Simultaneous determination of timolol maleate, rosuvastatin calcium and diclofenac sodium in pharmaceuticals and physiological fluids using HPLC-UV

A novel HPLC-UV method was developed for the simultaneous determination of timolol (TM), rosuvastatin (RST), and diclofenac sodium (DS) in pharmaceuticals, human plasma and aqueous humor using naproxen sodium as internal standard (IS). The target compounds were analyzed on Hypersil BDS C(18) column (250 mm × 4.6 mm, 5 μm), applying 0.2% triethylamine (TEA) and acetonitrile (ACN) (40:60, v/v), in isocratic mode as mobile phase, pH 2.75 adjusted with 85% phosphoric acid at a flow rate of 1 ml/min. The column oven temperature was kept at 45°C and the peak response was monitored at 284 nm after injecting a 50 μl sample into HPLC system. The direct liquid-liquid extraction procedure was applied to human plasma and bovine aqueous humor samples using mobile phase as an extraction solvent after deproteination with methanol. The different HPLC experimental parameters were optimized and the method was validated according to standard guidelines. The recoveries of the suggested method in human plasma were 98.72, 96.04, and 95.14%, for TM, RST, and DS, while in aqueous humor were 94.99, and 98.23%, for TM, and DS, respectively. The LOD values were found to be 0.800, 0.500, and 0.250 ng/ml, for TM, RST, and DS, respectively, while their respective LOQ values were 2.00, 1.50, and 1.00 ng/ml. The co-efficient of variation (CV) were in the range of 0.1492-1.1729% and 1.0516-4.0104%, for intra-day and inter-day studies, respectively. The method was found accurate in human plasma and bovine aqueous humor and will be applied for the quantification of these compounds in plasma, and aqueous humor samples using animal models and in pharmaceuticals.     

Binding of bis-ANS to Bacillus subtilis lipase: A combined computational and experimental investigation

8-Anilino-1-naphthalene sulfonate (ANS) and its covalent dimer bis-ANS are widely used for titrating hydrophobic surfaces of proteins. Interest to understand the nature of interaction of these dyes with proteins was seriously pursued. However as the techniques used in these studies varied, they often provided varied information regarding stoichiometry, binding affinity, actual binding sites etc. In the present study, we used combination of computation methods (docking and MD simulation) and experimental methods (mutations, steady-state and time-resolved fluorescence) to investigate bis-ANS interaction with Bacillus subtilis lipase. We identified seven binding sites for bis-ANS on lipase using computational docking and MD simulation and verified these data using a set of single amino acid substituted mutants. Docking and MD simulation studies indicated that the binding sites were various indentations and grooves on protein surface with hydrophobic characteristics. Both hydrophobic and ionic interactions were involved in each of these binding events. We further examine the fluorescence properties of bis-ANS bound to mutant lipases that either gained or lost a binding site. Our results indicated that neither gain nor loss of single binding site caused any change in fluorescence lifetimes (and their relative amplitudes) of mutant lipase-bound bis-ANS in comparison to that bound to wild type; hence, it suggested that nature of bis-ANS binding to each of the sites in lipase was very similar.     

Conservation of the Extended Substrate Specificity Profiles Among Homologous Granzymes Across Species

Granzymes are structurally related serine proteases involved in cell death and immunity. To date four out of five human granzymes have assigned orthologs in mice; however for granzyme H, no murine ortholog has been suggested and its role in cytotoxicity remains controversial. Here, we demonstrate that, as is the case for granzyme C, human granzyme H is an inefficient cytotoxin that together with their similar pattern of GrB divergence and functional similarity strongly hint to their orthologous relationship. Besides analyzing the substrate specificity profile of granzyme H by substrate phage display, substrate cleavage susceptibility of human granzyme H and mouse granzyme C was assessed on a proteome-wide level. The extended specificity profiles of granzymes C and H (i.e. beyond cleavage positions P4-P4′) match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes as granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can simply be conferred by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility.Several molecular mechanisms are in place to combat transformed malignant cells and virally infected cells. Granzymes (Gr)¹, a family of structurally related serine proteases found in the granules of many immune cells, play crucial roles in such cellular defense mechanisms. The granzyme family consists of five human proteases (granzymes A, B, H, K, and M) and 10 murine members (granzymes A to G, K, M, and N). To date for four human granzymes (A, B, K, and M) clear murine orthologs have been assigned, while the most probable murine ortholog of human granzyme H (GrH) is granzyme C (GrC) based on their 70% sequence similarity, 61% sequence identity, and identical chromosomal location relative to granzyme B (GrB). Furthermore, both granzymes are expressed by NK and CD4+ T-cells (1, 2): GrH is constitutively expressed at high levels in NK cells and less in CD4+ T cells, whereas GrC expression can be observed after stimulation. Thus, their overlapping expression profiles further support possible functional similarities between mouse GrC and human GrH.The physiological role of granzymes was presumed to be the induction of death in target cells. Granzyme B is a highly efficient cytotoxin (3) and other granzymes such as GrA, GrC, GrF, and GrK can cause cell death at high concentrations (4–8). Recently, granzymes A, K, and M were shown to steer inflammatory processes when used at physiological levels (9–11). Two previous studies identified GrH as an alternative cytotoxic effector protease. Although both studies showed typical hallmarks of apoptosis, including mitochondrial depolarization, reactive oxygen species (ROS) production, DNA degradation as well as chromatin condensation, Fellows et al. (12) found that, in contrast to GrB mediated cell death, GrH induced cell death did not result in caspase activation, cytochrome c release, or cleavage of Bid and/or ICAD. In sharp contrast however, Hou et al. (13) demonstrated that GrH induced apoptosis depended on caspase activation and that GrH cleaved ICAD and Bid, the latter ultimately resulting in mitochondrial cytochrome c release. Such discrepancies have been documented in other granzyme studies and are usually linked to the use of different granzyme delivery systems, sources of recombinantly produced granzymes, differences in the granzyme concentration and species-specific differences in substrate specificities (14).GrC induces cell death reminiscent of GrH induced cell death as observed by Fellows et al. (12), as both exert their cytotoxic functions independent of caspase activation, Bid or ICAD cleavage, or by mitochondrial release of cytochrome c (5). GrC induced apoptosis was characterized by the rapid externalization of phosphatidylserine, nuclear condensation and collapse, and single-stranded DNA nicking. Supporting evidence implying a role for GrC in lymphocyte induced cytotoxicity was inferred from the fact that GrB cluster-deficient mice (mice that do not express GrB and show a five- to sixfold reduced expression of GrC and GrF respectively) display a more pronounced defect in the clearance of allogeneic tumor cells when compared with GrB-only knockout mice (4). This suggests that GrC and/or GrF may be important for correct functioning of cytotoxic lymphocytes. Besides, in GrB-only knockout mice, a likely compensatory mechanism occurs, given that during cytotoxic lymphocyte activation, peak expression of GrC occurs earlier, giving rise to overall higher GrC levels as compared with wild-type mice (1, 4, 15). Of note, despite its implication in cytotoxicity, GrC was shown to be an inefficient cytotoxin, with a 2900-fold greater EC₅₀ value as compared with hGrB when delivered into P815 cells with recombinant mouse perforin (16).Positional Scanning Synthetic Combinatorial Libraries (PS-SCL) revealed the chymotrypsin-like activity of GrH, which it shares with granzyme M (17–19), with an optimal P4-P1 peptide substrate sequence Pro-Thr-Ser-Tyr. Less stringent specificities were observed at positions P4, P3, and P2 (19) where GrH seems to tolerate multiple amino acids with different chemical characteristics (especially neutral and aliphatic amino acids). Although GrH and GrM (optimal peptide identified as Lys-Val-Pro-Leu) share a P1 chymotryptic activity, GrH prefers bulkier, aromatic amino acids (Tyr and Phe) at P1 whereas GrM prefers Leu. Both further recognize Leu and Met at P1, implying that some substrates could be cleaved by both granzymes. GrM also shows broader specificities at P3, but at P4 and P2 it prefers basic residues and Pro respectively. Besides, GrC chymase activity could be inferred from N-terminal COFRADIC and substrate phage display screens, which defined the P4-P3′ substrate specificity of GrC as [Ile/Val]-X-[Phe-Tyr]-[Phe-Leu-Tyr-Met]↓X-[Gly-Ser]-[Asp-Glu] (16).Recently, the crystal structure of the D₁₀₂>N GrH variant in complex with a decapeptide substrate (PTSYAGDDSG) or inhibitor (Ac-PTSY-chloromethylketone) was resolved (20). The electron density maps clearly showed the full length protease adopting a canonical structure of 2 α-helices and 13 β-strands assembled into two juxtaposed β-barrel domains bridged by the catalytic triad (composed of His₅₇, Asp₁₀₂ and Ser₁₉₅). The S1 specificity pocket is built up from residues of 2 loops; loop 189 (from residue 183–196) and loop 220 (from residue 215–226) with the determinants being Thr₁₈₉, Gly₂₁₆, and Gly₂₂₆. From these, Gly₂₂₆ was assigned as the most important determinant for the preference of bulky aromatic amino acids (such as Tyr and Phe) at P1. Where GrH contains a Gly at position 226, GrB, GrC, and GrM harbor an Arg, Gln, and Pro residue respectively (supplemental Fig. S1). Mutation of Gly₂₂₆ in GrH to Arg₂₂₆ compromised binding of bulky aromatic residues and enabled interaction with negatively charged amino acids. Hydrogen bond formation between a P1 Tyr and Asn₂₁₇ of GrH could furthermore strengthen the observed preference of Tyr over Phe at P1 (19). The presence of Pro at position 226 in GrM instead of Gly narrows the S1 pocket and might be indicative for the preference of Leu instead of Phe and Tyr at P1. In addition, the structure revealed that the S4′ pocket formed by the backbones of the Arg₃₉-Lys₄₀-Arg₄₁ motif resulted in a preference for acidic residues at P4′, in addition to influencing the P3′ preference for acidic residues via salt bridge formation with this Lys₄₀. Although the Arg₃₉-Lys₄₀-Arg₄₁ motif is a unique GrH feature, GrB possesses a partially degenerate Leu-Lys-Arg motif in which Lys₄₀ also enables interaction with acidic residues in P3′. Next to P3′, proteome-wide screening for GrB substrates led to the identification of a clear preference for acidic residues at P4′ caused by salt bridge formation with Arg₄₁ (21), a characteristic that was previously assigned to Lys₄₀ of GrB (22). To validate these structural observations, similar to the P4′ Asp mutation in the GrB substrate PI-9 (22), both Asp residues found at P3′ and P4′ in the nuclear phosphoprotein La, previously identified as a macromolecular GrH substrate (23), were mutated to Ala. These mutations completely abolished GrH mediated proteolysis, a first indication that the P4-P1 specificity profile is not a sole determinant for substrate recognition by GrH. Next to the La phosphoprotein, cleavage of the viral adenovirus DNA binding protein (DBP) and the 100K assembly protein (L4–100K), the latter resulting in the relieve of GrB inhibition by L4–100K, has been observed, implicating GrH in host antiviral defense and indicative for a functional synergism between GrH and GrB (24).Elucidation of the crystal structure, with an electron density map showing residues Ile₁₆-His₂₄₄ (i.e. 94% of full length GrC and 99.6% of active GrC), furthermore showed that wild-type GrC can be restrained in its proteolytic function despite the presence of the catalytic triad residues His₅₇-Asp₁₀₂-Ser₁₉₅ (16). Comparing the crystal structures of GrC and GrA showed an unusual conformation of the active site, which could explain the inactivity of GrC. Apparently, the 190-strand, preceding Ser₁₉₅ of the catalytic triad, has undergone a register shift on the structural level leading to Phe₁₉₁ filling the S1 pocket. This pocket is furthermore covered by Glu₁₉₂, which forms a salt bridge with Arg₉₉ and a hydrogen bond with the backbone amide of Ser₁₉₅. Because of this unusual conformation, the Glu₁₉₂-Glu₁₉₃ peptide bond points away from the substrate, as such leading to an improperly formed oxyanion hole, which normally stabilizes the negatively charged substrate oxygen atom during catalysis. Mutation of Glu₁₉₂-Glu₁₉₃ to the corresponding amino acids in its closest related homolog GrB (Arg₁₉₂-Gly₁₉₃) disrupted the Glu₁₉₂-Ser₁₉₅ hydrogen bond and caused a shift of the 190-strand, thereby clearing the S1 pocket and giving rise to an active GrC mutant. These results indicate allosteric control of wild-type GrC in which binding of a substrate or cofactor might stabilize the 190-strand, which becomes extremely mobile due to breaking the Glu₁₉₂-Ser₁₉₅ hydrogen bond and turning the region surrounding the active site more rigid.To elucidate a possible functional homology between GrH and GrC, we performed differential degradome analyses using N-terminal COFRADIC in the species-matching proteome backgrounds. For these analyses, we made use of the active E₁₉₂E₁₉₃>RG GrC mutant as described in (16) (further referred to as mut GrC). These analyses, further complemented with phage display data on granzyme H, clearly show that both granzymes display a highly similar substrate specificity profile, analogous to other orthologous granzymes. Besides, and in contrast to GrA, a general conservation of the extended substrate specificity profiles among the homologous granzymes B, C, H, and M could be observed across species, highlighting the importance of the extended substrate specificity in steering substrate cleavage susceptibility.     

Analysis of Putative Apoplastic Effectors from the Nematode,Globodera rostochiensis,and Identification of an Expansin-Like Protein That Can Induce and Suppress Host Defenses

The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.     

Biological control of strawberry crown rot,root rot and grey mould by the beneficial fungus Aureobasidium pullulans

Utilization of biocontrol agents is a sustainable approach to reduce plant diseases caused by fungal pathogens. In the present study, we tested the effect of the candidate biocontrol fungus Aureobasidium pullulans (De Bary) G. Armaud on strawberry under in vitro and in vivo conditions to control crown rot, root rot and grey mould caused by Phytophthora cactorum (Lebert and Cohn) and Botrytis cinerea Pers, respectively. A dual plate confrontation assay showed that mycelial growth of P. cactorum and B. cinerea was reduced by 33–48% when challenged by A. pullulans as compared with control treatments. Likewise, detached leaf and fruit assays showed that A. pullulans significantly reduced necrotic lesion size on leaves and disease severity on fruits caused by P. cactorum and B. cinerea. In addition, greenhouse experiments with whole plants revealed enhanced biocontrol efficacy against root rot and grey mould when treated with A. pullulans either in combination with the pathogen or pre-treated with A. pullulans followed by inoculation of the pathogens. Our results demonstrate that A. pullulans is an effective biocontrol agent to control strawberry diseases caused by fungal pathogens and can be an effective alternative to chemical-based fungicides.

     

Highly Efficient Perovskite Solar Cells Enabled by Multiple Ligand Passivation

In the past decade, the efficiency of perovskite solar cells quickly increased from 3.8% to 25.2%. The quality of perovskite films plays vital role in device performance. The films fabricated by solution‐process are usually polycrystalline, with significantly higher defect density than that of single crystal. One kind of defect in the films is uncoordinated Pb²⁺, which is usually generated during thermal annealing process due to the volatile organic component. Another detrimental kind of defect is Pb⁰, which is often observed during the film fabrication process or solar cell operation. Because the open circuit voltage has a close relation with the defect density, it is thus desirable to passivate these two kinds of defects. Here, a molecule with multiple ligands is introduced, which not only passivates the uncoordinated Pb²⁺ defects, but also suppresses the formation of Pb⁰ defects. Meanwhile, such a treatment improves the energy level alignment between the valence band of perovskite and the highest occupied molecular orbital of spiro‐OMeTAD. As a result, the performance of perovskite solar cells significantly increases from 19.0% to 21.4%.