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Papers by Maria Portia Nagata

Journal of Applied Phycology, 2007

Nostoc commune Vauch. is one of the few freshwater cyanobacteria that has been used as human food... more Nostoc commune Vauch. is one of the few freshwater cyanobacteria that has been used as human food. But its commercial production has been elusive. This prompted us to investigate further on the chemical composition and morpho-cytological characteristics of the discoid and spherical forms of the alga from the Philippines and Japan. Light microscopy of the two forms revealed that they

Journal of Animal Science and Biotechnology, 2019

Background The application of cryopreservation and artificial insemination technology have contri... more Background The application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction. However, a substantial proportion of spermatozoa undergoes alterations and loses their fertility during cryopreservation, rendering the frozen-thawed semen impractical for routine use. Cryopreservation is known to reduce sperm lifespan and fertility. Variation in cryosurvival of spermatozoa from different sires and even with the individual sire is common in artificial insemination (AI) centers. Our goal is to improve post-thawed semen quality by optimization of cryopreservation technique through sperm selection prior to cryopreservation process. Results Our strategy of sperm selection based on rheotaxis and thermotaxis (SSRT) on macrosale in a rotating fluid flow demonstrated the ability to maintain the original pre-freezing structural integrity, viability and biological function related to fertilization competence. This strategy has a pos...

Andrology

The isolation and characterization of sperm subpopulations that can achieve fertilization is a ma... more The isolation and characterization of sperm subpopulations that can achieve fertilization is a major challenge of assisted reproduction methods. We focused on the microfluidic sperm sorter as a novel tool for collecting highly motile spermatozoa from heterogeneous semen samples.

Scientific Reports

We fabricated a simple microfluidic device for separation of bovine oocytes based on the oocyte q... more We fabricated a simple microfluidic device for separation of bovine oocytes based on the oocyte quality to improve the conception rate of in vitro fertilization (IVF) by using good quality oocytes. The microfluidic device separates oocytes based on sedimentation rate differences in a sucrose buffer, which is dependent on oocyte quality. The microfluidic device has a 700 µm width, 1 mm height, and 10 mm long separation channel. Oocytes were injected from the upper half of the separation channel, and they flowed while sinking. The outlets of the separation channel were divided into upper and lower chambers. Good quality oocytes settled faster than poor quality oocytes in sucrose buffer; therefore, good quality oocytes were collected from the lower outlet. We performed IVF after the microfluidic separation of oocytes. The developmental rate to blastocysts of oocytes collected from the lower outlet was significantly higher than those collected from the upper outlet (36.0% vs. 14.1%). This result was comparable to that in the BCB staining method performed as a comparison method (BCB+ : 35.7%, BCB−: 15.4%). These findings indicate that our microfluidic device could be applied to oocyte separation and contribute to improvement of in vitro embryo production system. Success of in vitro fertilization (IVF) depends highly on quality of oocytes 1. Oocyte quality has been evaluated based on morphological observations and the degree of adhesion of the cumulus cells 2. However, it is difficult to distinguish between good and fair quality oocytes, and results have been assigned based on the personal judgments of veterinarians or technicians. Therefore, it is necessary to develop a new sorting technique that can efficiently separate good and poor quality oocytes. Brilliant cresyl blue (BCB) staining is one oocyte selection method 3,4. Oocytes stained by BCB show better quality in manipulation than unstained oocytes. Alm et al. demonstrated 34.1% embryonic development with stained oocytes by BCB against 3.9% of unstained oocytes 3. However, this method uses visual observation and requires approximately 2 h to stain the oocytes. In addition, some recent studies do not recommend use of BCB stain for oocyte selection because of DNA damage 5 , chromosomal abnormality 6 , cleavage 7 , and apoptosis 8 of oocytes caused by BCB staining 9. In contrast, Yotsushima et al. reported that the speed of sedimentation of cumulus-oocyte complexes (COCs) and denuded oocytes in hypertonic solution correlated with the morphological quality of COCs 10. This report indicated that the sedimentation method could be a useful method to objectively select good quality oocytes. With this method, non-toxic solutions should be used to separate oocytes. Sucrose solution is one of the ideal separation solution candidates because of its attributes, such as non-damaging to oocytes and ease in controlling the solution density. Liu et al.

Proceedings of the National Academy of Sciences of the United States of America, Jan 3, 2018

Selection of functional spermatozoa plays a crucial role in assisted reproduction. Passage of spe... more Selection of functional spermatozoa plays a crucial role in assisted reproduction. Passage of spermatozoa through the female reproductive tract requires progressive motility to locate the oocyte. This preferential ability to reach the fertilization site confers fertility advantage to spermatozoa. Current routine sperm selection techniques are inadequate and fail to provide conclusive evidence on the sperm characteristics that may affect fertilization. We therefore developed a selection strategy for functional and progressively motile bovine spermatozoa with high DNA integrity based on the ability to cross laminar flow streamlines in a diffuser-type microfluidic sperm sorter (DMSS). The fluid dynamics, with respect to microchannel geometry and design, are relevant in the propulsion of spermatozoa and, consequently, ultrahigh-throughput sorting. Sorted spermatozoa were assessed for kinematic parameters, acrosome reaction, mitochondrial membrane potential, and DNA integrity. Kinematic ...

Journal of Biochemistry, 2010

Protein refolding is an important process to obtain active recombinant proteins from inclusion bo... more Protein refolding is an important process to obtain active recombinant proteins from inclusion bodies (protein aggregates). However, the conventional refolding method of dialysis or dilution is a time consuming procedure and often, recovering yields of active proteins are low. In this study, we used controllable diffusion through laminar flow in microchannels to control the denaturant concentration. The performance of the designed microfluidic chips was evaluated by the refolding of difficult-to-fold proteins (citrate synthase and the zeta-associated protein 70-kDa protein kinase domain). We demonstrated this by varying the flow rates of the diluting buffer stream(s) and multi-junctions which could control the different flow rate ratios of the buffer stream(s) and the denatured protein stream. By this strategy, we were able to improve the efficiency of protein refolding. Our method achieved refolding within a short period of time at room temperature without the need of any small molecules or chaperone proteins. Moreover, the efficiency of protein refolding by microfluidic chip was found higher than that prepared by dialysis or dilution. These results suggest that microfluidic chips employing this strategy may provide miniaturized tools for rapid and efficient recovery of active proteins from inclusion bodies.

ELECTROPHORESIS, 2009

Proteolysis is an important part of protein identification in proteomics analysis. The convention... more Proteolysis is an important part of protein identification in proteomics analysis. The conventional method of in-solution digestion of proteins is time-consuming and has limited sensitivity. In this study, trypsin-or a-chymotrypsin-immobilized microreactors prepared by a microfluidics-based enzyme-immobilization technique were studied for rapid sample preparation in proteomic analysis. The kinetic studies for hydrolysis of substrate by microreactors revealed that immobilized proteases had higher hydrolytic efficiency than those performed by in-solution digestion. The performance of the microreactors was evaluated by digesting cytochrome c and BSA. Protein digestion was achieved within a short period of time ($5 min) at 301C without any complicated reduction and alkylation procedures. The efficiency of digestion by trypsin-immobilized reactor was evaluated by analyzing the sequence coverage, which was 47 and 12% for cytochrome c and BSA, respectively. These values were higher than those performed by in-solution digestion. Besides, because of higher stability against high concentration of denaturant, the microreactors can be useful for immediate digestion of the denaturated protein. In the present study, we propose a protease-immobilized microreactor digestion method, which can utilize as a proteome technique for biological and clinical research.

Chemical Engineering Journal, 2010

We investigated a method for size homogenization of liposomes using microchannel laminar flow. Th... more We investigated a method for size homogenization of liposomes using microchannel laminar flow. This microchannel method combined with sonication produced the desired homogeneous liposome populations with size controlled by reassembly of liposomes in laminar flow, using simple operations that offer good reproducibility and organic-solvent free procedures. The liposome solution, which was prepared using the traditional method of film hydration, was loaded into a syringe. This liposome solution was sonicated while being transported into the capillary tubing using syringe injection. In both non-sonicated and sonicated batchwise preparations, liposomes displayed non-homogeneous and non-reproducible size profiles. On the other hand, homogeneous liposomes were obtained with good reproducibility using our microchannel method combined with sonication.

Analytical Biochemistry, 2009

This article reports the enhancement of thermal stability involving normal duplex and mutation-ca... more This article reports the enhancement of thermal stability involving normal duplex and mutation-carrying DNA duplexes in microchannel laminar flow. The application of an in-house temperature-controllable microchannel-type flow cell is demonstrated for improved discrimination of mismatch base pairs such as A-G and T-G that are difficult to distinguish due to the rather small thermal destabilizations. Enhancement in thermal stability is reflected by an increased thermal melting temperature achieved in microchannel laminar flow as compared with batch reactions. To examine the kinetics and thermodynamics of duplex-coil equilibrium of DNA oligomers, denaturation-renaturation hysteresis curves were measured. The influence of microchannel laminar flow on DNA base mismatch analysis was described from the kinetic and thermodynamic perspectives. An increasing trend was observed for association rate constant as flow rate increased. In contrast, an apparent decrease in dissociation rate constant was observed with increasing flow rate. The magnitudes of the activation energies of dissociation were nearly constant for both the batch and microchannel laminar flow systems at all flow rates. In contrast, the magnitudes of activation energies of association decreased as flow rate increased. These results clearly show how microchannel laminar flow induces change in reaction rate by effecting change in activation energy. We anticipate, therefore, that this approach based on microchannel laminar flow system holds great promise for improved mismatch discrimination in DNA analyses, particularly on single-base-pair mismatch, by pronouncedly enhancing thermal stability.

ABSTRACT We investigate the behaviour of a simple microfluidic device designed to separate partic... more ABSTRACT We investigate the behaviour of a simple microfluidic device designed to separate particles based on density. The device consists of a separation-channel with three inlet and two outlet channels. The particle samples were loaded in the middle of the main channel. The results of separation experiments using model particles provide clear evidence that this approach can be used to achieve good separation of particles of close density populations. Particle separation was found more favourable with low flow rates. Forces exerted on particles were modelled by Stokes' law and found to be consistent with experimental results. One advantage of this microfluidic system is low exposure of the sample to hydrodynamic shear stresses compared with conventional methods. This device may be adopted to precisely handle single cells and easily interface with other tools and separation techniques.

Advanced Science Letters, 2010

Page 1. Delivered by Ingenta to: ? IP : 93.91.26.12 Thu, 21 Apr 2011 19:21:39 Copyright © 2010 Am... more Page 1. Delivered by Ingenta to: ? IP : 93.91.26.12 Thu, 21 Apr 2011 19:21:39 Copyright © 2010 American Scientific Publishers All rights reserved Printed in the United States of America RESEARCH ARTICLE Advanced Science Letters Vol. 3, 273–281, 2010 ...