A Simple Colorimetric Assay of Bleomycin-Mediated DNA Cleavage Utilizing Double-Stranded DNA-Modified Gold Nanoparticles.

A colorimetric assay of DNA cleavage by bleomycin (BLM) derivatives was developed utilizing high colloidal stability on double-stranded (ds) DNA-modified gold nanoparticles (dsDNA-AuNPs) possessing a cleavage site. The assay was performed using dsDNA-AuNPs treated with inactive BLM or activated BLM (Fe(II)⋅BLM). A 10-min exposure in dsDNA-AuNPs with inactive BLM treatment resulted in a rapid color change from red to purple because of salt-induced non-crosslinking aggregation of dsDNA-AuNPs. In contrast, the addition of active Fe(II)⋅BLM retained the red color, probably because of the formation of protruding structures at the outermost phase of dsDNA-AuNPs caused by BLM-mediated DNA cleavage. Furthermore, the results of our model experiments indicate that oxidative base release and DNA-cleavage pathways could be visually distinguished with color change. The present methodology was also applicable to model screening assays using several drugs with different mechanisms related to antitumor activity. These results strongly suggest that this assay with a rapid color change could lead to simple and efficient screening of potent antitumor agents.

Changes in In-Hospital Survival and Long-Term Neurodevelopmental Outcomes of Extremely Preterm Infants: A Retrospective Study of a Japanese Tertiary Center.

OBJECTIVES: The objective of this study was to elucidate whether the survival and long-term neurodevelopmental outcomes of extremely preterm infants have improved in a Japanese tertiary center with an active treatment policy for infants born at 22-23 weeks of gestation. STUDY DESIGN: This single-centered retrospective cohort study enrolled extremely preterm infants treated at Saitama Medical Center, Saitama Medical University, from 2003 to 2014. Patients with major congenital abnormalities were excluded. Primary outcomes were in-hospital survival and severe neurodevelopmental impairment (NDI) at 6 years of age, which was defined as having severe cerebral palsy, severe cognitive impairment, severe visual impairment, or deafness. We assessed the changes in primary outcomes between the first (period 1; 2003-2008) and the second half (period 2; 2009-2014) of the study period and evaluated the association between birth-year and primary outcomes using multivariate logistic regression models. RESULTS: Of the 403 eligible patients, 340 (84%) survived to discharge. Among 248 patients available at 6 years of age, 43 (14%) were classified as having severe NDI. Between the 2 periods, in-hospital survival improved from 155 of 198 (78%) to 185 of 205 (90%), but severe NDI increased from 11 of 108 (10%) to 32 of 140 (23%). In multivariate logistic regression models adjusted for gestational age, birthweight, sex, singleton birth, and antenatal corticosteroids, the aOR (95% CI) of birth-year for in-hospital survival and severe NDI was 1.2 (1.1-1.3) and 1.1 (1.0-1.3), respectively. CONCLUSION: Mortality among extremely preterm infants has improved over the past 12 years; nevertheless, no significant improvement was observed in the long-term neurodevelopmental outcomes.

The safety and effectiveness of elective laparoscopic surgery for benign ovarian cysts during pregnancy-Comparison with emergency surgery.

AIM: Relatively small benign ovarian cysts are conservatively managed in early pregnancy. However, emergency surgery is required should acute abdomen occur. Our study aimed to examine and compare the outcomes of benign ovarian cysts treated with elective laparoscopic surgery or emergency surgery during pregnancy. METHODS: From 2004 to 2017, we treated 135 pregnant patients (110 elective and 25 emergencies) with benign ovarian cysts at our tertiary perinatal center and compared their surgical and perinatal outcomes. RESULTS: There was no significant difference in cyst diameter (7.6 ± 2.5 vs. 6.8 ± 2.1 cm), but cysts <6 cm were significantly more common in emergency (36%) than in elective (15%) cases. Mature teratomas were significantly more common in elective cases (89% vs. 52%) but corpus luteum cysts were more common in emergency cases (0% vs. 32%). The rates of laparoscopic surgery (98.2% vs. 52.0%) and ovarian conservation (99.1% vs. 80.0%) were significantly higher, and post-surgical hospitalization (4.6 ± 1.3 vs. 9.8 ± 10.5 days) was significantly shorter in elective than in emergency cases. There was no significant difference in the gestational age for delivery (38.9 ± 1.9 vs. 38.4 ± 2.7 weeks), preterm birth rate (12% vs. 20%), or birth weight (2939 ± 469 vs. 3019 ± 510 g). CONCLUSIONS: We cannot state that an emergency surgery during pregnancy is rarely required for small benign ovarian cysts. However, the surgical outcomes were significantly better for elective than for emergency surgery, with no difference in perinatal outcomes. If a benign ovarian cyst is found early in pregnancy, elective laparoscopic surgery may be considered with adequate informed consent.

Thermoresponsive Polyphosphoester via Polycondensation Reactions: Synthesis, Characterization, and Self-Assembly.

Using a novel strategy, amphiphilic polyphosphoesters based on poly(oxyethylene H-phosphonate)s (POEHP) with different poly(ethylene glycol) segment lengths and aliphatic alcohols with various alkyl chain lengths were synthesized using polycondensation reactions. They were characterized by 1H NMR, 13C {H} NMR 31P NMR, IR, and size exclusion chromatography (SEC). The effects of the polymer structure on micelle formation and stability, micelle size, and critical micelle temperature were studied via dynamic light scattering (DLS). The hydrophilic/hydrophobic balance of these polymers can be controlled by changing the chain lengths of hydrophilic PEG and hydrophobic alcohols. A solubilizing test, using Sudan III, revealed that hydrophobic substances can be incorporated inside the hydrophobic core of polymer associates. Loading capacity depends on the length of alkyl side chains. The results obtained indicate that these structurally flexible polymers have the potential as drug carriers.

Terminal cationization of poly(N-isopropylacrylamide) brush surfaces facilitates efficient thermoresponsive control of cell adhesion and detachment.

A variety of poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces have been reported for temperature-controlled cell adhesion/detachment. However, the surfaces reported to date need further improvement to achieve good outcomes for both cell adhesion and detachment, which are inherently contradictory behaviors. This study investigated the effects of terminal cationization and length of grafted PIPAAm chains on temperature-dependent cell behavior. PIPAAm brushes with three chain lengths were constructed on glass coverslips via surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization. Terminal substitution of the grafted PIPAAm chains with either monocationic trimethylammonium or nonionic isopropyl moieties was performed through the reduction of terminal RAFT-related groups and subsequent thiol-ene reaction with the corresponding acrylamide derivatives. Although the thermoresponsive properties of the PIPAAm brush surfaces were scarcely affected by the terminal functional moiety, the zeta potentials of the cationized PIPAAm surfaces were higher than those of the nonionized ones, both below and above the phase transition temperature of PIPAAm (30°C). When bovine endothelial cells were cultured on each surface at 37°C, the number of adherent cells decreased with longer PIPAAm. Notably, cell adhesion on the cationized PIPAAm surfaces was higher than that on the nonionized surfaces. This terminal effect on cell adhesion gradually weakened with increasing PIPAAm length. In particular, long-chain PIPAAm brushes virtually showed cell repellency even at 37°C, regardless of the termini. Interestingly, moderately long-chain PIPAAm brushes promoted cell detachment at 20°C, with negligible terminal electrostatic interruption. Consequently, both cell adhesion and detachment were successfully improved by choosing an appropriate PIPAAm length with terminal cationization.

Fabrication of Hybrid Capsules via CaCO3 Crystallization on Degradable Coacervate Droplets.

Organic-inorganic CaCO3 capsules were prepared by crystallization of CaCO3 on Pickering emulsion prepared using coacervate droplets made from thermoresponsive and degradable poly(2-methylene-1,3-dioxepane- co-2-hydroxyethyl acrylate) (poly(MDO- co-HEA)) in sole aqueous medium. The diameters of CaCO3-based Pickering emulsion could be controlled by varying several parameters: diameter of CaCO3 powders, initial polymer concentration, and copolymer composition. The CaCO3 Pickering emulsion was able to load low-molecular-weight hydrophobic substances at temperatures above the lower critical solution temperature (LCST) due to formation of polymer-concentrated phases, i.e., coacervate droplets. The diameter of CaCO3 capsules prepared by crystallization also depended on the diameter of the CaCO3 Pickering emulsion. The CaCO3 shell was composed of calcite-type crystals, the most stable polymorph among known CaCO3 crystals. The facially prepared CaCO3 capsules are valuable for use in functional biomaterials, such as drug delivery carriers and cell culture scaffolds for noninvasive bone-regenerative medicine.

Direct observation of DNA overwinding by reverse gyrase.

Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70 °C, has so far been studied solely by gel electrophoresis; thus, the reaction dynamics remain obscure. Here, we image the overwinding reaction at 71 °C under a microscope, using DNA containing consecutive 30 mismatched base pairs that serve as a well-defined substrate site. A single reverse gyrase molecule processively winds the DNA for >100 turns. Bound enzyme shows moderate temperature dependence, retaining significant activity down to 50 °C. The unloaded reaction rate at 71 °C exceeds five turns per second, which is >10(2)-fold higher than hitherto indicated but lower than the measured ATPase rate of 20 s(-1), indicating loose coupling. The overwinding reaction sharply slows down as the torsional stress accumulates in DNA and ceases at stress of mere ∼ 5 pN ⋅ nm, where one more turn would cost only sixfold the thermal energy. The enzyme would thus keep DNA in a slightly overwound state to protect, but not overprotect, the genome of hyperthermophiles against thermal melting. Overwinding activity is also highly sensitive to DNA tension, with an effective interaction length exceeding the size of reverse gyrase, implying requirement for slack DNA. All results point to the mechanism where strand passage relying on thermal motions, as in topoisomerase IA, is actively but loosely biased toward overwinding.

Analysis of serotonin concentrations in human milk by high-performance liquid chromatography with fluorescence detection.

Serotonin (5-hydroxytryptamine, 5-HT) plays an important role in milk volume homeostasis in the mammary gland during lactation; 5-HT in milk may also affect infant development. However, there are few reports on 5-HT concentrations in human breast milk. To address this issue, we developed a simple method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for measuring 5-HT concentrations in human breast milk. Breast milk samples were provided by four healthy Japanese women. Calibration curves for 5-HT in each sample were prepared with the standard addition method between 5 and 1000 ng/ml, and all had correlation coefficients >0.999. The recovery of 5-HT was 96.1%-101.0%, with a coefficient of variation of 3.39%-8.62%. The range of 5-HT concentrations estimated from the calibration curves was 11.1-51.1 ng/ml. Thus, the HPLC-FD method described here can effectively extract 5-HT from human breast milk with high reproducibility.

Microsensors based on a whispering gallery mode in AlGaN microdisks undercut by hydrogen-environment thermal etching.

AlGaN microdisks were fabricated via a top-down process using electron-beam lithography, inductively coupled plasma reactive-ion etching, and hydrogen-environment thermal etching from commercial epitaxial wafers with a 100-300 nm thick AlGaN layer grown on a c-plane GaN layer by metal-organic chemical vapor deposition. The hydrogen-environment thermal etching performed well in undercutting the AlGaN microdisks owing to the selective etching for the GaN layer. The AlGaN microdisks acted as the whispering gallery mode (WGM) optical microresonators, exhibiting sharp resonant peaks in room temperature photoluminescence spectra. The evanescent component of the whispering gallery mode (WGM) is influenced by the ambient condition of the microdisk, resulting in the shift of the resonant peaks. The phenomenon is considered to be used for microsensors. Using the WGM in the AlGaN microdisks, we demonstrated microsensors and a microsensor system, which can potentially be used to evaluate biological and chemical actions in a microscale area in real time.

Vector synthesis high-resolution electrocardiography, atrial natriuretic peptide and N-terminal prohormone brain natriuretic peptide for estimation of cardiac load in pregnancy.

AIM: We analyzed atrial natriuretic peptide (ANP), N-terminal pro-brain natriuretic natriuretic peptide (NT-proBNP) and vector synthesis high-resolution electrocardiography (ECG), to estimate cardiac load with circulatory dynamic change from pregnancy through the post-partum period. METHODS: The subjects were singleton pregnant women (n = 19), who were divided into three stages: stage 1, 34-36 weeks of gestation; stage 2, 2-6 post-partum days; and stage 3, 1-3 months after delivery. Vector synthesis high-resolution ECG, ANP and NT-proBNP were analyzed for all subjects. RESULTS: A pregnant woman with massive uterin liomyoma expressed largest the corrected recover time (RTc) dispersion in I + II of tow Dimensional (2D) color distribution map ANP and NT-proBNP were significantly higher in stage 2 than in stages 1 and 3. CONCLUSIONS: ANP, NT-proBNP and vector synthesis high-resolution ECG there might be able to evaluate cardiac load of normal pregnancy.

Cohesin removal precedes topoisomerase IIα-dependent decatenation at centromeres in male mammalian meiosis II.

Sister chromatid cohesion is regulated by cohesin complexes and topoisomerase IIα. Although relevant studies have shed some light on the relationship between these two mechanisms of cohesion during mammalian mitosis, their interplay during mammalian meiosis remains unknown. In the present study, we have studied the dynamics of topoisomerase IIα in relation to that of the cohesin subunits RAD21 and REC8, the shugoshin-like 2 (Schizosaccharomyces pombe) (SGOL2) and the polo-like kinase 1-interacting checkpoint helicase (PICH), during both male mouse meiotic divisions. Our results strikingly show that topoisomerase IIα appears at stretched strands connecting the sister kinetochores of segregating early anaphase II chromatids, once the cohesin complexes have been removed from the centromeres. Moreover, the number and length of these topoisomerase IIα-connecting strands increase between lagging chromatids at anaphase II after the chemical inhibition of the enzymatic activity of topoisomerase IIα by etoposide. Our results also show that the etoposide-induced inhibition of topoisomerase IIα is not able to rescue the loss of centromere cohesion promoted by the absence of the shugoshin SGOL2 during anaphase I. Taking into account our results, we propose a two-step model for the sequential release of centromeric cohesion during male mammalian meiosis II. We suggest that the cohesin removal is a prerequisite for the posterior topoisomerase IIα-mediated resolution of persisting catenations between segregating chromatids during anaphase II.

Surface-Functionalized Biodegradable Nanoparticles Consisting of Amphiphilic Graft Polymers Prepared by Radical Copolymerization of 2-Methylene-1,3-Dioxepane and Macromonomers.

Biodegradable polyester-based nanoparticles were prepared by the precipitation of amphiphilic graft copolymers, which were prepared by the ring-opening radical copolymerization of 2-methylene-1,3-dioxepane (MDO) and amphiphilic macromonomers. The diameter of the nanoparticles was controlled by the degree of grafting and the molecular weight of the grafting oligomer. PMDO-g-poly(ethylene glycol) nanoparticles were degraded by the alkaline hydrolysis of the polyester backbone. Although the colloidal stability of nanoparticles was retained due to the reorientation of the PEG chains during hydrolysis, the size of the nanoparticles decreased with increasing hydrolysis time. We also prepared PMDO-g-poly(N-isopropylacrylamide) nanoparticles, which show aggregation in response to increasing temperature.

Mih1/Cdc25 is negatively regulated by Pkc1 in Saccharomyces cerevisiae.

Mitotic cyclin-dependent kinase (CDK) is activated by Cdc25 phosphatase through dephosphorylation at the Wee1-mediated phosphorylation site. In Saccharomyces cerevisiae, regulation of Mih1 (Cdc25 homologue) remains unclear because inactivation/degradation of Swe1 (Wee1 homologue) is the main trigger for G2/M transition. By deleting all mitotic cyclins except Clb2, a strain was created where Mih1 became essential for mitotic entry at high temperatures. Using this novel assay, the essential domain of Mih1 was identified and Mih1 regulation was characterized. Mih1(3E1D) with phosphomimetic substitutions of four putative PKC target residues in Mih1 had a reduced complementation activity, whereas Mih1(4A) with those nonphosphorylatable substitutions was active. The band pattern of Mih1 by SDS-PAGE was similar to that of Mih1(4A) only after inactivation of Pkc1 in a pkc1(ts) mutant. Over-expression of GFP-tagged Mih1 or GFP-Mih1(4A) accumulated as dot-like structures in the nucleus, whereas GFP-Mih1(3E1D) was localized in the cytoplasm. Over-expression of an active form of Pkc1 excluded GFP-Mih1 from the nucleus, but had minimal effect on GFP-Mih1(4A) localization. Furthermore, addition of ectopic nuclear localization signal to the Mih1(3E1D) sequence recovered complementation activity and nuclear localization. These results suggest that Mih1 is negatively regulated by Pkc1-mediated phosphorylation, which excludes it from the nucleus under certain conditions.

Porcine sperm capacitation involves tyrosine phosphorylation and activation of aldose reductase.

Mammalian sperm must be activated in the tubal isthmus through capacitation to induce the acrosome reaction and subsequent fertilization. Although the molecular mechanisms involved in capacitation have yet to be fully elucidated, increased concentrations of reactive oxygen species (ROS) and the extent of tyrosine phosphorylation of proteins have been suggested to play central roles in the completion of capacitation. In this study, aldose reductase was for the first time identified as one of the tyrosine-phosphorylated proteins involved in the capacitation of porcine cauda epididymal sperm. Both tyrosine phosphorylation and activity of aldose reductase associated with the particulate fraction of sperm cells were significantly enhanced during capacitation. Alrestatin, a membrane-permeable and specific inhibitor of aldose reductase, plays a role in the inhibition of aldose reductase activity, elevation of intracellular levels of ROS, and induction of hyperactivated motility, all at similar dose dependencies. Alrestatin canceled both the increase in the tyrosine phosphorylation of aldose reductase and the decrease in the glutathione levels in sperm-induced during capacitation. The hyperactivated motility was induced to a higher extent in the presence of glucose than in the presence of fructose. These results indicate that aldose reductase plays an important role in induction of hyperactivation and capacitation of sperm through the elevation of ROS in sperm cells. Furthermore, aldose reductase was shown to be added to sperm during transit through the epididymis, suggesting that aldose reductase is one of the key proteins that support the functional maturation of sperm.

Thermoresponsive anionic copolymer brushes containing strong acid moieties for effective separation of basic biomolecules and proteins.

A thermoresponsive copolymer brush possessing the sulfonic acid group, poly(N-isopropylacrylamide (IPAAm)-co-2-acrylamido-2-methylpropanesulfonic acid (AMPS)-co-tert-butylacrylamide (tBAAm)), was grafted onto the surface of silica beads through surface-initiated atom transfer radical polymerization. Prepared copolymer and copolymer brushes on silica beads were characterized by observing the phase transition profile, CHNS elemental analysis, X-ray photoelectron spectroscopy, and gel permeation chromatography. The phase transition profile indicated that an appropriate AMPS composition for enabling thermally modulated property changes is 5 mol %, while excessive amounts of sulfonic acid groups prevented copolymer phase transition. Chromatographic elutions of catecholamine derivatives and basic proteins were observed, using the prepared copolymer brush-modified beads as chromatographic matrices, and the results suggest that the beads interact with these analytes through relatively strong electrostatic interactions. Thus, poly(IPAAm-co-AMPS-co-tBAAm) brush-modified beads will be useful for effective thermoresponsive chromatography matrices that separate basic biomolecules through strong electrostatic interactions.

Monolithic silica rods grafted with thermoresponsive anionic polymer brushes for high-speed separation of basic biomolecules and peptides.

Thermoresponsive anionic copolymer brushes, poly(N-isopropylacrylamide-co-acrylic acid-co-tert-butylacrylamide) [P(IPAAm-co-AAc-co-tBAAm)], were grafted onto a monolithic silica rod column through surface-initiated atom-transfer radical polymerization (ATRP) to prepare an effective thermoresponsive anionic chromatography matrix. An ATRP initiator was attached to the rod surface. N-Isopropylacrylamide (IPAAm), tert-butyl acrylate (tBA), tert-butylacrylamide (tBAAm), and the ATRP catalyst CuCl/CuCl2/tris[2-(N,N-dimethylamino)ethyl]amine were dissolved in 2-propanol, and the reaction mixture was pumped into the initiator-modified column. After grafting P(IPAAm-co-tBA-co-tBAAm) on the monolithic silica surfaces, deprotection of the tert-butyl group of tBA was performed. Chromatographic analysis showed that the prepared column was able to separate catecholamine derivatives and angiotensin subtypes within a shorter analysis time (5 min) than a silica-bead-packed column modified with the same copolymer brush could. These results indicated that the prepared copolymer-modified monolithic silica rod column may be a promising bioanalytical and bioseparation tool for rapid analysis of basic bioactive compounds and peptides.

Thermoresponsive copolymer brushes possessing quaternary amine groups for strong anion-exchange chromatographic matrices.

A thermoresponsive copolymer incorporating a quaternary amine group, poly(N-isopropylacrylamide-co-3-acrylamidopropyl trimethylammonium chloride (APTAC)-co-tert-butylacrylamide), was conjugated to the surface of silica beads through surface-initiated atom transfer radical polymerization. Prepared copolymer- and copolymer brush-modified beads were characterized by CHN elemental analysis, X-ray photoelectron spectroscopy, gel permeation chromatography, and observation of phase transition profiles. Phase transition profiles of the prepared copolymer indicated that 5 mol % APTAC is suitable for enabling thermally modulated property changes in the copolymer. Chromatographic elution behaviors of adenosine nucleotides and proteins were observed using prepared beads as chromatography matrices. Higher retention time of adenosine nucleotides and strong protein adsorption behavior were observed compared with those on beads with tertiary amine groups, because of the strong basic properties. Therefore, copolymer brush modified beads will be useful as thermoresponsive ion-exchange chromatographic matrices.

Preparation of Degradable and Transformable Core-Corona-Type Particles that Control Cellular Uptake by Thermal Shape Change.

Particle-cell interactions, such as cellular uptake, vary depending on the particle size, shape, and surface properties. By dynamic control of the physical properties of particles, microparticle-cell interactions can intentionally be altered. Particle degradability is also necessary for their application in the body. In this study, we aimed to prepare degradable core-corona-type particles that are deformed near the body temperature and investigated particle shape-dependent cellular uptake. Degradable and transformable particles consisting of poly(2-methylene-1,3-dioxepane)-co-poly(ethylene glycol) with three-armed poly(ε-caprolactone) (PCL) were prepared. The particle melting point was controlled by the chain length of the three-armed PCL. Particle degradation occurred under both acidic and alkaline conditions via ester group hydrolysis in the polymer backbones. The rod-shaped microparticles prepared by uniaxial stretching at a temperature above the melting point of the core showed less uptake into macrophages than did the spherical microparticles. Therefore, the degradable transformable particles enable macrophage interaction control via stimuli-regulated particle shapes and are expected to be applied as drug delivery carriers that can be decomposed and excreted from the body.

A pregnant woman with long-standing, retained intraabdominal glass shards who gave birth to a live infant with no complications: a case report.

BACKGROUND: Most cases of traumatic injury during pregnancy involve blunt trauma, with penetrating trauma being uncommonly rare. In glass shard injuries, fragments often penetrate deeply, and multiple injuries may occur simultaneously; attention must be paid to the possibility of organ injury from the residual fragments. However, no case of this occurring during pregnancy has been reported yet. CASE PRESENTATION: We present the case of a 34-year-old pregnant Cameroonian woman who retained intraabdominal glass shards following a penetrating injury at 13 weeks gestation and not diagnosed until 22 weeks gestation. Notably, this patient continued the pregnancy without complications and gave birth via cesarean section at 36 weeks gestation. CONCLUSION: In pregnant women sustaining a penetrating glass trauma during pregnancy, careful attention should be paid to the fragments; in that case, computed tomography is a useful modality for accurately visualizing any remaining fragments in the body. Essentially, the foreign bodies in glass shard injuries during pregnancy should be removed immediately, but conservative management for term delivery is an important choice for patients at risk for preterm delivery.

Thermoresponsive nanospheres with a regulated diameter and well-defined corona layer.

In the present work, we prepared core-corona-type nanospheres bearing a thermoresponsive polymer with a controlled chain length on their surface. The corona layers were composed of poly(N-isopropylacrylamide) (PNIPAAm) chains (Mn = 3000-18,000) with a narrow polydispersity index prepared by atom-transfer radical polymerization (ATRP). Nanospheres were prepared by dispersion copolymerization of styrene with the PNIPAAm macromonomer in a polar solvent. The obtained nanospheres were monodisperse in diameter. The diameter of the nanospheres was regulated either by the number or chain length of the PNIPAAm macromonomers. In fact, the nanosphere diameter was regulated from ca. 100 to 1000 nm. When two types of PNIPAAm macromonomers are used, the obtained nanospheres have two different kinds of PNIPAAm on their surface. The surface of the nanospheres was observed to be thermoresponsive nanosphere in 0, 50, 100 mmol L(-1) NaCl aqueous solution. The nanosphere diameter and the surface-grafted polymer were concurrently adjusted for use in biomedical applications.