Genetic and expression analysis of all non‐synonymous single nucleotide polymorphisms in the human deoxyribonuclease I‐like 1 and 2 genes

Members of the human DNase I family, DNase I‐like 1 and 2 (DNases 1L1 and 1L2), with physiological role(s) other than those of DNase I, possess three and one non‐synonymous SNPs in the genes, respectively. However, only limited population data are available, and the effect of these SNPs on the catalytic activity of the enzyme remains unknown. Genotyping of all the non‐synonymous SNPs was performed in three ethnic groups including six different populations using the PCR‐RFLP method newly developed. Asian and African groups including Japanese, Koreans, Ghanaians and Ovambos were typed as a single genotype at each SNP, but polymorphism at only SNP V122I in DNase 1L1 was found in Caucasian groups including Germans and Turks; thus a Caucasian‐specific allele was identified. The DNase 1L1 and 1L2 genes show relatively low genetic diversity with regard to these non‐synonymous SNPs. The level of activity derived from the V122I, Q170H and D227A substituted DNase 1L1 corresponding to SNPs was similar to that of the wild‐type, whereas replacement of the Asp residue at position 197 in the DNase 1L2 protein with Ala, corresponding to SNP D197A, reduced its activity greatly. Thus, SNP V122I in DNase 1L1 exhibiting polymorphism exerts no effect on the catalytic activity, and furthermore SNP D197A in DNase 1L2, affecting its catalytic activity, shows no polymorphism. These findings permit us to postulate that the non‐synonymous SNPs identified in the DNase 1L1 and 1L2 genes may exert no influence on the activity levels of DNases 1L1 and 1L2 in human populations.     

Global genetic analysis of all single nucleotide polymorphisms in exons of the human deoxyribonuclease I-like 3 gene and their effect on its catalytic activity

Deoxyribonucleases (DNases) have been suggested to be implicated in the pathophysiology of autoimmune diseases. In the DNASE1L3 gene encoding human DNase I‐like 3 (DNase 1L3), a member of the DNase I family, only two non‐synonymous (R178 H and R206C) single nucleotide polymorphisms (SNPs) have been examined [Ueki et al., Clin. Chim. Acta 2009, 407, 20–24]. Three other non‐synonymous (G82R, K96N, and I243M) and four synonymous (S17S, T84T, R92R, and A181A) SNPs, in addition to R206C and R178H, have been identified in DNASE1L3. We investigated the distribution of all these SNPs in exons of the gene in eight Asian, three African, and three Caucasian populations worldwide using newly devised genotyping methods. SNP T84T showed polymorphism in all the populations, and R92R was polymorphic in the three African and three Caucasian populations; R206C was distributed only in Caucasian populations. In contrast, no minor allele was found in five SNPs (S17S, G82R, K96N, A181A, and I243M) in DNASE1L3. Generally, the DNase 1L3 gene shows relatively low genetic diversity with regard to exonic SNPs. When the effect of amino acid/nucleotide substitutions resulting from the SNPs on DNase 1L3 activity was examined, none of the synonymous SNPs had any effect on the DNase 1L3 activity, whereas among non‐synonymous SNPs, SNP G82R diminished the activity of the enzyme, being similar to R206C. These findings permit us to assume that, although only R206 exhibits polymorphisms in a Caucasian‐specific manner, at least SNPs G82R and R206C in DNASE1L3 might be potential risk factors for autoimmune disease.     

Global analysis of single nucleotide polymorphisms in the exons of human deoxyribonuclease I-like 1 and 2 genes

Several SNPs in the deoxyribonuclease I-like 1 (DNase 1L1) and DNase 1L2 were investigated. In the present study, the genotype distributions of three synonymous SNPs (V59V, rs1050095; P67P, rs1130929; A277A, rs17849495) in the DNase 1L1 gene and four non-synonymous SNPs, V122I (rs34952165), Q170H (rs6643670), and D227A (rs5987256) in the DNase 1L1 gene, as well as D197A (rs62621282) in the DNase 1L2 gene were investigated in 13 populations. In all the populations, no variation was found in four SNPs (V59V, Q170H, D227A, and A277A) in DNASE1L1 or in D197A in DNASE1L2. As for V122I, only the German population showed a low degree of polymorphism. The SNP V122I in DNASE1L1 was monoallelic for the G-allele in all of the Asian and African populations examined, with no polymorphism being evident. Since the A-allele in SNP V122I was distributed in only the Caucasian populations, not in the other ethnic groups, it was confirmed that the A-allele in SNP V122I was Caucasian-specific. On the other hand, only P67P in DNASE1L1 was polymorphic among three synonymous SNPs. The effect of nucleotide substitution corresponding to polymorphic SNP P67P on DNase 1L1 activity was examined: the corresponding nucleotide substitution in polymorphic SNP P67P has little effect on the DNase activity.     

Seven nonsynonymous SNPs in the gene encoding human deoxyribonuclease II may serve as a functional SNP potentially implicated in autoimmune dysfunction

Many nonsynonymous SNPs in the human DNase II gene (DNASE2), potentially relevant to autoimmunity in conditions such as rheumatoid arthritis, have been identified, but only limited population data are available and no studies have evaluated whether such SNPs are functional. Genotyping of all the 15 nonsynonymous human DNase II SNPs was performed in three ethnic groups including 16 different populations using the PCR‐restriction fragment length polymorphism technique. A series of constructs corresponding to each SNP was examined. Fifteen nonsynonymous SNPs in the gene, except for p.Val206Ile in a Korean population, exhibited a mono‐allelic distribution in all of the populations. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, four activity‐abolishing and five activity‐reducing SNPs were confirmed to be functional. The amino acid residues in activity‐abolishing SNPs were conserved in animal DNase II. All the nonsynonymous SNPs that affected the catalytic activity of human DNase II showed extremely low genetic heterogeneity. However, a minor allele of seven SNPs producing a loss‐of‐function or extremely low activity‐harboring variant could serve as a genetic risk factor for autoimmune dysfunction. These functional SNPs in DNASE2 may have clinical implications in relation to the prevalence of autoimmune diseases.     

Effect of urea on analyte complexation by 2,6‐dimethyl‐β‐CD in peptide enantioseparations by CE

The aim of the present study was the investigation of the effect of urea on analyte complexation in CD‐mediated separations of peptide enantiomers by CE in the pH range of about 2–5. pH‐independent complexation and mobility parameters in the absence and presence of 2 M urea were obtained by three‐dimensional, non‐linear curve fitting of the effective analyte mobility as a function of pH and heptakis‐(2,6‐di‐O‐methyl)‐β‐CD concentration. Urea led to decreased binding strength of the CD towards the protonated and neutral analyte enantiomers as well as to decreased mobilities of the free analytes. In contrast, mobilities of the fully protonated enantiomer–CD complexes as well as the pK_a values of the free and complexed analytes increased. The effect of urea on separation efficiency varied with pH and CD concentration. In the case of Ala‐Tyr and Ala‐Phe, separations improved in the presence of urea at pH 2.2. In contrast, separations were impaired by urea at pH 3.8 and low concentrations of the CD. Decreased separation efficiency was noted for Asp‐PheOMe and Glu‐PheNH₂ at low CD concentrations when urea was added but separations improved at higher CD concentrations over the entire pH range studied. The effect of urea on analyte complexation appeared to be primarily non‐stereoselective. Furthermore, the pH‐dependent reversal of the enantiomer migration order observed for Ala‐Tyr and Ala‐Phe can be rationalized by the complexation and mobility parameters.     

Genetic and expression analysis of SNPs in the human deoxyribonuclease II: SNPs in the promoter region reduce its in vivo activity through decreased promoter activity

Five SNPs in the human DNase II gene have been reported to be associated with rheumatoid arthritis (RA). Genotype and haplotype analysis of 14 SNPs, nine SNPs of which reported in the NCBI dbSNP database in addition to these five SNPs, was performed in healthy subjects. The enzymatic activities of the amino acid substituted DNase II corresponding to each SNP and serum DNase II in healthy Japanese, and promoter activities derived from each haplotype of the RA‐related SNPs were measured. Significant correlations between genotype in each RA‐related SNP and enzymatic activity levels were found; alleles associated with RA exhibited a reduction in serum DNase II activity. Furthermore, the promoter activities of each reporter construct corresponding to predominant haplotypes in three SNPs in the promoter region of the gene exhibited significant correlation with levels of serum DNase II activity. These findings indicate these three SNPs could alter the promoter activity of DNASE2, leading to a decline in DNase II activity in the serum through gene expression. Since the three SNPs in the promoter region of the DNase II gene could affect in vivo DNase II activity through reduction of the promoter activity, it is feasible to identify these SNPs susceptible to RA.     

Analysis of variation in the ovine ultra‐high sulphur keratin‐associated protein KAP5‐4 gene using PCR‐SSCP technique

Keratin‐associated proteins (KAPs) are one of the main structural components of the wool fibre. Variation in the KAP genes (KRTAPs) may affect the structure of KAPs and hence wool characteristics. In this study, we used PCR‐SSCP to analyse ovine KRTAP5‐4, a gene encoding a member of the KAP5 family. Five different PCR‐SSCP patterns were detected in the 250 sheep that were analysed. Either one or a combination of two patterns was observed for each sheep, which was consistent with these sheep being either homozygous or heterozygous at this locus. DNA sequencing revealed that these patterns represent five different DNA sequences. One of the sequences was identical to a published ovine KRTAP5‐4 sequence. The remaining four were unique, but shared a high homology with the published ovine KRTAP5‐4 sequence, suggesting that these sequences represent allelic variants of KRTAP5‐4. There were a total of six SNPs and one length polymorphism in the sequences. Of the five SNPs found in the coding region, four were non‐synonymous SNPs and would result in amino acid changes. The length polymorphism would affect the cysteine content of the putative peptide and this along with the SNPs may have an impact on the structure of KAP5‐4, and hence affect wool traits.     

Confirmation that SNPs in the high mobility group‐A2 gene (HMGA2) are associated with adult height in the Japanese population; wide‐ranging population survey of height‐related SNPs in HMGA2

Adult height is a highly heritable trait in that multiple genes are involved. Recent genome‐wide association studies have identified a novel single‐nucleotide polymorphism (SNP) rs1042725 in the high mobility group‐A2 gene (HMGA2) and shown it to be associated with human height in Caucasian populations. We performed a replication study to examine the associations between SNPs in HMGA2 and adult height in the Japanese population based on autopsy cases. Although we could not confirm a significant association between rs1042725 in HMGA2 and adult height, another SNP, rs7968902, in the gene achieved significance for its association in the same populations, and the effect was the same as that documented previously. These findings permit us to conclude that the SNPs in HMGA2 are common variants influencing human height across different populations. Moreover, a worldwide population study of these SNPs using 14 different populations including Asians, Africans and Caucasians demonstrated that both haplotypes and genotypes for three height‐related SNPs (rs1042725, rs7968682 and rs7968902) in HMGA2 were distributed in an ethnicity‐dependent manner. This information will be useful for clarifying the genetic basis of human height.     

Design,Synthesis, and Evaluation of 3‐((4‐(t‐Butyl)‐2‐(2‐benzylidenehydrazinyl)thiazol‐5‐yl)methyl)quinolin‐2(1H)‐ones as Neuraminidase Inhibitors

A series of novel 3‐((4‐(t‐butyl)‐2‐(2‐benzylidenehydrazinyl)thiazol‐5‐yl)methyl)quinolin‐2(1H)‐ones ( 7a – 7z ) were designed, synthesized and evaluated for their ability of inhibiting neuraminidase (NA) of in?uenza H1N1 virus. Some compounds displayed moderate influenza NA inhibitory activity. Compound 7l with the scaffold of 2‐(2‐(2‐methoxybenzylidene)hydrazinyl)thiazole was the best one, exhibiting moderate NA inhibitory activity with IC₅₀ of 44.66 µmol/L. Structure‐activity relationship showed that compounds with methoxy or hydroxy groups at the ortho position, fluorine and nitro groups at the meta position and chlorine and bromine groups at the para position of phenyl ring were more active. Docking study indicated that compound 7l has important interactions with some key residues (including Asp151, Glu119, Arg292, Tyr406, and Asn347) and binds to 430‐cavity adjacent to NA active site.     

The self‐assembled of cyclic D,L‐α‐peptide systems: Insights into the structure and energetics

Cyclic D,L ‐α‐peptides are able to self‐assemble to nanotubes, although the inherent reason of the stability of this kind of nanotube as well as the intrinsic driving force of self‐assembly of the cyclic D ,L ‐α‐peptides still remain elusive. In this work, using several computational approaches, we investigated the structural and energy characteristics of a series of cyclo[(‐L ‐Phe‐D ‐Ala‐)₄] and cyclo[(‐L ‐Ala‐D ‐Ala‐)₄] oligomers. The results reveal that the thermodynamic stability, cooperativity, and self‐assembly patterns of cyclic D ,L ‐α‐peptide nanotubes are mainly determined by the interactions between cross‐strand side chains instead of those between backbones. For cyclo[(‐L ‐Phe‐D ‐Ala‐)₄] oligomers, the steric interaction between cross‐strand side chains, especially the electrostatic repulsion between the phenyls in Phe residues, brings anticooperative effect into parallel stacking mode, which is responsible for the preference of self‐assembling nanotube in antiparallel vs. parallel stacking orientation. Based on our results, a novel self‐assembling mechanism is put forward—it is the L ‐L antiparallel dimer of cyclo[(‐L ‐Phe‐D ‐Ala‐)₄], instead of the commonly presumed monomer, that acts as the basic building block in self assembly. It explains why these cyclic peptides uniquely self‐assemble to form antiparallel nanotubes. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010     

Synthesis of Sequential Polypeptides Containing O‐Phospho‐L‐Serine

The chemistry of phosphoserine [Ser(P)] containing peptides and polypeptides was extensively investigated to explore a new biomineralization material science. The selective cleavage of the O,O′‐diphenyl phospho‐protecting groups of Ser(PO₃Ph₂) was examined using hydrogenolysis catalysts. Among the catalysts examined, only PtO₂ in 50% trifluoroacetic acid (TFA)/AcOH successfully cleaved the protecting group of Ser(PO₃Ph₂) to give Ser(P). Based on these characteristic new findings, Ser(P)‐containing dipeptides such as Gly‐Ser(P), Ala‐Ser(P), Ser‐Ser(P), Asp‐Ser(P), Glu‐Ser(P), and Lys‐Ser(P), and tetrapeptide [Asp‐Ser(P)]₂ were synthesized by a facile method. When we used the Ser(PO₃Ph₂) residues at the C terminals, the amino functional groups of amino acids and peptides can be coupled by the unsymmetric mixed anhydride using isobutyl chloroformate but cannot be by the symmetric anhydride method using dicyclohexylcarbodiimide. Neither unsymmetric mixed anhydride nor symmetric anhydride can be coupled with p‐nitrophenol at their C terminals. High‐molecular‐weight sequential polypeptides containing Ser(P) such as poly[Ser(P)‐Xaa] (Xaa: Gly, Ala, Ser, Lys, Asp, Glu) and poly[Gly‐Ser(P)‐Gly] were first synthesized by the polycondensation of the di‐ and tripeptide p‐nitrophenyl active esters, followed by the quantitative elimination of the diphenyl protecting groups by PtO₂ in TFA/AcOH. The new strategy to synthesize Ser(P)‐containing peptides and model proteins may help the development of hybrid formulations of marine and biomimetic protein minerals.

     

Comparative α‐Helicity of Cyclic Pentapeptides in Water

Helix‐constrained polypeptides have attracted great interest for modulating protein–protein interactions (PPI). It is not known which are the most effective helix‐inducing strategies for designing PPI agonists/antagonists. Cyclization linkers (X₁–X₅) were compared here, using circular dichroism and 2D NMR spectroscopy, for α‐helix induction in simple model pentapeptides, Ac‐cyclo(1,5)‐[X₁‐Ala‐Ala‐Ala‐X₅]‐NH₂, in water. In this very stringent test of helix induction, a Lys1→Asp5 lactam linker conferred greatest α‐helicity, hydrocarbon and triazole linkers induced a mix of α‐ and 3₁₀‐helicity, while thio‐ and dithioether linkers produced less helicity. The lactam‐linked cyclic pentapeptide was also the most effective α‐helix nucleator attached to a 13‐residue model peptide.     

SNaPshot minisequencing to resolve mitochondrial macro‐haplogroups found in Africa

African mitochondrial DNA (mtDNA) haplogroups are divided into seven macro‐haplogroups (L0′1′2′3′4′5′6), while the rest of the world's lineages are classified as subgroups of macro‐haplogroups M, N and R. The most common approach to characterizing mtDNA variation is the sequencing of hypervariable segments I and II of the non‐coding control region of the molecule. Given the higher mutation rate within the control region compared with the coding regions of the molecule, recurrent mutations in the former can sometimes hide possible phylogenetic structure. The incorporation of haplogroup‐defining coding region mutations has helped in overcoming this limitation. By judiciously selecting 14 coding region SNPs and incorporating them into a multiplex minisequencing assay we were able to resolve mtDNA sequences from some sub‐Saharan African populations into ten macro‐haplogroups (L0–L6, M, N and R). We tested the efficacy of the panel by screening 699 individuals, consisting mostly of Khoe‐San, Bantu speakers and individuals with mixed ancestries (Coloreds) and found no inconsistencies compared with hypervariable segment sequencing results. The panel provided a fast and efficient means of classifying mtDNA into the ten mitochondrial macro‐haplogroups and provided a reliable screening to distinguish African from non‐African‐derived mtDNA lineages.     

cis‐trans Peptide‐Bond Isomerization in α‐Methylproline Derivatives

α‐Methyl‐L ‐proline is an α‐substituted analog of proline that has been previously employed to constrain prolyl peptide bonds in a trans conformation. Here, we revisit the cis‐trans prolyl peptide bond equilibrium in derivatives of α‐methyl‐L ‐proline, such as N‐Boc‐protected α‐methyl‐L ‐proline and the hexapeptide H‐Ala‐Tyr‐αMePro‐Tyr‐Asp‐Val‐OH. In Boc‐α‐methyl‐L ‐proline, we found that both cis and trans conformers were populated, whereas, in the short peptide, only the trans conformer was detected. The energy barrier for the cis‐trans isomerization in Boc‐α‐methyl‐L ‐proline was determined by line‐shape analysis of NMR spectra obtained at different temperatures and found to be 1.24 kcal/mol (at 298 K) higher than the corresponding value for Boc‐L ‐proline. These findings further illuminate the conformationally constraining properties of α‐methyl‐L ‐proline.     

The Catalytic Mechanism of the Marine‐Derived Macrocyclase PatGmac

Cyclic peptides are a class of compounds with high therapeutic potential, possessing bioactivities including antitumor and antiviral (including anti‐HIV). Despite their desirability, efficient design and production of these compounds has not been achieved to date. The catalytic mechanism of patellamide macrocyclization by the PatG macrocyclase domain has been computationally investigated by using quantum mechanics/molecular mechanics methodology, specifically ONIOM(M06/6‐311++G(2d,2p):ff94//B3LYP/6‐31G(d):ff94). The mechanism proposed herein begins with a proton transfer from Ser783 to His 618 and from the latter to Asp548. Nucleophilic attack of Ser783 on the substrate leads to the formation of an acyl–enzyme covalent complex. The leaving group Ala‐Tyr‐Asp‐Gly (AYDG) of the substrate is protonated by the substrate's N terminus, leading to the breakage of the P1?P1′ bond. Finally, the substrate's N terminus attacks the P1 residue, decomposing the acyl–enzyme complex forming the macrocycle. The formation and decomposition of the acyl–enzyme complex have the highest activation free energies (21.1 kcal mol^?1 and 19.8 kcal mol^?1 respectively), typical of serine proteases. Understanding the mechanism behind the macrocyclization of patellamides will be important to the application of the enzymes in the pharmaceutical and biotechnological industries.     

Structure‐Based Design and Synthesis of the First Weak Non‐Phosphate Inhibitors for IspF,an Enzyme in the Non‐Mevalonate Pathway of Isoprenoid Biosynthesis

In this paper, we describe the structure‐based design, synthesis, and biological evaluation of cytosine derivatives and analogues that inhibit IspF, an enzyme in the non‐mevalonate pathway of isoprenoid biosynthesis. This pathway is responsible for the biosynthesis of the C₅ precursors to isoprenoids, isopentenyl diphosphate (IPP, 1 ) and dimethylallyl diphosphate (DMAPP, 2 ; Scheme 1). The non‐mevalonate pathway is the sole source for 1 and 2 in the protozoan Plasmodium parasites. Since mammals exclusively utilize the alternative mevalonate pathway, the enzymes of the non‐mevalonate pathway have been identified as attractive new drug targets in the fight against malaria. Based on computer modeling (cf. Figs. 2 and 3), new cytosine derivatives and analogues (Fig. 1) were selected as potential drug‐like inhibitors of IspF protein, and synthesized (Schemes 2–5). Determination of the enzyme activity by ¹³C‐NMR spectroscopy in the presence of the new ligands showed inhibitory activities for some of the prepared cytosine and pyridine‐2,5‐diamine derivatives in the upper micromolar range (IC₅₀ values; Table). The data suggest that it is possible to inhibit IspF protein without binding to the polar diphosphate binding site and the side chain of Asp56′, which interacts with the ribose moiety of the substrate and substrate analogues. Furthermore, a new spacious sub‐pocket was discovered which accommodates aromatic spacers between cytosine derivatives or analogues (binding to ‘Pocket III’) and rings that occupy the flexible hydrophobic region of ‘Pocket II’. The proposed binding mode remains to be further validated by X‐ray crystallography.     

Enzyme Modification and Oxidative Cross‐linking Using Carboxylate‐, Phenol‐ and Catechol‐Conjugated 1,8‐Naphthalimides

The ground‐ and excited‐state interactions of β‐alanine, tyrosine and l ‐dopa substituted 1,8 naphthalimides (NI‐Ala, NI‐Tyr and NI‐Dopa) with lysozyme and mushroom tyrosinase were evaluated to understand the mechanism of oxidative modification. Photooxidative cross‐linking of lysozyme was observed for all three conjugates. The yield was significantly reduced for NI‐Tyr and NI‐Dopa due to intramolecular electron transfer to the excited singlet state of the 1,8‐naphthalimide. Incubation of NI‐Tyr and NI‐Dopa with mushroom tyrosinase resulted in an increased fluorescence from the naphthalimide, suggesting that the phenol and catechol portion of the conjugates are oxidized by the enzyme. This result demonstrates that the compounds bind in the active site of mushroom tyrosinase. The catalytic activity of mushroom tyrosinase to oxidize both tyrosine (monophenolase) and l ‐dopa (diphenolase) was modified by NI‐Tyr and NI‐Dopa. Monophenolase activity was inhibited, and the diphenolase activity was enhanced in the presence of these conjugates. Detailed Michaelis–Menten studies show that both V_max and K_m are modified, consistent with a mixed inhibition mechanism. Collectively, the results show that the compounds interact in the enzyme's active site, but also modify the distribution of the enzyme's oxidation states that are responsible for catalysis.     

The Propensity of α‐Aminoisobutyric Acid (=2‐Methylalanine; Aib) to Induce Helical Secondary Structure in an α‐Heptapeptide: A Computational Study

We present a molecular‐dynamics simulation study of an α‐heptapeptide containing an α‐aminoisobutyric acid (=2‐methylalanine; Aib) residue, Val¹‐Ala²‐Leu³‐Aib⁴‐Ile⁵‐Met⁶‐Phe⁷, and a quantum‐mechanical (QM) study of simplified models to investigate the propensity of the Aib residue to induce 3₁₀/α‐helical conformation. For comparison, we have also performed simulations of three analogues of the peptide with the Aib residue being replaced by L ‐Ala, D ‐Ala, and Gly, respectively, which provide information on the subtitution effect at C(α) (two Me groups for Aib, one for L ‐Ala and D ‐Ala, and zero for Gly). Our simulations suggest that, in MeOH, the heptapeptide hardly folds into canonical helical conformations, but appears to populate multiple conformations, i.e., C₇ and 3₁₀‐helical ones, which is in agreement with results from the QM calculations and NMR experiments. The populations of these conformations depend on the polarity of the solvent. Our study confirms that a short peptide, though with the presence of an Aib residue in the middle of the chain, does not have to fold to an α‐helical secondary structure. To generate a helical conformation for a linear peptide, several Aib residues should be present in the peptide, either sequentially or alternatively, to enhance the propensity of Aib‐containing peptides towards the helical conformation. A correction of a few of the published NMR data is reported.     

MDM2‐Associated Clusterization‐Triggered Emission and Apoptosis Induction Effectuated by a Theranostic Spiropolymer

Heteroatom‐containing spiropolymers were constructed in a facile manner by a catalyst‐free multicomponent spiropolymerization route. P1a2b as the most potent of these spiropolymers, demonstrates cluster‐triggered emission resulting from strong interactions with the MDM2 protein. By preventing the anti‐apoptotic p53/MDM2 interaction, P1a2b triggers apoptosis in cancerous cells, while demonstrating a good biocompatibility and non‐toxicity in non‐cancerous cells. The combined results from solution and cell‐based cluster‐triggered emission studies, docking, protein expression experiments and cytotoxicity data strongly support the MDM2‐binding hypothesis and indicate a potential application as a fluorescent cancer marker as well as therapeutic for this spiropolymer.     

CdSeTe@CdS@ZnS Quantum‐Dot‐Sensitized Macroporous Tio2 Film: A Multisignal‐Amplified Photoelectrochemical Platform

A macroporous TiO₂ film (M‐TiO₂), which was prepared by burning off the polystyrene microsphere (PS) template from a PS/TiO₂ composite film, can provide a large active surface, improve electron‐transport performance, and increase the photocurrent. Furthermore, core–shell–shell CdSeTe@CdS@ZnS quantum dots (QDs) were introduced to sensitize the M‐TiO₂ film, which can efficiently broaden the absorption spectra range, separate and transfer charge carriers, reduce recombination loss, and improve photovoltaic response, with a sensitization shell of CdS and a passivation shell of ZnS. A multisignal‐amplified photoelectrochemical platform was fabricated by further modifying this film with a combination of biotin–DEVD–peptide (Biotin–Gly–Asp–Gly–Asp–Glu–Val–Asp–Gly–Cys) (which is specifically cleaved by caspase‐3) and streptavidin‐labeled alkaline phosphatase (SA‐ALP). Under the enzymatic catalysis of ALP with the substrate 2‐phospho‐L ‐ascorbic acid trisodium salt (AAP), ascorbic acid (AA) was generated as a better electron donor, leading to increased photocurrent output. The activity of caspase‐3, which depends on the amount of residual peptide on the electrode, was inversely proportional to the amount of AA. By monitoring the variation of photocurrent caused by AA, caspase‐3 activity and the therapeutic effect of nilotinib (a special medicine of chronic myeloid leukemia, CML) were indirectly detected and evaluated. The photoelectrochemical platform can be used as a potential evaluation system for monitoring caspase‐3 activity and drug effects.