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[Display omitted] • PNA-nitrobiphenyl photosensitizer conjugates distortionlessly formed duplexes with DNA. • The singlet oxygen generated from the duplexes tended to be quenched by the PNA backbone. • The PNA conjugates on the sticky end of DNA photooxidized G in the double strand DNA region only. Nitrobiphenyl photosensitizer (NBP)-peptide nucleic acids (PNA) conjugates were synthesized to develop a tool for photo-knockdown of target DNAs. The presence of NBP hardly hindered duplex formation with the complementary single strand DNA as demonstrated by the comparison of T m values and CD spectra with those for standard PNA/DNA duplexes. However, the photooxidation of guanines in NBP-PNA/DNAs was significantly less effective than those of corresponding NBP-DNA/DNA. Production of singlet oxygen (1O 2) during the photooxidation was confirmed by consumption of furfuryl alcohol, a 1O 2 detector. The poor photooxidation efficiency was ameliorated with 1O 2 generated from an externally added NBP derivative. It was found that, when complexed with the sticky end of a double strand DNA, NBP-PNA was able to photooxidize G in the DNA/DNA duplex region, whereas G in the PNA/DNA duplex region was considerably unreactive. These results suggest that 1O 2 produced from NBP-PNA tends to quench during diffusion along the PNA/DNA backbone, whereas quenching is less likely during diffusion along DNA/DNA region. [ABSTRACT FROM AUTHOR]

To realize nucleic acid-targeting photodynamic therapy, a photosensitizer should be attached at the optimal position on a complementary oligonucleotide, where a guanine photooxidation is maximized. Here we show the photooxidation of 22 DNA duplexes with varied lengths between a 1O2-generating biphenyl photosensitizer attached at a midchain thymine in a strand and the single guanine reactant in the other strand. The best photooxidation efficiencies are achieved at 9, 10, and 21 base intervals, which coincides with the pitch of 10.5 base pairs per turn in a DNA duplex. The low efficiencies for near and far guanines are due to quenching of the biphenyl by guanine and dilution of 1O2 by diffusion, respectively. The 1O2-diffusion mapping along DNA duplex provides clues to the development of efficient and selective photosensitizer agents for nucleic acid-targeting photodynamic therapy, as well as an experimental demonstration of diffusion of a particle along cylindrical surface in molecular level. [ABSTRACT FROM AUTHOR]

[Display omitted] • Azobenzene is incorporated in the middle of acyl chain of α -galactosyl ceramide. • E -to- Z photoisomerization is facilitated in a buffer solution rather than in water. • Photoirradiation promoted CD1d-TCR binding and cytokine production. • Docking study suggests the importance of CD1d conformational change. α-Galactosyl ceramide (α-GalCer) is a promising adjuvant in cancer immunotherapy that activates invariant natural killer T (iNKT) cells in a CD1d-dependent manner, but an immunosuppression (anergic effect) due to frequent administration has been a problem. To prevent anergy by spatiotemporally limiting the adjuvant effect, we previously synthesized α-GalCer analogs with an E -azobenzene at the tip of acyl chain and conducted E -to- Z photoisomerization, which would ease binding of the acyl chain to an antigen-presenting protein, CD1d, and thus boost the formation of active and specific triplex {iNKT cell bearing T-cell receptor (iNKT-TCR)/α-GalCer/CD1d}. However, boosting was at most only 1.2 times. In this study, we synthesized eight α-GalCer analogs with an azobenzene incorporated into the middle of acyl chain to enhance the difference between E - and Z -isomers in their abilities of bindings to CD1d and thus of cytokine induction. Flow cytometric analysis indicated that three Z isomers were 3.2 to 6.8 folds more active than E isomers in the triplex formation. Docking simulations for α-GalCer/CD1d duplex formation, however, demonstrated only 1.1 to 1.2 folds favorability of Z -isomers over E -isomers. Difference of two results suggests that E -to- Z isomerization might cause conformational changes in CD1d protein to facilitate binding to iNKT-TCR. Cytokine productions were mostly more prominent with Z -isomers than E -isomers (0.73 ∼ 9.35 folds for IFN-γ; 1.23 ∼ 6.52 folds for IL-4), whereas the produced amounts of cytokines were 1.5-to-15 folds lower than those by α-GalCer. Interestingly, an α-GalCer analog with azobenzene at acyl chain tip that was blunt in photoswitching triplex formation showed distinctive photoinduced cytokine productions. This result might be due to promotion of interaction between iNKT-TCR and CD1d molecule. [ABSTRACT FROM AUTHOR]

Summary: Previous genomic studies have revealed the genomic landscape of myeloma cells. Although some of the genomic abnormalities shown are believed to be correlated to the molecular pathogenesis of multiple myeloma and/or clinical outcome, these correlations are not fully understood. The aim of this study is to elucidate the correlation between genomic abnormalities and clinical characteristics by targeted capture sequencing in the Japanese multiple myeloma cohort. We analysed 154 patients with newly diagnosed multiple myeloma. The analysis revealed that the study cohort consisted of a less frequent hyperdiploid subtype (37·0%) with relatively high frequencies of KRAS mutation (36·4%) and IGH‐CCND1 translocation (26·6%) compared with previous reports. Moreover, our targeted capture sequencing strategy was able to detect rare IGH‐associated chromosomal translocations, such as IGH‐CCND2 and IGH‐MAFA. Interestingly, all 10 patients harboured MAX mutations accompanied by 14q23 deletion. The patients with del(17p) exhibited an unfavourable clinical outcome, and the presence of KRAS mutation was associated with shorter survival in patients with multiple myeloma, harbouring IGH‐CCND1. Thus, our study provides a detailed landscape of genomic abnormalities, which may have potential clinical application for patients with multiple myeloma. [ABSTRACT FROM AUTHOR]

Fluorescent nucleosides with modified nucleobases are useful tools for detecting nucleic acids and probing their structures and functions. Nucleobases are suitable for modification because 1) intrinsically light-absorbing nucleobases can be converted to fluorescent chromophores by simple chemical modification, 2) attaching substituents to nucleobases at appropriately selected positions does not inhibit base pairing or duplex formation, and 3) duplex formation and protein interactions affect the environment of nucleobases, causing changes in their fluorescence intensities and/or wavelengths. This review summarizes recent fluorescent nucleosides and their photophysical properties, such as absorption wavelength, emission wavelength, and fluorescence quantum yield together with their solvent dependency. [ABSTRACT FROM AUTHOR]

The Escherichia coli (E. coli)-based protein synthesis using recombinant elements (PURE) system is a cell-free protein synthesis system reconstituted from purified factors essential for E. coli translation. The PURE system is widely used for basic and synthetic biology applications. One of the major challenges associated with the PURE system is that the protein yield of the system varies depending on the protein. Studies have reported that the efficiency of translation is significantly affected by nucleotide and amino acid sequences, especially in the N-terminal region. Here, we investigated the inherent effect of various N-terminal sequences on protein synthesis using the PURE system. We found that a single amino acid substitution in the N-terminal region significantly altered translation efficiency in the PURE system, especially at low temperatures. This result gives us useful suggestions for the expression of the protein of interest in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Lanthanide nanoparticles (LNPs) conjugated with monosaccharides were synthesized as a photon energy-upconverting nanodevice with affinity to cancer cells. The conjugates were designed to selectively damage the cancer cells containing protoporphyrin IX, a photosensitizer endogenously synthesized from priorly administrated 5-aminolevlunic acid (ALA), by a highly tissue-penetrative near-infrared (NIR) irradiation. First of all, the affinities of monosaccharides toward cells (HeLa, RAW264.7, and MKN45) were assessed by a novel cell aggregation assay with trivalent monosaccharide-citric acid conjugates. As a result, HeLa exhibited high affinity for glucose, while RAW264.7 for glucose, galactose, mannose, and fucose. A similar cell-monosaccharide affinity was microscopically observed when the cells were mixed with monosaccharide-LNP conjugates and rinsed, in which the high affinity LNP probes luminesced on the cells. The high affinity monosaccharide-LNPs showed greater photodamaging effects than the unmodified LNP toward the corresponding cells, when the cells were pretreated with ALA and irradiated by NIR. This study demonstrates that carbohydrates can be used as selective ligands for cancer cells in a photodynamic therapy with LNP. [ABSTRACT FROM AUTHOR]

Here, we serendipitously found that photoirradiation of nitrobenzene derivatives with alkyl thiols produced sulfonamides in low yields without the addition of other reagents. We proposed a new photoreaction mechanism, in which the photo-excited nitrobenzene biradical abstracts a hydrogen atom from H-S, the resultant thiyl radical undergoes the coupling with the nitrogen atom in the nitro group, and the two oxygen atoms in the nitro group are transferred onto the sulfur atom. The photoreaction by-produced p-sulfanyl, aniline, and o-sulfonyl derivatives presumably through SRN1 mechanism from the p-substituted nitrobenzene, photo-desulfonylation, and photo-Fries rearrangement from the sulfonamide, respectively. Discovery of this photoreaction will expand the scope of the chemistry of nitro and thiol compounds, since the sulfonamides are expected to find extensive applications. [ABSTRACT FROM AUTHOR]

Ribosome display utilizes formation of the mRNA–ribosome–polypeptide ternary complex in a cell-free protein synthesis system to link genotype (mRNA) to phenotype (polypeptide). However, the presence of intrinsic components, such as nucleases in the cell-extract-based cell-free protein synthesis system, reduces the stability of the ternary complex, which would prevent attainment of reliable results. We have developed an efficient and highly controllable ribosome display system using the PURE ( P rotein synthesis U sing R ecombinant E lements) system. The mRNA–ribosome–polypeptide ternary complex is highly stable in the PURE system, and the selected mRNA can be easily recovered because activities of nucleases and other inhibitory factors are very low in the PURE system. We have applied the PURE ribosome display to antibody engineering approaches, such as epitope mapping and affinity maturation of antibodies, and obtained results showing that the PURE ribosome display is more efficient than the conventional method. We believe that the PURE ribosome display can contribute to the development of useful antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. [ABSTRACT FROM AUTHOR]

Abstract: Single- and double-stranded oligodeoxynucleotides (ODNs) incorporating both 2-aminopurine (2AP) and an indole-fused cytosine analog (PPI) were prepared and studied for their fluorescence properties. PPI and 2AP can be excited simultaneously by irradiation at 300nm, with emission observed at 500nm for PPI and 370nm for 2AP. We demonstrated the utility of these properties in the dual fluorescence labeling of ODNs giving well-separated emission peaks. In addition, both of the fluorescence signals of a doubly modified ODN changed independently, reflecting the local duplex formation at the regions containing 2AP or PPI. Potential applications of this strategy for the dual fluorescence labeling of oligonucleotides with 2AP and PPI include monitoring local structure alterations of functional nucleic acids and the multiplex detection of biologically important nucleic acids. [Copyright &y& Elsevier]

Protein folding is often hampered by protein aggregation, which can be prevented by a variety of chaperones in the cell. A dataset that evaluates which chaperones are effective for aggregation-prone proteins would provide an invaluable resource not only for understanding the roles of chaperones, but also for broader applications in protein science and engineering. Therefore, we comprehensively evaluated the effects of the major Escherichia coli chaperones, trigger factor, DnaK/DnaJ/GrpE, and GroEL/GroES, on ∼800 aggregationprone cytosolic E. coli proteins, using a reconstituted chaperone-free translation system. Statistical analyses revealed the robustness and the intriguing properties of chaperones. The DnaK and GroEL systems drastically increased the solubilities of hundreds of proteins with weak biases, whereas trigger factor had only a marginal effect on solubility. The combined addition of the chaperones was effective for a subset of proteins that were not rescued by any single chaperone system, supporting the synergistic effect of these chaperones. The resource, which is accessible via a public database, can be used to investigate the properties of proteins of interest in terms of their solubilities and chaperone effects. [ABSTRACT FROM AUTHOR]

Nascent chain-mediated translation arrest serves as a mechanism of gene regulation. A class of regulatory nascent polypeptides undergoes elongation arrest in manners controlled by the dynamic behavior of the growing chain; Escherichia coli SecM monitors the Sec protein export pathway and Bacillus subtilis MifM monitors the YidC membrane protein integration/folding pathway. We show that MifM and SecM interact with the ribosome in a species specific manner to stall only the ribosome from the homologous species. Despite this specificity. MifM is not exclusively designed to monitor membrane protein integration because it can be converted into a secretion monitor by replacing the N-terminal transmembrane sequence with a secretion signal sequence. These results show that a regulatory nascent chain is composed of two modular elements, one devoted to elongation arrest and another devoted to subcellular targeting, and they imply that physical pulling force generated by the latter triggers release of the arrest executed by the former. The combinatorial nature may assure common occurrence of nascent chain-mediated regulation. [ABSTRACT FROM AUTHOR]

[Display omitted] We have previously developed a glucose-linked biphenyl photosensitizer that can pass through glucose transporters, aiming for cancer-selective photodynamic therapy (PDT). Its small size (MW: 435) will allow oral administration and a fast clearance avoiding photosensitivity. However, its fluorescence efficiency was quite low, causing difficulty in monitoring cellular uptake. We thus synthesized a series of monosaccharide-linked biphenyl derivatives with a sulfur atom replacing an oxygen atom, in search of a photosensitizer with a brighter fluorescence. Among them, 4′-nitrobiphenyl thioglucoside showed a fluorescence emission extending to near infra-red region with a strength three times greater than that of the previous compound. This compound was found to have a higher 1O 2 -producing efficiency (Φ Δ : 0.75) than the previous compound (Φ Δ : 0.65). The thioglucoside indicated a significant photodamaging effect (IC 50 : 250 μM) against cancer cells. Although the galactose and mannose analogs exerted similar photodamaging effects, they were moderately toxic in the dark at a concentration of 300 μM. The thioglucoside and thiomannoside were at least partially uptaken through glucose transporters as demonstrated by inhibition with cytochalasin B, whereas no inhibition was observed for the galactoside. The behavior of d -glucose toward the cellular uptakes of these photosensitizers was bipolar: inhibitory at a low concentration and recovery or acceleratory at a higher concentration. These results indicate that 4′-nitrobiphenyl thioglucoside is the smallest (MW: 393) cancer-targeting photosensitizer with a trackable fluorescence property. [ABSTRACT FROM AUTHOR]

Abstract: We have developed a partially recombinant, cell-free, protein-synthesis system reconstituted solely from those essential elements of the Escherichia coli translation system, termed protein synthesis using recombinant elements (PURE). It provides higher reaction controllability in comparison to crude cell-free protein-synthesis systems for translation studies and biotechnology applications. The PURE system stands out among translation methods in that it provides not only a simple and unique “reverse” purification method of separating the synthesized protein from reaction mixture, but also that the system can be tailor-made according to individual protein requirements. In this paper, two new approaches to obtaining active proteins are described: the use of molecular chaperones, and modification of the reaction conditions. Several possible applications of the PURE system are also discussed. [Copyright &y& Elsevier]

Mitochondria contain the translocator of the outer mitochondrial membrane (TOM) for protein entry into the organelle, and its subunit Tom40 forms a protein-conducting channel. Here we report the role of Tom40 in protein translocation across the membrane. The site-specific photocrosslinking experiment revealed that translocating unfolded or loosely folded precursor segments of up to 90 residues can be associated with Tom40. Purified Tom40 bound to non-native proteins and suppressed their aggregation when they are prone to aggregate. A denatured protein bound to the Tom40 channel blocked the protein import into mitochondria. These results indicate that, in contrast to the nonstick tunnel of the ribosome for polypeptide exit, the Tom40 channel offers an optimized environment to translocating non-native precursor proteins by preventing their aggregation. [ABSTRACT FROM AUTHOR]

Discusses a study on the two distinct mechanisms that drive protein translocation across the mitochondrial outer membrane in the last step of the cytochrome b2 import pathway. Biological significance of the transmembrane movements driven by the anchor diffusion and the Brownian ratchet; Methodology of the study; Results and discussion; Conclusion.

Focuses on a study which mapped the interaction between translocase of the outer membrane (TOM) proteins and a mitochondrial precursor protein at two distinct stages in the translocation across the outer membrane. How the precursor protein is accumulated across the outer membrane; Relative geometry between the presequence and the TOM complex; Discussion on the model for protein translocation across the outer membrane.

Tests amino acids with photoreactive benzophene side chains of various lengths for their ability to be site-specifically incorporated into proteins. Arrest on the import pathways of the fusion proteins; Photocrosslinking of translocation intermediates to the components mediating mitochondrial protein import.

Thermal analysis of a thin-film thermal print bead was carried out with the aim of obtaining guidelines for the design of a print head for high speed and high-resolution printing. The analysis showed that if current is passed through the heating resistors for a shod period, there will be a time lag between the temperature increase in the resistors and the temperature increase in the heat sensitive paper. Furthermore, although a short pulse is prefer- able for high-resolution printing, there is a point beyond which further decrease in the duration of the pulse has little effect. It was estimated that changes to the beat insulation layer and protective film alone would not lead to an improvement in the performance of the print head as a whole. The printing test yielded the same results as those provided by the thermal analysis. For example, a pulse duration of 5 ms is not preferable for a resolution of 16 dots/mm. However, the contrast obtained with a pulse duration of 0.3 ms may also be obtained with a pulse duration of 0.8 ms. [ABSTRACT FROM AUTHOR]

This paper describes the result of thermal analysis of the inner region of the thin film thermal print head and the effect of the shape of the meander type heating resistor. The thermal head allowing short pulse driving for higher speed with higher reliability has been demanded recently. The influence of the pulse duration on the temperature profile of the inner region of the head and the heating resistor shape have not been studied. We have studied various conditions affecting the temperature profile of the inner region of the thin film thermal print head and the shape of the meander type heating resistor, and obtained the following results: 1 As the pulse applied to the heating resistor gets shorter, the necessary power for pulse application increases and the temperature slope in the heat insulating layer near the heating resistor becomes steeper. 2. To lessen the temperature slope in the heat insulating layer, thinning the protective layer, increasing the thermal conductivity of the protective layer and preheating the whole inner region are effective. 3. In surface temperature distribution, corners of the meander type heating register become hotter as the width of the pulse applied to it becomes shorter. Widening the heating register at corners, narrowing the straight parts and increasing the number of meanderings are effective. [ABSTRACT FROM AUTHOR]

α-Galactosylceramide (α-GalCer) is recognized by the CD1d proteins on antigen-presenting cells at the ceramide moiety and the galactose moiety is presented to iNKT cells, which stimulates the immune responses. However, the immune suppression by repeated injections of α-GalCer has discouraged its development as an anti-cancer agent. To overcome the shortcoming by spatiotemporal restriction of its exposure, we synthesized the photochromic azobenzene-incorporated analogues and tested the photo-immunoregulation effect in its binding to CD1d. FACS analyses indicated that some of these analogues enhanced the affinity to CD1d on photo-irradiation by about 20%. A docking simulation suggests that the photochromic molecule should be bulkier for a clearer discrimination between on and off states. [ABSTRACT FROM AUTHOR]

Progressive multifocal leukoencephalopathy is a rare but fatal demyelinating disease of the central nervous system with reactivation of JC virus. Herein, we describe a case of progressive multifocal leukoencephalopathy associated with a therapeutic regimen of daratumumab, lenalidomide, and dexamethasone for multiple myeloma. A 69‐year‐old man with multiple myeloma was admitted to the neurological care unit due to progressive cognitive dysfunction after seven cycles of the regimen. His CD4+ T cell count was 190 cells/μL. Diffusion‐weighted magnetic resonance imaging revealed nodular diffusion restrictions in the frontal and parietal lobes. He was diagnosed with progressive multifocal leukoencephalopathy following a positive polymerase chain reaction test for JC virus DNA in the cerebrospinal fluid. After cessation of multiple myeloma treatment, his cognition and magnetic resonance imaging findings improved within 3 months. We should recognize this potentially fatal complication in patients receiving this regimen. [ABSTRACT FROM AUTHOR]

Attempts to create complex molecular systems that mimic parts of cellular systems using a bottom-up approach have become important in the field of biology. Among various molecular systems, in vitro protein synthesis inside lipid vesicles (liposomes), which we refer to as the artificial cell, has become an attractive system because it possesses two fundamental features of living cells: central dogma, and compartmentalization. Here, we investigated the effect of altering the amount or concentration of four constituents of the artificial cell consisting of a commercially available reconstituted in vitro transcription-translation (IVTT) system. As this IVTT system is available worldwide, the results will be useful to the scientific community when shared, unlike those from a lab-made IVTT system. We succeeded in revealing the effect and trend of altering each parameter and identified a suitable condition for preparing liposomes that are unilamellar and can synthesize proteins equally as well as the original IVTT system. Because the commercially available reconstituted IVTT system is an important standardization tool and the constituents can be adjusted as desired, our results will be useful for the bottom-up creation of more complex molecular systems. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is an incurable B‐cell malignancy often accompanied by profound immunodeficiency. Lenalidomide (Len) is an immunomodulatory drug that exerts promising therapeutic effects on MM through the immune system. However, predictive markers related to the effects of Len treatment are not fully understood. This study aimed to identify candidate biomarkers for predicting the clinical efficacy of Len and dexamethasone (Ld) therapy through a comprehensive analysis of serum cytokines. The levels of 48 cytokines in the serum of patients with MM just before Ld therapy (n = 77), at the time of best response (n = 56), and at disease progression (n = 49) were measured and evaluated. Patients with high IL‐18 and M‐CSF levels showed significantly shorter progression‐free survival and overall survival (OS). In contrast, patients with high PDGF‐BB levels had longer survival. Moreover, low levels of G‐CSF, IL‐7, IL‐8, and SDF‐1α were associated with shorter OS after Ld therapy. During Ld therapy, pro‐inflammatory cytokines such as IL‐2Rα, IL‐18, and TNF‐α were decreased, while IFN‐γ was increased. IL‐4 and IL‐6 levels increased during disease progression. In conclusion, this study provides a better understanding of the association between cytokines and the efficacy of Ld therapy as well as the unique changes in cytokines related to inflammatory and immune responses during Ld therapy. [ABSTRACT FROM AUTHOR]

Afuresertib (AFU), a novel inhibitor of the serine/threonine kinase AKT, has clinical efficacy as a monotherapy against hematological malignancies and is expected to be used in combination with standard therapies for multiple myeloma (MM). To develop a more effective and less toxic combination of immunomodulatory drugs (IMiDs) for therapy, the antitumor effect of sub‑optimal doses of AFU, pomalidomide plus dexamethasone (PD), and the AFU‑PD combination on MM cells were examined in the present study. Two MM cell lines, XG‑7 and U266, with low sensitivity to both PD and AFU monotherapies, were subjected to these combinations and analyzed. Although the cell lines showed a slight reduction in viability with the sub‑optimal doses of each monotherapy, the combination of the treatments resulted in a reduction in cell viability and the progression of apoptosis. Co‑treatment with sub‑optimal doses of PD and AFU enhanced caspase activation and highly suppressed the expression of IKZF1 and IKZF3. In addition, this combination promoted the dephosphorylation and stabilization of 4EBP1, an inhibitor of eIF4E activation, which led to the impairment of eIF4E‑mediated translational activity. Furthermore, AFU showed a sufficient inhibitory effect on the phosphorylation of FOXO1, a tumor suppressor, in monotherapy or in combination with PD, which may be attributable to the activation of FOXO1, the subsequent inhibition of tumor growth, and the induction of cell death. In conclusion, the combination therapy with sub‑optimal doses of PD and AFU exhibited potent antitumor activity in MM cells and may provide a novel strategy for the treatment of patients who experienced intolerable toxicity or insufficient response during IMiD therapy. [ABSTRACT FROM AUTHOR]

Abstract: 2′-O-[N-(4-Aminobutylcarbamoyl)]uridine (Uabcm) was synthesized and incorporated into oligonucleotides. The oligonucleotides incorporating Uabcm formed more stable duplexes with their complementary and mismatched RNAs than those containing 2′-O-carbamoyluridine (Ucm). The stability of duplex with a Uabcm-rG base pair showed higher thermostability than the duplex having unmodified U-rG base pair. The Uabcm residue showed enhanced resistance to snake venome phosphodiesterase. [Copyright &y& Elsevier]

• Patients with Werner syndrome are likely to acquire TP53 mutations/deletions. • Patients with Werner syndrome and MDS/AML have complex chromosomal abnormalities. • Werner syndrome hematopoietic stem cells acquire competitive fitness by inactivating p53. Werner syndrome (WS) is a progeroid syndrome caused by mutations in the WRN gene, which encodes the RecQ type DNA helicase for the unwinding of unusual DNA structures and is implicated in DNA replication, DNA repair, and telomere maintenance. patients with WS are prone to develop malignant neoplasms, including hematological malignancies. However, the pathogenesis of WS-associated hematological malignancies remains uncharacterized. Here we investigated the somatic gene mutations in WS-associated myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Whole-exome sequencing (WES) of 4 patients with WS with MDS/AML revealed that all patients had somatic mutations in TP53 but no other recurrent mutations in MDS/AML. TP53 mutations were identified at low allele frequencies at more than one year before the MDS/AML stage. All 4 patients had complex chromosomal abnormalities including those that involved TP53. Targeted sequencing of nine patients with WS without apparent blood abnormalities did not detect recurrent mutations in MDS/AML except for a PPM1D mutation. These results suggest that patients with WS are apt to acquire TP53 mutations and/or chromosomal abnormalities involving TP53 , rather than other MDS/AML-related mutations. TP53 mutations are frequently associated with prior exposure to chemotherapy; however, all four patients with WS with TP53 mutations/deletions had not received any prior chemotherapy, suggesting a pathogenic link between WRN mutations and p53 insufficiency. These results indicate that WS hematopoietic stem cells with WRN insufficiency acquire competitive fitness by inactivating p53, which may cause complex chromosomal abnormalities and the subsequent development of myeloid malignancies. These findings promote our understanding of the pathogenesis of myeloid malignancies associated with progeria. [ABSTRACT FROM AUTHOR]

Based on the results of site-specific photocrosslinking of translocation intermediates, we have identified Tim50, a component of the yeast TIM23 import machinery, which mediates translocation of presequence-containing proteins across the mitochondrial inner membrane. Tim50 is anchored to the inner mitochondrial membrane, exposing the C-terminal domain to the intermembrane space. Tim50 interacts with the N-terminal intermembrane space domain of Tim23. Functional defects of Tim50 either by depletion of the protein or addition of anti-Tim50 antibodies block the protein translocation across the inner membrane. A translocation intermediate accumulated at the TOM complex is crosslinked to Tim50. We suggest that Tim50, in cooperation with Tim23, facilitates transfer of the translocating protein from the TOM complex to the TIM23 complex [Copyright &y& Elsevier]

Many studies of the reconstitution of the Escherichia coli small ribosomal subunit from its individual molecular parts have been reported, but contrastingly, similar studies of the large ribosomal subunit have not been well performed to date. Here, we describe protocols for preparing the 33 ribosomal proteins of the E. coli 50S subunit and demonstrate successful reconstitution of a functionally active 50S particle that can perform protein synthesis in vitro. We also successfully reconstituted both ribosomal subunits (30S and 50S) and 70S ribosomes using a full set of recombinant ribosomal proteins by integrating our developed method with the previously developed fully recombinant-based integrated synthesis, assembly and translation. The approach described here makes a major contribution to the field of ribosome engineering and could be fundamental to the future studies of ribosome assembly processes. [ABSTRACT FROM AUTHOR]

5-[3-(2-Aminopyrimidin-4-yl)aminopropyn-1-yl]uracil (Ura Pyr ) was designed as a new nucleobase to recognize Ade-Thy base pair in double-stranded DNA. We successfully synthesized the dexoynucleoside phosphoramidite having Ura Pyr and incorporated it into triplex forming oligonucleotides (TFOs). Melting temperature analysis revealed that introduction of Ura Pyr into TFOs could effectively stabilize their triplex structures without loss of base recognition capabilities. [ABSTRACT FROM AUTHOR]

We developed new DNA triplexes that contain four base triads T-A·T, A-ψ·CBr, G-PIC·YO, and C-G·Py+, where CBr, YO, Py, ψ, and PIC are 5-bromocytosine, 5-methyl-4-pyrimidone, 2-aminopyridine, the aglycons of deoxypseudouridine, and deoxypseudoisocytidine, respectively. DNA duplex incorporating T-A, A-ψ, G-PIC, and C-G, and triplex forming oligonucleotide incorporating T, CBr, YO, and Py formed the triplex as evaluated by Tm measurements. The triplex formation was successfully applied to the inhibition of transcription of the DNA duplex incorporating T7-promoter sequence modified by the above modified bases. [ABSTRACT FROM AUTHOR]

In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features. In this study, we integrated all of the processes required for Escherichia coli small ribosomal subunit assembly. In our method, termed fully Recombinant-based integrated Synthesis, Assembly, and Translation (R-iSAT), assembly and evaluation of the small ribosomal subunits are coupled with ribosomal RNA (rRNA) synthesis in a reconstituted cell-free protein synthesis system. By changing the components of R-iSAT, including recombinant ribosomal protein composition, we coupled ribosomal assembly with ribosomal protein synthesis, enabling functional synthesis of ribosomal proteins and subsequent subunit assembly. In addition, we assembled and evaluated subunits with mutations in both rRNA and ribosomal proteins. The study demonstrated that our scheme provides new ways to comprehensively analyze any elements of the small ribosomal subunit, with the goal of improving our understanding of ribosomal biogenesis, function, and engineering. Shimojo et al. demonstrate the use of individually purified ribosomal proteins added into iSAT (integrated ribosomal synthesis, assembly, and translation) system to enable assembly of functional 30S subunits. They further show that while some 30S r-proteins must be full synthesized before transcription, others may be co-transcriptionally produced, to enable the assembly of 30S particles. [ABSTRACT FROM AUTHOR]

While the development of low‐molecular‐weight drugs is saturating, agents for photodynamic therapies (PDTs) may become alternative seeds in pharmaceutical industry. Among them, orally administrative, cancer‐selective, and side effect‐free photosensitizers (PSs) that can be activated by tissue‐penetrative near‐infrared (NIR) lights are strongly demanded. We discovered such a PS from scratch by focusing on a twist‐assisted spin‐orbit charge transfer intersystem crossing (ISC) mechanism in a biphenyl derivative, which was demonstrated by thorough photophysical studies. The unique ISC mechanism enables the PS to be small and slim so as to pass through glucose transporters and exert a PDT effect selectively on a cancer cell line. The smallness will allow for oral administration and fast clearance, which have been agenda of approved PSs with larger molecular weights. We also demonstrated that our PS was able to be activated with an NIR pulse laser through two‐photon excitation. [ABSTRACT FROM AUTHOR]

IgG is an indispensable biological experimental tool as well as a widely-used therapeutic protein. However, cell culture-based expression of monoclonal IgG is costly and time-consuming, making this process difficult to use for high-throughput screening in early-stage evaluation of biologics. With the goal of establishing a fast, simple, and robust high-throughput expression system for IgG, we implemented the synthesis of functional aglycosylated IgG by constructive approach based on a reconstituted prokaryotic cell-free protein synthesis system (PURE system). Optimization of the PURE system revealed that the following factors and reaction conditions were needed for IgG synthesis: (1) inclusion of the disulfide bond isomerase DsbC, (2) adjustment of the GSH/GSSG ratio, (3) inclusion of the molecular chaperone DnaK and its cofactors, and (4) use of an extended incubation time. Synthesis temperature and template DNA ratio (light chain-/heavy chain-encoding) also had been optimized for each IgG. Under optimal conditions, peak production of the anti-HER2 antibody trastuzumab reached 124 µg/mL. Furthermore, the active forms of other IgGs, including IgG1, IgG2, and IgG4 subclasses, also were synthesized. These results provide basic information for the development of novel high-throughput expression and functional screening systems for IgG, as well as useful information for understanding the IgG synthesis process. [ABSTRACT FROM AUTHOR]

This study aimed to synthesize triplex-forming oligonucleotides (TFOs) containing 2′-deoxy-6-thioxanthosine (s 6 X) and 2′-deoxy-6-thioguanosine (s 6 Gs) residues and examined their triplex-forming ability. Consecutive arrangement of s 6 X and s 6 Gs residues increased the triplex-forming ability of the oligonucleotides more than 50 times, compared with the unmodified TFOs. Moreover, the stability of triplex containing a mismatched pair was much lower than that of the full-matched triplex, though s 6 X could form a s 6 X-GC mismatched pair via tautomerization of s 6 X. The present results reveal excellent properties of modified TFOs containing s 6 Xs and s 6 Gs residues, which may be harnessed in gene therapy and DNA nanotechnology. [ABSTRACT FROM AUTHOR]

6- O -(2-Nitrobenzyl)guanosine and 4- O -(2-nitrobenzyl)uridine triphosphates ( NB GTP, NB UTP) were synthesized, and their biochemical and photophysical properties were evaluated. We synthesized NB UTP using the canonical triphosphate synthesis method and NB GTP from 2′,3′- O -TBDMS guanosine via a triphosphate synthesis method by utilizing mild acidic desilylation conditions. Deprotection of the nitrobenzyl group in NB GTP and NB UTP proceeded within 60 s by UV irradiation at 365 nm. Experiments using NB GTP or NB UTP in T7-RNA transcription reactions showed that NB GTP could be useful for the photocontrol of transcription by UV irradiation. [ABSTRACT FROM AUTHOR]

The article describes the possibility of new nucleotide pairs applicable to DNA triplex. The authors designed oligonucleotide duplexes incorporating 5-aryl deoxycytidine derivatives. The authors state that the results had indicated the potential utility of new nucleotide triplets stabilized by stacking interaction.

Enzymatic synthesis and the reverse transcription of RNAs containing 2′-O-carbamoyl uridine were evaluated. A mild acidic deprotection procedure allowed the synthesis of 2′-O-carbamoyl uridine triphosphate (UcmTP). UcmTP was incorporated correctly into long RNAs, and its fidelity during reverse transcription using SuperScript III was sufficient for RNA aptamer selection. [ABSTRACT FROM AUTHOR]

7-(Benzofuran-2-yl)-7-deazadeoxyguanosine (BFdG) was synthesized and incorporated into an oligodeoxynucleotide (ODN). The single-stranded ODN containing BFdG shows 91-fold fluorescence enhancement upon binding of single-strand DNA binding protein. [ABSTRACT FROM AUTHOR]

In Escherichia coli, elongation factor G (EF-G), a key protein in translational elongation, is particularly susceptible to oxidation. We demonstrated previously that EF-G is inactivated upon formation of an intramolecular disulphide bond. However, the details of the mechanism by which the oxidation of EF-G inhibits the function of EF-G on the ribosome remain to be elucidated. When we oxidized EF-G with hydrogen peroxide, neither the insertion of EF-G into the ribosome nor single-cycle translocation activity in vitro was affected. However, the GTPase activity and the dissociation of EF-G from the ribosome were suppressed when EF-G was oxidized. The synthesis of longer peptides was suppressed to a greater extent than that of a shorter peptide when EF-G was oxidized. Thus, the formation of the disulphide bond in EF-G might interfere with the hydrolysis of GTP that is coupled with dissociation of EF-G from the ribosome and might thereby retard the turnover of EF-G within the translational machinery. When we added thioredoxin to the suppressed translation system that included oxidized EF-G, translational activity was almost immediately restored. We propose that oxidation of EF-G might provide a regulatory mechanism for transient and reversible suppression of translation in E. coli under oxidative stress. [ABSTRACT FROM AUTHOR]

U1 snRNA is an interesting biological tool for splicing correction and regulation of gene expression. However, U1 snRNA has never been chemically synthesized. In this study, the first chemical synthesis of U1snRNA and its analogues was carried out. Moreover, it was found that the binding affinity of the modified U1 snRNA with an ethylene glycol linkage to snurportin 1 (nuclear import adaptor) was as high as that of the unmodified RNA. [ABSTRACT FROM AUTHOR]

Abstract: To increase base recognition capability and sensitivity, we propose the separation of a commonly used single-probe system for oligonucleotide analysis into a set of three probes: a fluorophore-labeled probe, a promoter probe, and a short probe. In this study, we found that the probes of only 4nt in length can selectively bind the corresponding gap site on complexes consisting of the target, fluorophore-labeled probe, and promoter probe, exhibiting a more than 14-fold difference in ligation between the matched and mismatched sequences. Moreover, we demonstrated that the immobilized short probes accurately recognized the sequences of the gap sites. [Copyright &y& Elsevier]

Abstract: Development of protein therapeutics or biosensors often requires in vitro affinity maturation. Here we report a robust affinity engineering strategy using a custom designed library. The strategy consists of two steps beginning with identification of beneficial single amino acid substitutions then combination. A high quality combinatorial library specifically customized to a given binding-interface can be rapidly designed by high-throughput mutational scanning of single substitution libraries. When applied to the optimization of a model antibody Fab fragment, the strategy created a diverse panel of high affinity variants. The most potent variant achieved 2110-fold affinity improvement to an equilibrium dissociation constant (Kd) of 3.45pM with only 7 amino acid substitutions. The method should facilitate affinity engineering of a wide variety of protein–protein interactions due to its context-dependent library design strategy. [Copyright &y& Elsevier]

Elongation factor G (EF-G), a key protein in translational elongation, is known to be particularly susceptible to oxidation in Escherichia coli. However, neither the mechanism of the oxidation of EF-G nor the influence of its oxidation on translation is fully understood. In the present study, we investigated the effects of oxidants on the chemical properties and function of EF-G using a translation system in vitro derived from E. coli. Treatment of EF-G with 0.5 mM H2O2 resulted in the complete loss of translational activity. The inactivation of EF-G by H2O2 was attributable to the oxidation of two specific cysteine residues, namely, Cys114 and Cys266, and subsequent formation of an intramolecular disulfide bond. Replacement of Cys114 by serine rendered EF-G insensitive to oxidation and inactivation by H2O2. Furthermore, generation of the translation system in vitro with the mutated EF-G protected the entire translation system from oxidation, suggesting that EF-G might be a primary target of oxidation within the translation system. Oxidized EF-G was reactivated via reduction of the disulfide bond by thioredoxin, a ubiquitous protein that mediates dithiol-disulfide exchange. Our observations indicate that the translational machinery in E. coli is regulated, in part, by the redox state of EF-G, which might depend on the balance between the supply of reducing power and the degree of oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: A complete reconstitution system for membrane integration of the simplest protein was developed by means of defined factors. A mutant version of Pf3 coat protein, 3L-Pf3 coat, requires neither signal recognition particle/Sec factors nor a membrane potential for its integration into the cytoplasmic membrane of Escherichia coli. Although 3L-Pf3 coat is spontaneously integrated into liposomes composed of phospholipids, diacylglycerol completely blocks such spontaneous integrations at a physiological level. Under the conditions where spontaneous integration does not occur, 3L-Pf3 coat integration was absolutely dependent on a novel integration-stimulating factor. Combination of the PURE system, an in vitro translation system composed of the purified factors involved in translation in E. coli, with liposomes containing the highly purified integration-stimulating factor revealed multiple cycles of 3L-Pf3 coat integration, achieving the complete reconstitution of membrane integration. Based on the function of the factor, we propose that the factor is named MPIase (Membrane Protein Integrase). [Copyright &y& Elsevier]

Mismatch binding protein MutS binding to bulge structure in DNA duplexes was controlled by UV irradiation. 4- O -(2-Nitrobenzyl)thymidine or 4- O -[2-(2-nitrophenyl)propyl]thymidine was incorporated into DNA duplexes a bulged position. The MutS did not bind to the caged DNA duplexes but bound after removing the 2-nitrobenzyl or 2-(2-nitrophenyl)propyl group by photo-irradiation. By using photo-caged DNA duplex, we revealed that binding of MutS to the uncaged DNA downstream of the T7 RNA promoter weakly inhibited transcription by T7 RNA polymerase. [ABSTRACT FROM AUTHOR]

Ribosome display is a powerful technology for selecting ligand-binding peptides or proteins. We demonstrate here that the ribosome display using the reconstituted cell-free protein synthesis system can be applied for the epitope mapping of monoclonal antibodies (mAbs). Using this technology, we selected peptides that specifically bind to three mAbs from random peptide library. When selection was performed against the anti-FLAG M2 antibody, selected peptides contained previously characterized consensus epitope, indicating that the methodology can be applied for the epitope mapping. When the selection was carried out against two anti-β-Catenin (anti-β-Cat) mAbs, selected peptides had a homology for the partial peptide sequences of β-Cat. Western blot analysis showed that these putative epitopes had affinity for the corresponding mAbs and β-Cat mutants that lack these regions did not bind to the antibodies, indicating we correctly mapped the epitope for these mAbs. The study shown here provides a way for the quick identification of the epitope of mAbs. [ABSTRACT FROM PUBLISHER]

Protein translocation across the outer mitochondrial membrane is mediated by the translocator called the TOM (translocase of the outer mitochondrial membrane) complex. The TOM complex possesses two presequence binding sites on the cytosolic side (the cis site) and on the intermembrane space side (the trans site). Here we analyzed the requirement of presequence elements and subunits of the TOM complex for presequence binding to the cis and trans sites of the TOM complex. The N-terminal 14 residues of the presequence of subunit 9 of F0-ATPase are required for binding to the trans site. The interaction between the presequence and the cis site is not sufficient to anchor the precursor protein to the TOM complex. Tom7 constitutes or is close to the trans site and has overlapping functions with the C-terminal intermembrane space domain of Tom22 in the mitochondrial protein import. [ABSTRACT FROM AUTHOR]