In vitro and in vivo activities of a trithiolato-diRuthenium complex conjugated with sulfadoxine against the apicomplexan parasite Toxoplasma gondii.

Organometallic compounds, including Ruthenium complexes, have been widely developed as anti-cancer chemotherapeutics, but have also attracted much interest as potential anti-parasitic drugs. Recently hybrid drugs composed of organometallic Ruthenium moieties that were complexed to different antimicrobial agents were synthesized. One of these compounds, a trithiolato-diRuthenium complex (RU) conjugated to sulfadoxine (SDX), inhibited proliferation of Toxoplasma gondii tachyzoites grown in human foreskin fibroblast (HFF) monolayers with an IC50 < 150 nM, while SDX and the non-modified RU complex applied either individually or as an equimolar mixture were much less potent. In addition, conjugation of SDX to RU lead to decreased HFF cytotoxicity. RU-SDX did not impair the in vitro proliferation of murine splenocytes at concentrations ranging from 0.1 to 0.5 µM but had an impact at 2 µM, and induced zebrafish embryotoxicity at 20 µM, but not at 2 or 0.2 µM. RU-SDX acted parasitostatic but not parasiticidal, and induced transient ultrastructural changes in the mitochondrial matrix of tachyzoites early during treatment. While other compounds that target the mitochondrion such as the uncouplers FCCP and CCCP and another trithiolato-Ruthenium complex conjugated to adenine affected the mitochondrial membrane potential, no such effect was detected for RU-SDX. Evaluation of the in vivo efficacy of RU-SDX in a murine T. gondii oocyst infection model comprised of non-pregnant outbred CD1 mice showed no effects on the cerebral parasite burden, but reduced parasite load in the eyes and in heart tissue.

A Versatile Broadband Attached Proton Test Experiment for Routine 13C Nuclear Magnetic Resonance Spectroscopy.

The proposed broadband attached proton test sequence allows the user to easily record 13C nuclear magnetic resonance multiplicity-edited and quaternary-carbon-only spectra. Compared to earlier attached proton test experiments, it preserves both a tolerance for wide ranges of one-bond-coupling constant values and the effective suppression of residual CHn signals in the quaternary-carbon-only spectra. The recording of edited spectra or quaternary-carbon-only spectra is made easy by a single, user-controllable constant. These attributes make the broadband attached proton test experiment attractive for the 13C analysis of small molecules, including spectral editing, particularly in high-throughput analysis laboratories.

Carbon source-dependent capsule thickness regulation in Streptococcus pneumoniae.

Background: The polysaccharide capsule of Streptococcus pneumoniae plays a major role in virulence, adherence to epithelial cells, and overall survival of the bacterium in the human host. Galactose, mannose, and N-acetylglucosamine (GlcNAc) are likely to be relevant for metabolization in the nasopharynx, while glucose is the primary carbon source in the blood. In this study, we aim to further the understanding of the influence of carbon sources on pneumococcal growth, capsule biosynthesis, and subsequent adherence potential. Methods: We tested the growth behavior of clinical wild-type and capsule knockout S. pneumoniae strains, using galactose, GlcNAc, mannose, and glucose as carbon source for growth. We measured capsule thickness and quantified capsule precursors by fluorescein isothiocyanate (FITC)-dextran exclusion assays and 31P-nuclear magnetic resonance measurements, respectively. We also performed epithelial adherence assays using Detroit 562 cells and performed a transcriptome analysis (RNA sequencing). Results: We observed a reduced growth in galactose, mannose, and GlcNAc compared to growth in glucose and found capsular size reductions in mannose and GlcNAc compared to galactose and glucose. Additionally, capsular precursor measurements of uridine diphosphate-(UDP)-glucose and UDP-galactose showed less accumulation of precursors in GlcNAc or mannose than in glucose and galactose, indicating a possible link with the received capsular thickness measurements. Epithelial adherence assays showed an increase in adherence potential for a pneumococcal strain, when grown in mannose compared to glucose. Finally, transcriptome analysis of four clinical isolates revealed not only strain specific but also common carbon source-specific gene expression. Conclusion: Our findings may indicate a careful adaption of the lifestyle of S. pneumoniae according to the monosaccharides encountered in the respective human niche.

In vitro antibacterial activity of dinuclear thiolato-bridged ruthenium(II)-arene compounds.

IMPORTANCE: The in vitro assessment of diruthenium(II)-arene compounds against Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus showed a significant antibacterial activity of some compounds against S. pneumoniae, with minimum inhibitory concentration (MIC) values ranging from 1.3 to 2.6 µM, and a medium activity against E. coli, with MIC of 25 µM. The nature of the substituents anchored on the bridging thiols and the compounds molecular weight appear to significantly influence the antibacterial activity. Fluorescence microscopy showed that these ruthenium compounds enter the bacteria and do not accumulate in the cell wall of gram-positive bacteria. These diruthenium(II)-arene compounds exhibit promising activity against S. aureus and S. pneumoniae and deserve to be considered for further studies, especially the compounds bearing larger benzo-fused lactam substituents.

Liposomal aggregates sustain the release of rapamycin and protect cartilage from friction.

Liposomes show promise as biolubricants for damaged cartilage, but their small size results in low joint and cartilage retention. We developed a zinc ion-based liposomal drug delivery system for local osteoarthritis therapy, focusing on sustained release and tribological protection from phospholipid lubrication properties. Our strategy involved inducing aggregation of negatively charged liposomes with zinc ions to extend rapamycin (RAPA) release and improve cartilage lubrication. Liposomal aggregation occurred within 10 min and was irreversible, facilitating excess cation removal. The aggregates extended RAPA release beyond free liposomes and displayed irregular morphology influenced by RAPA. At nearly 100 µm, the aggregates were large enough to exceed the previously reported size threshold for increased joint retention. Tribological assessment on silicon surfaces and ex vivo porcine cartilage revealed the system's excellent protective ability against friction at both nano- and macro-scales. Moreover, RAPA was shown to attenuate the fibrotic response in human OA synovial fibroblasts. Our findings suggest the zinc ion-based liposomal drug delivery system has potential to enhance OA therapy through extended release and cartilage tribological protection, while also illustrating the impact of a hydrophobic drug like RAPA on liposome aggregation and morphology.

Synthesis and Antiparasitic Activity of New Trithiolato-Bridged Dinuclear Ruthenium(II)-arene-carbohydrate Conjugates.

Eight novel carbohydrate-tethered trithiolato dinuclear ruthenium(II)-arene complexes were synthesized using CuAAC 'click' (Cu(I)-catalyzed azide-alkyne cycloaddition) reactions, and there in vitro activity against transgenic T. gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) and in non-infected human foreskin fibroblasts, HFF, was determined at 0.1 and 1 µM. When evaluated at 1 µM, seven diruthenium-carbohydrate conjugates strongly impaired parasite proliferation by >90%, while HFF viability was retained at 50% or more, and they were further subjected to the half-maximal inhibitory concentration (IC50) measurement on T. gondii ß-gal. Results revealed that the biological activity of the hybrids was influenced both by the nature of the carbohydrate (glucose vs. galactose) appended on ruthenium complex and the type/length of the linker between the two units. 23 and 26, two galactose-based diruthenium conjugates, exhibited low IC50 values and reduced effect on HFF viability when applied at 2.5 µM (23: IC50 = 0.032 µM/HFF viability 92% and 26: IC50 = 0.153 µM/HFF viability 97%). Remarkably, compounds 23 and 26 performed significantly better than the corresponding carbohydrate non-modified diruthenium complexes, showing that this type of conjugates are a promising approach for obtaining new antiparasitic compounds with reduced toxicity.

New Nucleic Base-Tethered Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Compounds: Synthesis and Antiparasitic Activity.

Aiming toward compounds with improved anti-Toxoplasma activity by exploiting the parasite auxotrophies, a library of nucleobase-tethered trithiolato-bridged dinuclear ruthenium(II)-arene conjugates was synthesized and evaluated. Structural features such as the type of nucleobase and linking unit were progressively modified. For comparison, diruthenium hybrids with other type of molecules were also synthesized and assessed. A total of 37 compounds (diruthenium conjugates and intermediates) were evaluated in a primary screening for in vitro activity against transgenic Toxoplasma gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) at 0.1 and 1 µM. In parallel, the cytotoxicity in non-infected host cells (human foreskin fibroblasts, HFF) was determined by alamarBlue assay. Twenty compounds strongly impairing parasite proliferation with little effect on HFF viability were subjected to T. gondii ß-gal half maximal inhibitory concentration determination (IC50) and their toxicity for HFF was assessed at 2.5 µM. Two promising compounds were identified: 14, ester conjugate with 9-(2-oxyethyl)adenine, and 36, a click conjugate bearing a 2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl substituent, with IC50 values of 0.059 and 0.111 µM respectively, significantly lower compared to pyrimethamine standard (IC50 = 0.326 µM). Both 14 and 36 exhibited low toxicity against HFF when applied at 2.5 µM and are candidates for potential treatment options in a suitable in vivo model.

Synthesis, Photophysical Properties and Biological Evaluation of New Conjugates BODIPY: Dinuclear Trithiolato-Bridged Ruthenium(II)-Arene Complexes.

The synthesis, photophysical properties and antiparasitic efficacy against Toxoplasma gondii ß-gal (RH strain tachyzoites expressing ß-galactosidase) grown in human foreskin fibroblast monolayers (HFF) of a series of 15 new conjugates BODIPY-trithiolato-bridged dinuclear ruthenium(II)-arene complexes are reported (BODIPY=4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, derivatives used as fluorescent markers). The influence of the bond type (amide vs. ester), as well as that of the length and nature (alkyl vs. aryl) of the spacer between the dye and the diruthenium(II) complex moiety, on fluorescence and biological activity were evaluated. The assessed photophysical properties revealed that despite an important fluorescence quenching effect observed after conjugating the BODIPY to the diruthenium unit, the hybrids could nevertheless be used as fluorescent tracers. Although the antiparasitic activity of this series of conjugates appears limited, the compounds demonstrate potential as fluorescent probes for investigating the intracellular trafficking of trithiolato-bridged dinuclear Ru(II)-arene complexes in vitro.

Cellular and Molecular Targets of Nucleotide-Tagged Trithiolato-Bridged Arene Ruthenium Complexes in the Protozoan Parasites Toxoplasma gondii and Trypanosoma brucei.

Toxoplasma gondii is an apicomplexan parasite that infects and proliferates within many different types of host cells and infects virtually all warm-blooded animals and humans. Trypanosoma brucei is an extracellular kinetoplastid that causes human African trypanosomiasis and Nagana disease in cattle, primarily in rural sub-Saharan Africa. Current treatments against both parasites have limitations, e.g., suboptimal efficacy and adverse side effects. Here, we investigate the potential cellular and molecular targets of a trithiolato-bridged arene ruthenium complex conjugated to 9-(2-hydroxyethyl)-adenine (1), which inhibits both parasites with IC50s below 10-7 M. Proteins that bind to 1 were identified using differential affinity chromatography (DAC) followed by shotgun-mass spectrometry. A trithiolato-bridged ruthenium complex decorated with hypoxanthine (2) and 2-hydroxyethyl-adenine (3) were included as controls. Transmission electron microscopy (TEM) revealed distinct ultrastructural modifications in the mitochondrion induced by (1) but not by (2) and (3) in both species. DAC revealed 128 proteins in T. gondii and 46 proteins in T. brucei specifically binding to 1 but not 2 or 3. In T. gondii, the most abundant was a protein with unknown function annotated as YOU2. This protein is a homolog to the human mitochondrial inner membrane translocase subunit Tim10. In T. brucei, the most abundant proteins binding specifically to 1 were mitochondrial ATP-synthase subunits. Exposure of T. brucei bloodstream forms to 1 resulted in rapid breakdown of the ATP-synthase complex. Moreover, both datasets contained proteins involved in key steps of metabolism and nucleic acid binding proteins.

The DEPTQ+ Experiment: Leveling the DEPT Signal Intensities and Clean Spectral Editing for Determining CHn Multiplicities.

We propose a new 13C DEPTQ+ NMR experiment, based on the improved DEPTQ experiment, which is designed to unequivocally identify all carbon multiplicities (Cq, CH, CH2, and CH3) in two experiments. Compared to this improved DEPTQ experiment, the DEPTQ+ is shorter and the different evolution delays are designed as spin echoes, which can be tuned to different 1JCH values; this is especially valuable when a large range of 1JCH coupling constants is to be expected. These modifications allow (i) a mutual leveling of the DEPT signal intensities, (ii) a reduction in J cross-talk in the Cq/CH spectrum, and (iii) more consistent and cleaner CH2/CH3 edited spectra. The new DEPTQ+ is expected to be attractive for fast 13C analysis of small-to medium sized molecules, especially in high-throughput laboratories. With concentrated samples and/or by exploiting the high sensitivity of cryogenically cooled 13C NMR probeheads, the efficacy of such investigations may be improved, as it is possible to unequivocally identify all carbon multiplicities, with only one scan, for each of the two independent DEPTQ+ experiments and without loss of quality.

The quest of the best - A SAR study of trithiolato-bridged dinuclear Ruthenium(II)-Arene compounds presenting antiparasitic properties.

A structure activity relationship (SAR) study of a library of 56 compounds (54 ruthenium and 2 osmium derivatives) based on the trithiolato-bridged dinuclear ruthenium(II)-arene scaffold (general formula [(η6-arene)2Ru2(µ2-SR)3]+, symmetric and [(η6-arene)2Ru2(µ2-SR1)2(µ2-SR2)]+, mixed, respectively) is reported. The 56 compounds (of which 34 are newly designed drug candidates) were synthesized by introducing chemical modifications at the level of bridge thiols, and they were grouped into eight families according to their structural features. The selected fittings were guided by previous results and focused on a fine-tuning of the physico-chemical and steric properties. Newly synthesized complexes were characterized by NMR spectroscopy, mass spectrometry and elemental analysis, and four single-crystal X-ray structures were obtained. The in vitro biological assessment of the compounds was realized by applying a three-step screening cascade: (i) evaluation of the activity against Toxoplasma gondii RH strain tachyzoites expressing ß-galactosidase (T. gondii-ß-gal) grown in human foreskin fibroblast monolayers (HFF) and assessment of toxicity in non-infected HFF host cells; (ii) dose-response assays using selected compound, and (iii) studies on the effects in murine splenocytes. A primary screening was performed at 1 and 0.1 µM, and resulted in the selection of 39 compounds that inhibited parasite proliferation at 1 µM by more than 95% and reduced the viability of HFF by less than 49%. In the secondary screening, dose-response assays showed that the selected compounds exhibited half maximal inhibitory concentration (IC50) values for T. gondii-ß-gal between 0.01 µM and 0.45 µM, with 30 compounds displaying an IC50 lower than 0.1 µM. When applied to non-infected HFF monolayers at 2.5 µM, 8 compounds caused more than 90% and 31 compounds more than 30% viability impairment. The tertiary screening included 14 compounds that did not cause HFF viability loss higher than 50% at 2.5 µM. These derivatives were assessed for potential immunosuppressive activities. First, splenocyte viability was assessed after treatment of cells with concanavalin A (ConA) and lipopolysaccharide (LPS) with compounds applied at 0.1 and 0.5 µM. Subsequently, the 5 compounds exhibiting the lowest splenocyte toxicity were further evaluated for their potential to inhibit B and T cell proliferation. Overall, compound 55 [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-o-CF3)2(µ2-SC6H4-p-OH)]Cl exhibited the most favorable features, and will be investigated as a scaffold for further optimization in terms of anti-parasitic efficacy and drug-like properties.

Driving the Emission Towards Blue by Controlling the HOMO-LUMO Energy Gap in BF2 -Functionalized 2-(Imidazo[1,5-a]pyridin-3-yl)phenols.

Several boron compounds with 2-(imidazo[1,5-a]pyridin-3-yl)phenols, differentiated by the nature of the substituent (R) in the para position of the hydroxy group, have been synthesized and thoroughly characterized both in solution (1 H, 13 C, 11 B, 19 F NMR) and in the solid state (X-ray). All derivatives displayed attractive photophysical properties like very high Stokes shift, high fluorescence quantum yields and a good photostability in solution. Time-Dependent Density Functional Theory (TD-DFT) calculations allowed to define the main electronic transitions as intra ligand transitions (1 ILT), which was corroborated by the Natural Transition Orbitals (NTOs) shapes. The HOMO-LUMO energy gap was correlated to the electronic properties of the substituent R on the phenolic ring, as quantified by its σp Hammett constant.

Carbon Source-Dependent Changes of the Structure of Streptococcus pneumoniae Capsular Polysaccharide with Serotype 6F.

The structure of the exopolysaccharide capsule of Streptococcus pneumoniae is defined by the genetic arrangement of the capsule operon allowing the unequivocal identification of the pneumococcal serotype. Here, we investigated the environment-dependent composition of the polysaccharide structure of S. pneumoniae serotype 6F. When grown in a chemically defined medium (CDM) with glucose versus galactose, the exopolysaccharide capsule of the serotype 6F strains reveals a ratio of 1/0.6 or 1/0.3 for galactose/glucose in the capsule by 1H-NMR analyses, respectively. Increased production of the capsule precursor UDP-glucose has been identified by 31P-NMR in CDM with glucose. Flow cytometric experiments using monoclonal antibodies showed decreased labelling of Hyp6AG4 (specific for serotype 6A) antibodies when 6F is grown in glucose as compared to galactose, which mirrors the 1H-NMR results. Whole-genome sequencing analyses of serotype 6F isolates suggested that the isolates evolved during two different events from serotype 6A during the time when the 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced. In conclusion, this study shows differences in the capsular structure of serotype 6F strains using glucose as compared to galactose as the carbon source. Therefore, 6F strains may show slightly different polysaccharide composition while colonizing the human nasopharynx (galactose rich) as compared to invasive locations such as the blood (glucose rich).

Probing the Interactions of Porphyrins with Macromolecules Using NMR Spectroscopy Techniques.

Porphyrinic compounds are widespread in nature and play key roles in biological processes such as oxygen transport in blood, enzymatic redox reactions or photosynthesis. In addition, both naturally derived as well as synthetic porphyrinic compounds are extensively explored for biomedical and technical applications such as photodynamic therapy (PDT) or photovoltaic systems, respectively. Their unique electronic structures and photophysical properties make this class of compounds so interesting for the multiple functions encountered. It is therefore not surprising that optical methods are typically the prevalent analytical tool applied in characterization and processes involving porphyrinic compounds. However, a wealth of complementary information can be obtained from NMR spectroscopic techniques. Based on the advantage of providing structural and dynamic information with atomic resolution simultaneously, NMR spectroscopy is a powerful method for studying molecular interactions between porphyrinic compounds and macromolecules. Such interactions are of special interest in medical applications of porphyrinic photosensitizers that are mostly combined with macromolecular carrier systems. The macromolecular surrounding typically stabilizes the encapsulated drug and may also modify its physical properties. Moreover, the interaction with macromolecular physiological components needs to be explored to understand and control mechanisms of action and therapeutic efficacy. This review focuses on such non-covalent interactions of porphyrinic drugs with synthetic polymers as well as with biomolecules such as phospholipids or proteins. A brief introduction into various NMR spectroscopic techniques is given including chemical shift perturbation methods, NOE enhancement spectroscopy, relaxation time measurements and diffusion-ordered spectroscopy. How these NMR tools are used to address porphyrin-macromolecule interactions with respect to their function in biomedical applications is the central point of the current review.

Simplifying LR-HSQC spectra using a triple-quantum filter: The LR-HTQC experiment.

Long-range heteronuclear single quantum correlation (LR-HSQC) experiments may be applied for detecting long-range correlations but suffer from two disadvantages, common to all heteronuclear long-range correlation experiments: (i) The information density in LR-HSQC spectra may be too high to be used directly without "filtering out" shorter range correlations, and (ii) often, substantial differences in intensity among cross peaks exist, potentially hampering the visualization of weak, often crucial cross peaks. In this contribution, we propose a modified LR-HSQC experiment, the LR-HTQC experiment (Long-Range Heteronuclear Triple Quantum Correlation) that partially solves the problems aforementioned. We show theoretically and experimentally that the LR-HTQC experiment removes the intense cross peaks of CH spin pairs, substantially reduces the medium intensity of cross peaks originating from CHH' spin systems, whereas the typically weak intensity of cross peaks of CHH'H″ and C(H)n, n > 3 spin systems is less affected. Consequently, the LR-HTQC experiment affords simplified long-range heteronuclear shift correlation spectra and scales down large intensity differences among different types of cross peaks, although a certain general reduction of signal intensities has to be accepted.

Conjugates Containing Two and Three Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Units as In Vitro Antiparasitic and Anticancer Agents.

The synthesis, characterization, and in vitro antiparasitic and anticancer activity evaluation of new conjugates containing two and three dinuclear trithiolato-bridged ruthenium(II)-arene units are presented. Antiparasitic activity was evaluated using transgenic Toxoplasmagondii tachyzoites constitutively expressing ß-galactosidase grown in human foreskin fibroblasts (HFF). The compounds inhibited T.gondii proliferation with IC50 values ranging from 90 to 539 nM, and seven derivatives displayed IC50 values lower than the reference compound pyrimethamine, which is currently used for treatment of toxoplasmosis. Overall, compound flexibility and size impacted on the anti-Toxoplasma activity. The anticancer activity of 14 compounds was assessed against cancer cell lines A2780, A2780cisR (human ovarian cisplatin sensitive and resistant), A24, (D-)A24cisPt8.0 (human lung adenocarcinoma cells wild type and cisPt resistant subline). The compounds displayed IC50 values ranging from 23 to 650 nM. In A2780cisR, A24 and (D-)A24cisPt8.0 cells, all compounds were considerably more cytotoxic than cisplatin, with IC50 values lower by two orders of magnitude. Irrespective of the nature of the connectors (alkyl/aryl) or the numbers of the di-ruthenium units (two/three), ester conjugates 6-10 and 20 exhibited similar antiproliferative profiles, and were more cytotoxic than amide analogues 11-14, 23, and 24. Polynuclear conjugates with multiple trithiolato-bridged di-ruthenium(II)-arene moieties deserve further investigation.

Interactions of Cationic Diruthenium Trithiolato Complexes with Phospholipid Membranes Studied by NMR Spectroscopy.

To apprehend the possible mechanisms involved in the cellular uptake and the membrane interactions of cytotoxic dinuclear p-cymene trithiolato ruthenium(II) complexes, the interactions of the complexes [(η6-p-MeC6H4Pri)2Ru2(R1)2(R2)]+ (R1 = R2 = SC6H4-m-Pri:1; R1 = SC6H4-p-OMe, R2 = SC6H4-p-OH:2; R1 = SCH2C6H4-p-OMe, R2 = SC6H4-p-OH:3) with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles were studied using nuclear magnetic resonance (NMR) spectroscopy. 1H NMR, nuclear Overhauser effect (NOE), diffusion ordered spectroscopy (DOSY), and T1 and T2 relaxation data provided information on interactions between the complexes and the model membranes and on the submolecular localization of the complexes at the membrane interface. The results suggest that (a) the interaction takes place without new covalent adduct formation, (b) the cationic diruthenium complexes interact with DOPC head groups most likely involving electrostatic interactions while remaining structurally unchanged, (c) the changes indicating interactions are more pronounced for the most lipophilic complex 1, and (d) the diruthenium complexes remain at the exterior vesicle surface and are unlikely inserted between the phospholipid chains. The complexes also interact with micellar/free DHPC and seem to induce micellization or aggregation in solutions below critical micelle concentration (CMC). Our study suggests high affinity of the Ru complexes for the membrane surface that likely plays a key role in cellular uptake and possibly also in redistribution in mitochondria.

Examining and expanding the friction ridge value decision.

The first step of a friction ridge examination involves determining the suitability-or value-of an impression. Often, this is interpreted as whether the impression is suitable for comparison. However, examiners tend to be variable in their suitability determinations, and suitability itself can be a multi-faceted decision, comprising suitability for comparison, suitability for exclusion, suitability for identification, suitability for AFIS entry, complexity, and others. We undertook a white box study to explore the different facets of suitability determinations and to measure the specific categories of information upon which examiners most heavily rely when reaching these decisions. Although minutiae count was the best indicator of a value determination, clarity and distortion were better predictors of complexity determinations. Examiners were found to be highly variable in their determinations, as well as in their annotations of what information they relied upon. Some unanimous decisions were reached for only high-quality impressions; there was never unanimity on "no value" determinations. Examiners tended to use high-confidence minutiae markers, even when there was connective ambiguity or low clarity. Several new suitability categorizations were introduced and had good usage from study participants, indicating that they might have some value for inclusion in routine casework.

Coumarin-Tagged Dinuclear Trithiolato-Bridged Ruthenium(II)⋠Arene Complexes: Photophysical Properties and Antiparasitic Activity.

The synthesis, characterization, photophysical and biological properties of 13 new conjugate coumarin-diruthenium(II)⋅arene complexes against Toxoplasma gondii are presented. For all conjugate organometallic unit/coumarins, an almost complete loss of fluorescence efficacy was observed. However, the nature of the fluorophore, the type of bonding, the presence and length of a linker between the coumarin dye and the ruthenium(II) moiety, and the number of dye units influenced their biological properties. The in vitro activity against a transgenic T. gondii strain grown in human foreskin fibroblasts (HFF) leads to IC50 values for T. gondii ß-gal from 105 to 735 nM. Of note is that nine compounds displayed lower IC50 than the standard drug pyrimethamine. One compound applied at its IC50 did not affect B-cell proliferation but had an impact on T-cell proliferation in murine splenocyte cultures. Transmission electron microscopy of T. gondii ß-gal-infected HFF showed that treatment predominantly affected the parasites' mitochondrion.

Dinuclear thiolato-bridged arene ruthenium complexes: from reaction conditions and mechanism to synthesis of new complexes.

Several dinuclear thiophenolato-bridged arene ruthenium complexes [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-R)3]+ (R = H, NO2, F) could so far only be obtained in fair yields using the synthetic route established in the early 2000s. With much less reactive aliphatic thiols or with bulky thiols, the reactions become even less efficient and the desired complexes are obtained with low yields or not at all. We employed density functional theory (DFT) calculations to gain a fundamental understanding of the reaction mechanisms leading to the formation of dithiolato and trithiolato complexes starting from the dichloro(p-cymene)ruthenium(ii) dimer [(η6-p-MeC6H4Pri)Ru(µ2-Cl)Cl]2. The results of the DFT study enabled us to rationalise the experimental results and allowed us, via a modified synthetic route, to synthesise previously unreported and hitherto considered as unrealistic complexes. Our study opens up possibilities for the synthesis of so far inaccessible thiolato-bridged dinuclear arene ruthenium(ii) complexes but more generally, also the synthesis of other thiolato-bridged dinuclear group 8 and 9 metal complexes could be reexamined.