrons coding proteins with known sequences]

Proceeding from the amino acid sequence of a number of proteins, with the help of a special computer program we have determined the frequency of pyrimidine isopliths of different length, the degree of clustering and the degree of asymmetry of complementary chains of the corresponding DNA cistrons, as well as the range of variation of these parametres which depends on the code degeneracy. The degree of asymmetry of the chains of DNA cistrons (H/L), calculated for 255 proteins of a known composition, may vary from 0.7 to 1.8. For 90% of these proteins the mean Py/Pu ratio in the coding chain of DNA is above 1. The conclusion has been made that the majority of amino acids contained in the proteins is coded for by purine triplets. It was found that the distribution of pyrimidine isopliths between DNA cistrons coding for different proteins is other than random and has a "DNA-like" character. The degree of clustering of pyrimidines (beta) in cistrons of different proteins may vary from 6.0 to 14.3. The cistrons of some proteins were found to contain long lyrimidine fragments with about 24 residues. A positive correlation (r2 = 0.74) was found to exist between the degree of clustering of pyrimidines and the degree of asymmetry of the chains corresponding to different proteins of DNA cistrons.     

Study of oral microbiota diversity among groups of families originally from different countries

The diversity of oral microbiota is affected by diets habits, gender, age, ethnic group, and environment. The acquisition of oral microbiota and the role of family on oral microbiota development is poorly understood. This study aims to characterize and compare the oral bacterial microbiota among families using 16S rRNA gene sequencing. This work was conducted in Jeddah city from 2020 to 2021, in which four families composed of 20 members of different ethnicity and lifestyle were recruited. After the collection of saliva samples, the DNA was extracted and processed for 16S rRNA gene metagenomics sequencing. Among 378 OUTs generated, 39 (10.3%) were unique in group A, 13 (3.4%) unique in group B, and 11 (2.9%) were unique in groups C and D. We observed a significant variation at the level of top abundance phylum (14), families (23), genera (24), and species (22) of bacteria among family members. Within family groups, different bacterial species were reported to be more dominant among certain family members than the other; Prevotella melaninogenica, Prevotella histicola and Haemophilus parainfluenzae, Veillonella atypica, Porphyromonas pasteri and Haemophilus pittmaniae were more dominant in parents of some families than the other family member. In summary, this study highlights the precise and perceptible association of oral microbial between family members. Our findings documented the clustering of certain bacterial species in family groups, supporting the role of community in the development of oral microbiota.     

δ18O‐derived incubation temperatures of oviraptorosaur eggs

In order to determine the incubation temperature of eggs laid by non‐avian dinosaurs, we analysed the oxygen isotope compositions of both eggshell carbonate (δ¹⁸O_c) and embryo bone phosphate (δ¹⁸O_p) from seven oviraptorosaur eggs with preserved in ovo embryo bones. These eggs come from the Upper Cretaceous Nanxiong Formation of Jiangxi Province, China. Oviraptorosaur theropods were selected because of their known brooding behaviour as evidenced by preserved adult specimens fossilized in brooding posture on their clutch. Incubation temperature of these embryos was estimated based on the following considerations: eggshell δ¹⁸O_c value reflects the oxygen isotope composition of egg water fluid; embryo bones precipitate from the same egg fluid; and oxygen isotope fractionation between phosphate and water is controlled by the egg temperature. A time‐dependent model predicting the δ¹⁸O_p evolution of the embryo skeleton during incubation as a function of egg temperature was built, and measured δ¹⁸O_c and δ¹⁸O_p values used as boundary conditions. According to the model outputs, oviraptorosaurs incubated their eggs within a 35–40°C range, similar to extant birds and compatible with the known active brooding behaviour of these theropod dinosaurs. Provided that both eggshell and embryo bones preserved their original oxygen isotope compositions, this method could be extended to investigate some reproductive traits of other extinct groups of oviparous amniotes.     

Gallium(III) complexes with 2-acetylpyridine-derived thiosemicarbazones: antimicrobial and cytotoxic effects and investigation on the interactions with tubulin

Complexes [Ga(2Ac4pFPh)₂]NO₃ (1), [Ga(2Ac4pClPh)₂]NO₃ (2), [Ga(2Ac4pIPh)₂]NO₃ (3), [Ga(2Ac4pNO₂Ph)₂]NO₃·3H₂O (4) and [Ga(2Ac4pT)₂]NO₃ (5) were obtained with 2-acetylpyridine N(4)-para-fluorophenyl-(H2Ac4pFPh), 2-acetylpyridine N(4)-para-chlorophenyl-(H2Ac4pClPh), 2-acetylpyridine N(4)-para-iodophenyl-(H2Ac4pIPh), 2-acetylpyridine N(4)-para-nitrophenyl-(H2Ac4pNO₂Ph) and 2-acetylpyridine N(4)-para-tolyl-(H2Ac4pT) thiosemicarbazone. 1–5 presented antimicrobial and cytotoxic properties. Coordination to gallium(III) proved to be an effective strategy for activity improvement against Pseudomonas aeruginosa and Candida albicans. The complexes were highly cytotoxic against malignant glioblastoma and breast cancer cells at nanomolar concentrations. The compounds induced morphological changes characteristic of apoptotic death in tumor cells and showed no toxicity against erythrocytes. 2 partially inhibited tubulin assembly at high concentrations and induced cellular microtubule disorganization, but this does not appear to be the main mechanism of cytotoxic activity.