Anticarcinogenic Potency of EF24: An Overview of Its Pharmacokinetics, Efficacy, Mechanism of Action, and Nanoformulation for Drug Delivery.

EF24, a synthetic monocarbonyl analog of curcumin, shows significant potential as an anticancer agent with both chemopreventive and chemotherapeutic properties. It exhibits rapid absorption, extensive tissue distribution, and efficient metabolism, ensuring optimal bioavailability and sustained exposure of the target tissues. The ability of EF24 to penetrate biological barriers and accumulate at tumor sites makes it advantageous for effective cancer treatment. Studies have demonstrated EF24's remarkable efficacy against various cancers, including breast, lung, prostate, colon, and pancreatic cancer. The unique mechanism of action of EF24 involves modulation of the nuclear factor-kappa B (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways, disrupting cancer-promoting inflammation and oxidative stress. EF24 inhibits tumor growth by inducing cell cycle arrest and apoptosis, mainly through inhibiting the NF-κB pathway and by regulating key genes by modulating microRNA (miRNA) expression or the proteasomal pathway. In summary, EF24 is a promising anticancer compound with a unique mechanism of action that makes it effective against various cancers. Its ability to enhance the effects of conventional therapies, coupled with improvements in drug delivery systems, could make it a valuable asset in cancer treatment. However, addressing its solubility and stability challenges will be crucial for its successful clinical application.

Kv2.1 Channels Prevent Vasomotion and Safeguard Myogenic Reactivity in Rat Small Superior Cerebellar Arteries.

Vascular smooth muscle voltage-gated potassium (Kv) channels have been proposed to contribute to myogenic autoregulation. Surprisingly, in initial experiments, we observed that the Kv2 channel inhibitor stromatoxin induced vasomotion without affecting myogenic tone. Thus, we tested the hypothesis that Kv2 channels contribute to myogenic autoregulation by fine-tuning the myogenic response. Expression of Kv2 channel mRNA was determined using real-time PCR and 'multiplex' single-cell RT-PCR. Potassium currents were measured using the patch-clamp technique. Contractile responses of intact arteries were studied using isobaric myography. Expression of Kv2.1 but not Kv2.2 channels was detected in intact rat superior cerebellar arteries and in single smooth muscle cells. Stromatoxin, a high-affinity inhibitor of Kv2 channels, reduced smooth muscle Kv currents by 61% at saturating concentrations (EC50 36 nmol/L). Further, stromatoxin (10-100 nmol/L) induced pronounced vasomotion in 48% of the vessels studied. In vessels not exhibiting vasomotion, stromatoxin did not affect myogenic reactivity. Notably, in vessels exhibiting stromatoxin-induced vasomotion, pressure increases evoked two effects: First, they facilitated the occurrence of random vasodilations and/or vasoconstrictions, disturbing the myogenic response (24% of the vessels). Second, they modified the vasomotion by decreasing its amplitude and increasing its frequency, thereby destabilizing myogenic tone (76% of the vessels). Our study demonstrates that (i) Kv2.1 channels are the predominantly expressed Kv channels in smooth muscle cells of rat superior cerebellar arteries, and (ii) Kv2.1 channels provide a novel type of negative feedback mechanism in myogenic autoregulation by preventing vasomotion and thereby safeguarding the myogenic response.

Anti-Idiotype scFv Localizes an Autoepitope in the Globular Domain of C1q.

We addressed the issue of C1q autoantigenicity by studying the structural features of the autoepitopes recognized by the polyclonal anti-C1q antibodies present in Lupus Nephritis (LN) sera. We used six fractions of anti-C1q as antigens and selected anti-idiotypic scFv antibodies from the phage library "Griffin.1". The monoclonal scFv A1 was the most potent inhibitor of the recognition of C1q and its fragments ghA, ghB and ghC, comprising the globular domain gC1q, by the lupus autoantibodies. It was sequenced and in silico folded by molecular dynamics into a 3D structure. The generated 3D model of A1 elucidated CDR similarity to the apical region of gC1q, thus mapping indirectly for the first time a globular autoepitope of C1q. The VH CDR2 of A1 mimicked the ghA sequence GSEAD suggested as a cross-epitope between anti-DNA and anti-C1q antibodies. Other potential inhibitors of the recognition of C1q by the LN autoantibodies among the selected recombinant antibodies were the monoclonal scFv F6, F9 and A12.

New reductive rearrangement of N-arylindoles triggered by the Grubbs-Stoltz reagent Et3SiH/KO t Bu.

N-Arylindoles are transformed into dihydroacridines in a new type of rearrangement, through heating with triethylsilane and potassium tert-butoxide. Studies indicate that the pathway involves (i) the formation of indole radical anions followed by fragmentation of the indole C2-N bond, and (ii) a ring-closing reaction that follows a potassium-ion dependent hydrogen atom transfer step. Unexpected behaviors of 'radical-trap' substrates prove very helpful in framing the proposed mechanism.

Et3SiH + KO t Bu provide multiple reactive intermediates that compete in the reactions and rearrangements of benzylnitriles and indolenines.

The combination of potassium tert-butoxide and triethylsilane is unusual because it generates multiple different types of reactive intermediates simultaneously that provide access to (i) silyl radical reactions, (ii) hydrogen atom transfer reactions to closed shell molecules and to radicals, (iii) electron transfer reductions and (iv) hydride ion chemistry, giving scope for unprecedented outcomes. Until now, reactions with this reagent pair have generally been explained by reference to one of the intermediates, but we now highlight the interplay and competition between them.

Draft Genome Sequence of Escherichia coli K-12 (ATCC 10798).

Here, we present the draft genome sequence of Escherichia coli ATCC 10798. E. coli ATCC 10798 is a K-12 strain, one of the most well-studied model microorganisms. The size of the genome was 4,685,496 bp, with a G+C content of 50.70%. This assembly consists of 62 contigs and the F plasmid.

Mutations Decreasing Intrinsic ß-Lactam Resistance Are Linked to Cell Division in the Nosocomial Pathogen Acinetobacter baumannii.

Transposon mutagenesis was used to identify novel determinants of intrinsic ß-lactam resistance in Acinetobacter baumannii An EZ-Tn5 transposon insertion in a gene corresponding to the A1S_0225 sequence resulted in a 4-fold decrease in resistance to ampicillin, cefotaxime, imipenem, and ceftriaxone but did not alter resistance to other classes of antibiotics. Based on this phenotype, the gene was designated blhA (ß-lactam hypersusceptibility). The blhA::EZ-Tn5 mutation conferred a similar phenotype in A. baumannii strain ATCC 17978. The wild-type blhA gene complemented the blhA::EZTn5 insertion and restored ß-lactam resistance levels back to wild-type levels. The blhA mutation also increased ß-lactam susceptibility in an adeB adeJ double mutant, indicating that the blhA mutation acted independently of these efflux systems to mediate susceptibility. In addition, mRNA levels for the blaOXA and blaADC ß-lactamase genes were not altered by the blhA mutation. The blhA mutation resulted in a prominent cell division and morphological defect, with cells exhibiting a highly elongated phenotype, combined with large bulges in some cells. The blhA gene is unique to Acinetobacter and likely represents a novel gene involved in cell division. Three additional mutations, in zipA, zapA, and ftsK, each of which encode predicted cell division proteins, also conferred increased ß-lactam susceptibility, indicating a common link between cell division and intrinsic ß-lactam resistance in A. baumannii.

In focus: Fe3O4 nanoparticles and human mesenteric artery interaction in vitro.

AIM: The growing implementation of iron oxide nanoparticles in medicine requires a thorough investigation of their physiological influence. Therefore, effects of Fe3O4 nanoparticles on isometric contractions of healthy human mesenteric artery in vitro were investigated. MATERIALS & METHODS: The effect of increasing concentrations (0.023, 0.069, 0.23, 0.69 and 2.31 µg/µl) of Fe3O4 nanoparticles (50-100 nm) on the contractility of mesenteric artery ring preparations was studied using wire myography technique. RESULTS & CONCLUSION: A lack of effects of Fe3O4 nanoparticles (50-100 nm) on isometric contractions of human mesenteric artery segments both in conditions of basal tension and precontraction was found. The observed unresponsiveness of human mesenteric arteries in vitro to Fe3O4 nanoparticles could be attributed to their safe mode of use in biomedicine.

Phase-Variable Control of Multiple Phenotypes in Acinetobacter baumannii Strain AB5075.

UNLABELLED: Acinetobacter baumannii strain AB5075 produces colonies with two opacity phenotypes, designated opaque and translucent. These phenotypes were unstable and opaque and translucent colony variants were observed to interconvert at high frequency, suggesting that a phase-variable mechanism was responsible. The frequency of phase variation both within colonies and in broth cultures increased in a cell density-dependent manner and was mediated by the accumulation of an extracellular factor. This factor was distinct from the known A. baumannii signaling molecule 3-OH C12-homoserine lactone. Opaque and translucent colony variants exhibited a number of phenotypic differences, including cell morphology, surface motility, biofilm formation, antibiotic resistance, and virulence in a Galleria mellonella model. Additional clinical isolates exhibited a similar phase-variable control of colony opacity, suggesting that this may be a common feature of A. baumannii. IMPORTANCE: A novel phase-variable mechanism has been identified in Acinetobacter baumannii that results in an interconversion between opaque and translucent colony phenotypes. This phase variation also coordinately regulates motility, cell shape, biofilm formation, antibiotic resistance, and virulence. The frequency of phase variation is increased at high cell density via a diffusible extracellular signal. To our knowledge, this report presents the first example of phase variation in A. baumannii and also the first example of quorum sensing-mediated control of phase variation in a bacterium. The findings are important, as this phase-variable mechanism can be identified only via changes in colony opacity using oblique light; therefore, many researchers studying A. baumannii may unknowingly be working with different colony variants.

Cystathionine gamma-lyase of perivascular adipose tissue with reversed regulatory effect in diabetic rat artery.

The aim of this study is to reveal the regulatory role of cystathionine gamma-lyase (CSE), the main source of hydrogen sulphide (H2S) in perivascular adipose tissue (PVAT), of diabetic rats. Diabetes was induced in male rats by a single intraperitoneal injection of streptozotocin. Animals with glucose levels above 20 mmol/L were determined as diabetic. The rat gracilis arteries (a. gracilis) were dissected with or without PVAT. In all in vitro experiments endothelium-denuded preparations were used for isometric contraction measurements. Increasing concentrations of 5-hydroxytryptamine (5-HT) from 10-10 to 10-5 mol/L were applied to induce gradual increase in force of contractions of circular artery segments. The relaxing effect of CSE was inhibited by DL-propargyl glycine (PGG). The presence of PVAT decreases the contractile response to 5-HT of a. gracilis from control rats. This response is reversed in contraction studies in the same rat artery from diabetic rats. DL-PPG (1 mmol/L) induced significant increase of the force of contraction in artery preparations with PVAT from control rats in the whole range of 5-HT. In contrast, PGG had a relaxing effect in high concentrations of 5-HT (10-6 and 10-5 mol/L) in diabetic rat arteries with PVAT. It is concluded that in skeletal muscle artery from diabetic rats, a mediator related to H2S is released from PVAT. This paracrine mediator increases the maximal force of contraction of endothelium-denuded preparations at higher concentrations of 5-HT.

Photophysical characterisation and studies of the effect of palladium(II) 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrin on isometric contraction of isolated human mesenteric artery: good news for photodynamic therapy.

BACKGROUND: Considering the important roles of porphyrins in biological systems and their promising use in photodynamic therapy (PDT), the present work investigated the photophysical properties of palladium(II) 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrin (PdTSPP) and the effects of non-activated by light form of this porphyrin on contractile behaviour of isolated healthy endothelium-denuded human mesenteric arteries. METHODS: The photophysical characterisation of PdTSPP: the formation of the triplet states and the singlet oxygen were studied using laser flash photolysis. The effect of PdTSPP on the isometric contraction of artery segments from human mesentery was assessed utilising the precise method of artery isometric tension recording using Mulvany-Halpern wire myograph. RESULTS: We found that PdTSPP had a high lifetime of the triplet states τT=270µs. The calculated Stern Volmer rate constant kq=1.7×10(9)M(-1)s(-1) showed an efficient quenching by oxygen that indicated formation of singlet oxygen, O2((1)Δg). The photophysical parameters of PdTSPP, in particular its ability to generate O2((1)Δg) has defined it as an exceptionally interesting molecule for PDT. The results of the contraction study showed that PdTSPP applied in increasing concentrations (1-100µM) had no effect on the basal tone of human mesenteric artery under isometric condition. Furthermore, PdTSPP failed to potentiate or to attenuate the isometric contraction of the artery preparations precontracted with high extracellular potassium (42mM KCl) or with 1nM endothelin-1. CONCLUSIONS: The excellent photophysical properties of PdTSPP as well as the lack of an effect on the contractility of human vasculature in vitro characterise PdTSPP as a suitable compound for potential medical applications.

DNA replication initiation patterns and spatial dynamics of the human ribosomal RNA gene loci.

Typically, only a fraction of the ≥600 ribosomal RNA (rRNA) gene copies in human cells are transcriptionally active. Expressed rRNA genes coalesce in specialized nuclear compartments - the nucleoli - and are believed to replicate during the first half of S phase. Paradoxically, attempts to visualize replicating rDNA during early S phase have failed. Here, I show that, in human (HeLa) cells, early-replicating rDNA is detectable at the nucleolar periphery and, more rarely, even outside nucleoli. Early-replicated rDNA relocates to the nucleolar interior and reassociates with the transcription factor UBF, implying that it predominantly represents expressed rDNA units. Contrary to the established model for active gene loci, replication initiates randomly throughout the early-replicating rDNA. By contrast, mostly silent rDNA copies replicate inside the nucleoli during mid and late S phase. At this stage, replication origins are fired preferentially within the non-transcribed intergenic spacers (NTSs), and ongoing rDNA transcription is required to maintain this specific initiation pattern. I propose that the unexpected spatial dynamics of the early-replicating rDNA repeats serve to ensure streamlined efficient replication of the most heavily transcribed genomic loci while simultaneously reducing the risk of chromosome breaks and rDNA hyper-recombination.

Replication stress induces 53BP1-containing OPT domains in G1 cells.

Chromosomal deletions and rearrangements in tumors are often associated with common fragile sites, which are specific genomic loci prone to gaps and breaks in metaphase chromosomes. Common fragile sites appear to arise through incomplete DNA replication because they are induced after partial replication inhibition by agents such as aphidicolin. Here, we show that in G1 cells, large nuclear bodies arise that contain p53 binding protein 1 (53BP1), phosphorylated H2AX (γH2AX), and mediator of DNA damage checkpoint 1 (MDC1), as well as components of previously characterized OPT (Oct-1, PTF, transcription) domains. Notably, we find that incubating cells with low aphidicolin doses increases the incidence and number of 53BP1-OPT domains in G1 cells, and by chromatin immunoprecipitation and massively parallel sequencing analysis of γH2AX, we demonstrate that OPT domains are enriched at common fragile sites. These findings invoke a model wherein incomplete DNA synthesis during S phase leads to a DNA damage response and formation of 53BP1-OPT domains in the subsequent G1.

Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells.

The discovery of interchromosomal interactions in higher eukaryotes points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of interchromosomal interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of transcriptional interactions using the mouse globin genes in erythroid tissues. Our results show that the active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes. We show that the transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized transcription factories. Our results establish a new gene expression paradigm, implying that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hot spots optimized for efficient and coordinated transcriptional control.

Visualization of DNA replication sites in mammalian nuclei.

DNA replication takes place at discrete sites in the cell nucleus, named replication foci. The spatial arrangements of these foci change in the course of S phase in a temporally regulated and reproducible fashion forming five distinct and highly conserved replication patterns. The organization of nuclear replication sites can be studied by electron and light microscopy techniques. This chapter describes several procedures for detection of replication foci in mammalian nuclei via indirect immunofluorescence microscopy.

Contractile effect of ghrelin on isolated guinea-pig renal arteries.

Ghrelin, a 28-amino acid peptide, known to exist in both acylated and des-acylated varieties, was identified as the first endogenous ligand of growth hormone secretagogue receptor in 1999. Various arteries are known to express ghrelin receptors, but the direct action of ghrelin on blood vessels has been unclear. In the present study we show that ghrelin concentration-dependently potentiates endothelin-1 (ET-1) induced tension development of guinea-pig renal artery, as measured using a wire-type isometric myography of vascular segments. In vascular smooth muscle cells (SMC) ghrelin caused activation of potassium outward currents via phospholipase C (PLC)-->inositol-1,4,5-trisphosphate (IP3) and PLC-->protein kinase C (PKC) signalling cascade, resulting in hyperpolarizaton of the cell membrane. On a tissue level ghrelin by itself had no effect on isometric tone, but augmented ET-1 induced contraction by a mechanism, involving PLC, Rho-kinase and intracellular IP3 -sensitive Ca2+ release, and not nucleotide-sensitive protein kinases or PKC. Together with our previous findings the data in this study suggest that ghrelin exerts its contractile activity on guinea-pig renal artery by facilitation of ET-1 triggered intracellular signalling in SMC, and/or by stimulating the release of a yet unknown contractile mediator from endothelium.

Nuclear transcription is essential for specification of mammalian replication origins.

I have demonstrated that nuclear transcription modulates the distribution of replication origins along mammalian chromosomes. Chinese Hamster Ovary (CHO) cells were exposed to transcription inhibitors in early G1 phase and replication origin sites in the dihydrofolate reductase (DHFR) gene locus were mapped several hours later. DNA within nuclei prepared from control and transcription-deficient G1-phase cells was replicated with similar efficiencies when introduced into Xenopus egg extracts. Replication initiated in the intergenic region within control late-G1 nuclei, but randomly within transcriptionally repressed nuclei. Random initiation was not a consequence of inability to produce an essential protein(s), since initiation was site-specific within cells exposed to the translation inhibitor cycloheximide during the same interval of G1 phase. Furthermore, in vivo inhibition of transcription within late-G1-phase cells reduced the frequency of usage of pre-established DHFR replication origin sites. Transcription rates in the DHFR domain were very low and did not change throughout G1 phase. This implies that, although ongoing nuclear transcription is required, local expression of the genes in the DHFR locus alone is not sufficient to create a site-specific replication initiation pattern. I conclude that epigenetic factors, including general nuclear transcription, play a role in replication origin selection in mammalian nuclei.

Alkaloids from the roots of Senecio macedonicus Griseb.

The new alkaloids 7-,9-diangeloylplatynecine (1) and 8-episarracine N-oxide (2), were isolated and identified from the roots of Senecio macedonicus. Another one, 8-epineosarracine was detected by GC/MS analyses of the crude alkaloid mixture. The cytotoxicity and biological activity of the alkaloids were tested on normal murine spleen lymphocytes and P3U1 mouse myeloma.

The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells.

We investigated the organization of DNA replication sites in primary (young or presenescent), immortalized and transformed mammalian cells. Four different methods were used to visualize replication sites: in vivo pulse-labeling with 5-bromo-2'-deoxyuridine (BrdU), followed by either acid depurination, or incubation in nuclease cocktail to expose single-stranded BrdU-substituted DNA regions for immunolabeling; biotin-dUTP labeling of nascent DNA by run-on replication within intact nuclei and staining with fluorescent streptavidin; and, finally, immunolabeling of the replication fork proteins PCNA and RPA. All methods produced identical results, demonstrating no fundamental differences in the spatio-temporal organization of replication patterns between primary, immortal or transformed mammalian cells. In addition, we did not detect a spatial coincidence between the early firing replicons and nuclear lamin proteins, the retinoblastoma protein or the nucleolus in primary human and rodent cells. The retinoblastoma protein does not colocalize in vivo with members of the Mcm family of proteins (Mcm2, 3 and 7) at any point of the cell cycle and neither in the chromatin-bound nor in the soluble nucleoplasmic fraction. These results argue against a direct role for the retinoblastoma or nuclear lamin proteins in mammalian DNA synthesis under normal physiological conditions.

Mammalian nuclei become licensed for DNA replication during late telophase.

Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized at various times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites of initiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in the preservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place during telophase, several hours prior to the specification of replication origins at the DHFR locus.