Daisuke Kuroda

Three-dimensional hole drilling of silica glass from the rear surface with femtosecond laser pulses

By moving silica glass in a preprogrammed structure, we directly produced three-dimensional holes with femtosecond laser pulses in single step. When distilled water was introduced into a hole drilled from the rear surface of the glass, the effects of blocking and redeposition of ablated material were greatly reduced and the aspect ratio of the depth of the hole was increased. Straight holes of 4-mu;m diameter were more than 200 microm deep. Three-dimensional channels can be micromachined inside transparent materials by use of this method, as we have demonstrated by drilling a square-wave-shaped hole inside silica glass.

Computer-aided antibody design

Recent clinical trials using antibodies with low toxicity and high efficiency have raised expectations for the development of next-generation protein therapeutics. However, the process of obtaining therapeutic antibodies remains time consuming and empirical. This review summarizes recent progresses in the field of computer-aided antibody development mainly focusing on antibody modeling, which is divided essentially into two parts: (i) modeling the antigen-binding site, also called the complementarity determining regions (CDRs), and (ii) predicting the relative orientations of the variable heavy (VH) and light (VL) chains. Among the six CDR loops, the greatest challenge is predicting the conformation of CDR-H3, which is the most important in antigen recognition. Further computational methods could be used in drug development based on crystal structures or homology models, including antibody–antigen dockings and energy calculations with approximate potential functions. These methods should guide experimental studies to improve the affinities and physicochemical properties of antibodies. Finally, several successful examples of in silico structure-based antibody designs are reviewed. We also briefly review structure-based antigen or immunogen design, with application to rational vaccine development.

Structural classification of CDR-H3 revisited: a lesson in antibody modeling.

Among the six complementarity‐determining regions (CDRs) in the variable domains of an antibody, the third CDR of the heavy chain (CDR‐H3), which lies in the center of the antigen‐binding site, plays a particularly important role in antigen recognition. CDR‐H3 shows significant variability in its length, sequence, and structure. Although difficult, model building of this segment is the most critical step in antibody modeling. Since our first proposal of the “H3‐rules,” which classify CDR‐H3 structure based on amino acid sequence, the number of experimentally determined antibody structures has increased. Here, we revise these H3‐rules and propose an improved classification scheme for CDR‐H3 structure modeling. In addition, we determine the common features of CDR‐H3 in antibody drugs as well as discuss the concept of “antibody druggability,” which can be applied as an indicator of antibody evaluation during drug discovery. Proteins 2008.

Fabrication of Fresnel zone plate embedded in silica glass by femtosecond laser pulses

We fabricated the Fresnel zone plate by embedding voids in silica glass. We investigated the focusing properties by launching a He-Ne laser beam into the zone plate. The spot size of the primary focal point was 7.0 mum and agreed with the theoretical value of 6.1 mum. The diffraction efficiency was 2.0 %. This technique enables us to make alignment free micro-scale lenses inside bulk materials.

Systematic classification of CDR‐L3 in antibodies: Implications of the light chain subtypes and the VL–VH interface

Antibody modeling is widely used for the analysis of antibody–antigen interactions and for the design of potent antibody drugs. The antibody combining site is composed of six complementarity determining regions (CDRs). The CDRs, except for CDR‐H3, which is the most diverse CDR, form limited numbers of canonical structures, which can be identified from the amino acid sequences. A method to classify the CDR‐H3 structure from its amino acid sequence was previously proposed. However, since those CDR structures were classified, many more antibody crystal structures have been determined. We performed systematic analyses of the CDR‐L3 structures and found novel canonical structures, and we also classified a previously identified canonical structure into two subtypes. In addition, two differently defined canonical structures in the κ and λ subtypes were classified into the same canonical structure. We also identified a key residue in CDR‐L3, which determines the conformation of CDR‐H3. Several analyses of CDR‐L3 loops longer than nine residues were performed. These new findings should be useful for structural modeling and are eventually expected to accelerate the design of antibody drugs. Proteins 2009.

Use of amino acid composition to predict epitope residues of individual antibodies

We identified specific amino acid propensities at the interfaces of antigen-antibody interactions in non-redundant qualified antigen-antibody complex structures from Protein Data Bank. Propensities were expressed by the frequency of each of the 20 x 20 standard amino acid pairs that appeared at the interfaces of the complexes and were named the antibody-specific epitope propensity (ASEP) index. Using this index, we developed a novel method of predicting epitope residues for individual antibodies by narrowing down candidate epitope residues which was predicted by the conventional method. The 74 benchmarked antigens were used in ASEP prediction. The efficiency of this method was assessed using the leave-one-out approach. On elimination of residues with ASEP indices in the lowest 10% of all measured, true positives were enriched for 49 antigens. On subsequent elimination of residues with ASEP indices in the lowest 50%, true positives were enriched for 40 of the 74 antigens assessed. The ASEP index is the first benchmark proposed to predict epitope residues for an individual antibody. Used in combination with mutation experiments, this index has the potential to markedly increase the success ratio of epitope analysis.

Bridging the gap between single-template and fragment based protein structure modeling using Spanner

Background: As the coverage of experimentally determined protein structures increases, fragment-based structural modeling approaches are expected to play an ever more important role in structural modeling. Here we introduce a structural modeling method by which an initial structural template can be extended by the addition of structural fragments to more closely match an aligned query sequence. A database of pro-tein fragments indexed by their internal coordinates was created and a novel methodology for their retrieval was implemented. After fragment selection and assembly, sidechains are replaced and the all-atom model is refined by restrained energy minimization. We implemented the proposed method in the program Span-ner and benchmarked it using a previously published set of 367 immunoglobulin (Ig) loops, 206 historical query-template pairs and alignments from the Critical Assessment of protein Structure Prediction (CASP) experiment, and 217 structural alignments between remotely homologous query-template pairs. The con-straint-based modeling software MODELLER and previously reported results for RosettaAntibody, were used as references. Results: The error in the modeled structures was assessed by root-mean square deviation (RMSD) from the native structure, as a function of the query-template sequence identity. For the Ig benchmark set, for which a single fragment was used to model each loop, the average RMSD for Spanner (3 +/- 1.5 A) was found to lie midway between that of MODELLER (4 +/- 2 A) and RosettaAntibody (2 +/- 1 A). For the CASP and structural alignment benchmarks, for which gaps represent a small fraction of the modeled residues, the difference between Spanner and MODELLER were much smaller then the standard deviations of either program. The Spanner web server and source code are available at http://sysimm.ifrec.osaka-u.ac.jp/Spanner/. Conclusions: For typical homology modeling, Spanner is at least as good, on average as the template-free constraint-driven approach used by MODELLER. The Ig model results suggest that when gap regions represent a significant fraction of the alignment, Spanner’s efficient use of fragment libraries, along with local sequence and secondary structural information, significantly improve model accuracy without a dra-matic increase in computational cost.

Control of positions and shapes of voids in transparent materials with femtosecond laser

When femtosecond laser pulses are tightly focused inside the bulk of transparent materials, the intensity in a focal volume becomes high enough to produce submicrometer-scale structural modifications. This damage was shown to be a cavity or a void surrounded by densified material. An array of voids can be used as optical data storages or gratings. We showed the control experiment of the positions and shapes of voids insider transparent materials with femtosecond laser pulses. We have demonstrated the experiments involving optical movement of a void along the optical axis by translation of the focal spot with femtosecond laser pulses. Irradiation of femtosecond laser pulses moves a void inside calcium fluoride and silica glass without any mechanical translations of the optical system up to 2 micron. In this paper, we show that the shapes of voids can be controlled by the spatial profile of incident laser pulses. Finally we show that the fabrication of a Fresnel lens inside silica glass.

Fabrication of birefringent microstructures in transparent materials with femtosecond laser pulses

When femtosecond laser pulses are tightly focused inside the bulk of transparent materials, the intensity in a focal volume become high enough to produce submicrometer-scale structural modifications. The modifications has been applied to fabricate 3D photonic structures. Tightly-focused femtosecond laser pulses create voids, which are surrounded by densified material. In this paper we show that the shapes of voids can be controlled by the spatial profile of incident laser pulse. We also show that the diffraction intensities due to the fabricated arrays of voids depend on the polarization-states of the readout beam. Finally, we demonstrate that irradiation of femtosecond laser pulses moves a void inside calcium fluoride and silica glass without any mechanical translations of the optical system. In situ observation revealed that a void moves towards incident direction of laser pulses as long as 2 micron.

Movement of a bubble inside silica glass and calcium fluoride by irradiation of femtosecond laser pulses

Many researchers have investigated the interaction of femtosecond laser pulses with a wide variety of materials. The structural modifications both on the surface and inside the bulk of transparent materials have been demonstrated. When femtosecond laser pulses are focused into glasses with a high numerical aperture objective, voids are formed. We report first observation that a bubble, which is called void moves under irradiation of femtosecond laser pulses inside silica glass and calcium fluoride. In situ observation reveals that the void moves towards incident direction of laser pulses as long as 5 micron by successive laser pulses without any mechanical translations.