Unique Features of Alarmone Metabolism in Clostridioides difficile.

The "magic spot" alarmones (pp)pGpp, previously implicated in Clostridioides difficile antibiotic survival, are synthesized by the RelA-SpoT homolog (RSH) of C. difficile (RSHCd) and RelQCd. These enzymes are transcriptionally activated by diverse environmental stresses. RSHCd has previously been reported to synthesize ppGpp, but in this study, we found that both clostridial enzymes exclusively synthesize pGpp. While direct synthesis of pGpp from a GMP substrate, and (p)ppGpp hydrolysis into pGpp by NUDIX hydrolases, have previously been reported, there is no precedent for a bacterium synthesizing pGpp exclusively. Hydrolysis of the 5' phosphate or pyrophosphate from GDP or GTP substrates is necessary for activity by the clostridial enzymes, neither of which can utilize GMP as a substrate. Both enzymes are remarkably insensitive to the size of their metal ion cofactor, tolerating a broad array of metals that do not allow activity in (pp)pGpp synthetases from other organisms. It is clear that while C. difficile utilizes alarmone signaling, its mechanisms of alarmone synthesis are not directly homologous to those in more completely characterized organisms. IMPORTANCE Despite the role of the stringent response in antibiotic survival and recurrent infections, it has been a challenging target for antibacterial therapies because it is so ubiquitous. This is an especially relevant consideration for the treatment of Clostridioides difficile infection (CDI), as exposure to broad-spectrum antibiotics that harm commensal microbes is a major risk factor for CDI. Here, we report that both of the alarmone synthetase enzymes that mediate the stringent response in this organism employ a unique mechanism that requires the hydrolysis of two phosphate bonds and synthesize the triphosphate alarmone pGpp exclusively. Inhibitors targeted against these noncanonical synthetases have the potential to be highly specific and minimize detrimental effects to stringent response pathways in commensal microbes.

Enantioselective Catalytic Cyclopropanation-Rearrangement Approach to Chiral Spiroketals.

A highly enantioselective synthesis of chiral heterobicyclic spiroketals is reported via a "one-pot" cyclopropanation-rearrangement (CP-RA) cascade reaction that is sequentially catalyzed by a chiral Rh(II) catalyst and tetrabutylammonium fluoride (TBAF). Exocyclic vinyl substrates form spirocyclopropanes with tert-butyldimethylsilyl-protected enoldiazoacetates in excellent yields and with excellent enantioselectivities when catalyzed by chiral dirhodium(II) carboxylates, and following desilylation with simultaneous rearrangement in the presence of TBAF, they give (S)-spiroketals in high yields with excellent chirality retention (>95% ee).

Next generation sequencing and functional pathway analysis to understand the mechanism of action of copper-tolfenamic acid against pancreatic cancer cells.

Anti-cancer activity of tolfenamic acid (TA) in preclinical models for pancreatic cancer (PaCa) is well established. Since the dosage for anti-cancer actions of TA is rather high, we recently demonstrated that IC50 values of Copper-TA are 30-80% less than TA in 12 cancer cell lines. This study elucidates the underlying mechanisms of Copper-TA in PaCa cells. Control and Copper-TA (IC50) treated PaCa cells were processed by next-generation sequencing (NGS) to determine differentially expressed genes using HTG EdgeSeq Oncology Biomarker panel. Ingenuity Pathway Analysis (IPA®) was used to identify functional significance of altered genes. The conformational studies for assessing the expression of key regulators and genes were conducted by Western blot and qPCR. IPA® identified several networks, regulators, as well as molecular and cellular functions associated with cancer. The top 5 molecular and cellular functions affected by Cu-TA treatment were cell death and survival, cellular development, cell growth and proliferation, cell cycle and cellular movement. The expression of top upstream regulators was confirmed by Western blot analysis while qPCR results of selected genes demonstrated that Copper-TA is efficacious at lower doses than TA. Results suggest that Copper-TA alters genes/key regulators associated with cancer and potentially serve as an effective anti-cancer agent.

Synthesis, characterization, DNA binding, topoisomerase inhibition, and apoptosis induction studies of a novel cobalt(III) complex with a thiosemicarbazone ligand.

In this study, 9-anthraldehyde-N(4)-methylthiosemicarbazone (MeATSC) 1 and [Co(phen)2(O2CO)]Cl·6H2O 2 (where phen = 1,10-phenanthroline) were synthesized. [Co(phen)2(O2CO)]Cl·6H2O 2 was used to produce anhydrous [Co(phen)2(H2O)2](NO3)33. Subsequently, anhydrous [Co(phen)2(H2O)2](NO3)33 was reacted with MeATSC 1 to produce [Co(phen)2(MeATSC)](NO3)3·1.5H2O·C2H5OH 4. The ligand, MeATSC 1 and all complexes were characterized by elemental analysis, FT IR, UV-visible, and multinuclear NMR (1H, 13C, and 59Co) spectroscopy, along with HRMS, and conductivity measurements, where appropriate. Interactions of MeATSC 1 and complex 4 with calf thymus DNA (ctDNA) were investigated by carrying out UV-visible spectrophotometric studies. UV-visible spectrophotometric studies revealed weak interactions between ctDNA and the analytes, MeATSC 1 and complex 4 (Kb = 8.1 × 105 and 1.6 × 104 M-1, respectively). Topoisomerase inhibition assays and cleavage studies proved that complex 4 was an efficient catalytic inhibitor of human topoisomerases I and IIα. Based upon the results obtained from the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay on 4T1-luc metastatic mammary breast cancer cells (IC50 = 34.4 ±â€¯5.2 µM when compared to IC50 = 13.75 ±â€¯1.08 µM for the control, cisplatin), further investigations into the molecular events initiated by exposure to complex 4 were investigated. Studies have shown that complex 4 activated both the apoptotic and autophagic signaling pathways in addition to causing dissipation of the mitochondrial membrane potential (ΔΨm). Furthermore, activation of cysteine-aspartic proteases3 (caspase 3) in a time- and concentration-dependent manner coupled with the ΔΨm, studies implicated the intrinsic apoptotic pathway as the major regulator of cell death mechanism.

Tolerance of High Oral Doses of Nonradioactive and Radioactive Caesium Chloride in the Pale Grass Blue Butterfly Zizeeria maha.

The biological effects of the Fukushima nuclear accident have been examined in the pale grass blue butterfly, Zizeeria maha (Lepidoptera: Lycaenidae). In previous internal exposure experiments, larvae were given field-collected contaminated host plant leaves that contained up to 43.5 kBq/kg (leaf) of radioactive caesium. Larvae ingested up to 480 kBq/kg (larva), resulting in high mortality and abnormality rates. However, these results need to be compared with the toxicological data of caesium. Here, we examined the toxicity of both nonradioactive and radioactive caesium chloride on the pale grass blue butterfly. Larvae were fed a caesium-containing artificial diet, ingesting up to 149 MBq/kg (larva) of radioactive caesium (137Cs) or a much higher amount of nonradioactive caesium. We examined the pupation rate, eclosion rate, survival rate up to the adult stage, and the forewing size. In contrast to previous internal exposure experiments using field-collected contaminated leaves, we could not detect any effect. We conclude that the butterfly is tolerant to ionising radiation from 137Cs in the range tested but is vulnerable to radioactive contamination in the field. These results suggest that the biological effects in the field may be mediated through ecological systems and cannot be estimated solely based on radiation doses.

Copper-tolfenamic acid: evaluation of stability and anti-cancer activity.

The non-steroidal anti-inflammatory drug, Tolfenamic acid (TA) acts as an anti-cancer agent in several adult and pediatric cancer models. Copper (Cu) is an important element with multiple biological functions and has gained interest in medical applications. Recently, [Cu(TA)2(bpy)] (Cu-TA) has been synthesized in order to enhance therapeutic activity. In this study, we synthesized Cu-TA using an established method, characterized it by UV visible spectroscopy and Fourier-transform infrared spectroscopy (FTIR), and tested its anti-cancer activity using twelve cell lines representing various cancers, such as Ewing sarcoma, glioblastoma, medulloblastoma, neuroblastoma, pancreatic and prostate. The anti-proliferative activity of Cu-TA was determined at 48 h post-treatment and compared with the parental compound, TA. The IC50 values were calculated using GraphPad Prism software. The biological stability of Cu-TA was evaluated using twelve-month-old powder and six-month-old stock solution. Cardiomyocytes (H9C2) were used to test the cytotoxicity in non-malignant cells. Cu-TA showed higher anti-proliferative activity, and the IC50 values were 30 to 80% lower when compared with TA. H9C2 cells were non-responsive to Cu-TA, suggesting that it is selective towards malignant cells. Comparison of the twelve-month-old powder and six-month-old stock solution using the Panc1 cell line showed similar IC50 values (<5% variation), confirming the stability of Cu-TA either in powder or solution form. These findings demonstrate the potential of Cu-TA as an effective anti-cancer agent. Further studies to delineate the detailed mechanism of action of Cu-TA for specific cancer model are underway.

Novel Survivin Inhibitor for Suppressing Pancreatic Cancer Cells Growth via Downregulating Sp1 and Sp3 Transcription Factors.

BACKGROUND/AIMS: Targeting survivin, an anti-apoptotic protein and mitotic regulator, is considered as an effective therapeutic option for pancreatic cancer (PaCa). Tolfenamic acid (TA) showed anti-cancer activity in pre-clinical studies. A recent discovery demonstrated a copper(II) complex of TA (Cu-TA) can result in higher activity. In this study, the ability of Cu-TA to inhibit survivin and its transcription factors, Specificity protein (Sp) 1 and 3 in PaCa cell lines and tumor growth in mouse xenograft model were evaluated. METHODS: Cell growth inhibition was measured in MIA PaCa-2 and Panc1 cells for 2 days using CellTiter-Glo kit. Sp1, Sp3 and survivin expression (by Western blot and qPCR), apoptotic cells and cell cycle phase distribution (by flow cytometry) were evaluated. A pilot study was performed using athymic nude mice [treated with vehicle/Cu-TA (25 or 50 mg/kg) 3 times/week for 4 weeks. RESULTS: The IC50 value for Cu-TA was about half than TA.Both agents repressed the protein expression of Sp1/Sp3/survivin, Cu-TA was more effective than TA. Especially effect on survivin inhibition was 5.2 (MIA PaCa-2) or 6.4 (Panc1) fold higher and mRNA expression of only survivin was decreased. Apoptotic cells increased with Cu-TA treatment in both cell lines, while Panc1 showed both effect on apoptosis and cell cycle (G2/M) arrest. Cu-TA decreased the tumor growth in mouse xenografts (25 mg/kg: 48%; 50 mg/kg: 68%). Additionally, there was no change observed in mice body weights, indicating no overt toxicity was occurring. CONCLUSION: These results show that Cu-TA can serve as an effective survivin inhibitor for inhibiting PaCa cell growth.

Comparative Morphological Analysis of the Immature Stages of the Grass Blue Butterflies Zizeeria and Zizina (Lepidoptera: Lycaenidae).

The pale grass blue butterfly has been used to assess the biological effects of the Fukushima nuclear accident. Zizeeria and Zizina are two closely related genera of grass blue butterflies that are widely distributed in tropical to temperate Asia, Australia, and Africa, making them suitable environmental indicators for these areas. However, the morphological features of the immature stages have been examined only in fragmentary fashion. Here, we reared Zizeeria maha argia, Zizeeria maha okinawana, Zizeeria karsandra karsandra, Zizina emelina emelina, Zizina otis labradus, and Zizina otis riukuensis using a standard rearing method that was developed for Zizeeria maha, and comparatively identified morphological traits to effectively classify the immature stages of species or subspecies. Morphological information on these and other subspecies including Zizeeria knysna knysna and Zizina otis antanossa from Africa was also collected from literature. The subspecies were all reared successfully. The subspecies all had dorsal nectary and tentacle organs with similar morphology. For the subspecies of Zizeeria maha, only minor morphological differences were noted. Similarly, the subspecies of Zizina otis shared many traits. Most importantly, Zizeeria and Zizina differed in the shape of the sensory hairs that accompany the dorsal nectary organ; Zizeeriahad pointed hairs, and Zizina had blunt or rounded hairs. However, Zizina emelina exhibited several intermediate features between these two genera. Overall, the morphological traits did not completely reflect the conventional systematic relationships. This comparative study describes the efficient rearing of the grass blue butterflies and provides a morphological basis for the use of these species as environmental indicators.

Distal-less induces elemental color patterns in Junonia butterfly wings.

BACKGROUND: The border ocellus, or eyespot, is a conspicuous color pattern element in butterfly wings. For two decades, it has been hypothesized that transcription factors such as Distal-less (Dll) are responsible for eyespot pattern development in butterfly wings, based on their expression in the prospective eyespots. In particular, it has been suggested that Dll is a determinant for eyespot size. However, functional evidence for this hypothesis has remained incomplete, due to technical difficulties. RESULTS: Here, we show that ectopically expressed Dll induces ectopic elemental color patterns in the adult wings of the blue pansy butterfly, Junonia orithya (Lepidoptera, Nymphalidae). Using baculovirus-mediated gene transfer, we misexpressed Dll protein fused with green fluorescent protein (GFP) in pupal wings, resulting in ectopic color patterns, but not the formation of intact eyespots. Induced changes included clusters of black and orange scales (a basic feature of eyespot patterns), black and gray scales, and inhibition of cover scale development. In contrast, ectopic expression of GFP alone did not induce any color pattern changes using the same baculovirus-mediated gene transfer system. CONCLUSIONS: These results suggest that Dll plays an instructive role in the development of color pattern elements in butterfly wings, although Dll alone may not be sufficient to induce a complete eyespot. This study thus experimentally supports the hypothesis of Dll function in eyespot development.

A novel cluster of mariner-like elements belonging to mellifera subfamily from spiders and insects: implications of recent horizontal transfer on the South-West Islands of Japan.

Mariner-like elements (MLEs) have been isolated from various eukaryotic genomes and they are divided into 15 subfamilies, including main five subfamilies: mauritiana, cecropia, mellifera/capitata, irritans, and elegans/briggsae. In the present study, MLEs belonging to mellifera subfamily were isolated from various spiders and insects (Hymenoptera and Lepidoptera) inhabiting the South-West Islands of Japan and neighboring regions. MLEs isolated from 15 different species formed a distinct novel cluster in mellifera subfamily. MLEs obtained from three different species [i.e., the bee Amegilla senahai subflavescens (Amsmar1), the wasp Campsomeris sp. (Casmar1), and the swallowtail butterfly Pachliopta aristolochiae (Paamar1)] contained an intact open reading frame that encoded a putative transposase. These transposases exhibited high similarity of 97.9% among themselves. In case of Casmar1, the presence of an intact ORF was found in high frequencies (i.e., 11 out of 12 clones). In addition, these transposases also showed the presence of a terminal inverted repeat-binding motif, DD(34)D and two highly conserved amino acid motifs, (W/L)(I/L)PHQL and YSP(D/N)L(A/S)P. These two motifs differed from previously known motifs, WVPHEL and YSPDLAP. MLEs isolated from these three different species may have been inserted into their genomes by horizontal transfer. Furthermore, the presence of an intact ORF suggests that they are still active in habitats along these isolated islands.