Imaging and Quantitative Analysis on the Etching of Diatom Frustules via Digital Holographic Microscopy.

Frustules, whose length spans from a few micrometers to more than a hundred micrometers, have been the subject of various modifications to improve their physical properties because of their complex porous silica structure. However, three-dimensional measurements of these changes can be challenging because of the complex 3D architecture and limitations of known methods. In this study, we present a new method that applies digital holographic microscopy (DHM) to analyze controlled etched frustules and observe real-time degradation of frustules at the single-cell level. Frustules obtained from Craspedostauros sp. diatoms were etched in 1 N NaOH for 5 min at 25 and 60 °C, respectively, and the frustule's valve was analyzed using DHM. DHM uses a combination of holography and tomography to reconstruct a 3D refractive index image of the frustule. Measurements of the width, volume, and surface area are achieved. Results showed that at 60 °C of etching, a significant difference with the unetched frustule was observed for all measurements but with high fluctuation values. Finally, real-time observation of the degradation of the frustule is observed when immersed in a high concentration of NaOH. This is the first time the real-time etching of the frustule is observed at the single-cell level. This research provides an easy estimation of the 3D measurements of frustules that may provide new fundamental information and applications.

Correction to "Fabrication of a Floatable Micron-Sized Enzyme Device Using Diatom Frustules".

[This corrects the article DOI: 10.1021/acsomega.3c02104.].

Fabrication of a Floatable Micron-Sized Enzyme Device Using Diatom Frustules.

Immobilization of enzymes has been widely reported due to their reusability, thermal stability, better storage abilities, and so on. However, there are still problems that immobilized enzymes do not have free movements to react to substrates during enzyme reactions and their enzyme activity becomes weak. Moreover, when only the porosity of support materials is focused, some problems such as enzyme distortion can negatively affect the enzyme activity. Being a solution to these problems, a new function "floatability" of enzyme devices has been discussed. A "floatable" micron-sized enzyme device was fabricated to enhance the free movements of immobilized enzymes. Diatom frustules, natural nanoporous biosilica, were used to attach papain enzyme molecules. The floatability of the frustules, evaluated by macroscopic and microscopic methods, was significantly better than that of four other SiO2 materials, such as diatomaceous earth (DE), which have been widely used to fabricate micron-sized enzyme devices. The frustules were fully suspended at 30 °C for 1 h without stirring, although they settled at room temperature. When enzyme assays were performed at room temperature, 37, and 60 °C with or without external stirring, the proposed frustule device showed the highest enzyme activity under all conditions among papain devices similarly prepared using other SiO2 materials. It was confirmed by the free papain experiments that the frustule device was active enough for enzyme reactions. Our data indicated that the high floatability of the reusable frustule device, and its large surface area, is effective in maximizing enzyme activity due to the high probability to react to substrates.

Hybridization of papain molecules and DNA-wrapped single-walled carbon nanotubes evaluated by atomic force microscopy in fluids.

Although various conjugates of single-walled carbon nanotubes (SWNTs) and biomolecules, such as nanobiosensors and nanobiodevices, have been reported, the conjugation of papain and SWNTs have not been reported because of the formation of unexpected aggregates. In this study, atomic force microscopy (AFM) in liquid was used to investigate the interactions between papain and DNA-wrapped SWNTs (DNA-SWNTs) at two different pH values (pH 3.0 and 10.5). The direct AFM observation of the mixture of papain and DNA-SWNTs confirmed the aggregation of papain molecules with DNA-SWNTs in the buffer solutions. The numerous and non-uniform adsorption of papain molecules onto DNA-SWNTs was more pronounced at pH 3.0 than that at pH 10.5. Furthermore, thick conjugates appeared when papain and DNA-SWNTs were simultaneously mixed. The near-infrared photoluminescence spectra of the SWNTs drastically changed when the papain molecules were injected into the DNA-SWNT suspension at pH 3.0. Thus, the regulation of electrostatic interactions is a key aspect in preparing optimal conjugates of papain and DNA-SWNTs. Furthermore, although previous papers reported AFM images of dried samples, this study demonstrates the potential of AFM in liquid in evaluating individual bioconjugates of SWNTs.

Attachment of DNA-Wrapped Single-Walled Carbon Nanotubes (SWNTs) for a Micron-Sized Biosensor.

We fabricated a micron-sized biodevice based on the near-infrared photoluminescence (PL) response of single-walled carbon nanotubes (SWNTs). Various biosensors using the unique optical responses of SWNTs have been proposed by many research groups. Most of these employed either colloidal suspensions of dispersed SWNTs or SWNT films on flat surfaces, such as electrodes. In this study, we attached DNA-wrapped SWNTs (DNA-SWNTs) to frustule (micron-sized nanoporous biosilica) surfaces, which were purified from cultured isolated diatoms. After the injection of an oxidant and a reductant, the SWNTs on the frustules showed prominent PL responses. This suggests that the biodevice functions as a micron-sized redox sensor. Frustules can be easily suspended in aqueous solutions because of their porous structures and can easily be collected as pellets by low-speed centrifugation. Thus, the removal of unbound SWNTs and the recovery of the fabricated DNA-SWNT frustules for reuse were achieved by gentle centrifugation. Our proposal for micron-sized SWNT biodevices would be helpful for various biological applications.

An Efficient Method of Observing Diatom Frustules via Digital Holographic Microscopy.

Herein, we propose a convenient method to enable pretreatment of target objects using digital holographic microscopy (DHM). As a test sample, we used diatom frustules (Nitzschia sp.) as the target objects. In the generally used sample preparation method, the frustule suspension is added dropwise onto a glass substrate or into a glass chamber. While our work confirms good observation of purified frustules using the typical sample preparation method, we also demonstrate a new procedure to observe unseparated structures of frustules prepared by baking them on a mica surface. The baked frustules on the mica surface were transferred to a glass chamber with 1% sodium dodecyl sulfate solution. In this manner, the unseparated structures of the diatom frustules were clearly observed. Furthermore, metal-coated frustules prepared by sputtering onto them on a mica surface were also clearly observed using the same procedure. Our method can be applied for the observation of any target object that is pretreated on a solid surface. We expect our proposed method to be a basis for establishing DHM techniques for microscopic observations of biomaterials.

Distinguishing Antioxidant Molecules with Near-Infrared Photoluminescence of DNA-Wrapped Single-Walled Carbon Nanotubes.

In this study, two biomolecule solutions were distinguished using the capacity difference in the near-infrared photoluminescence (PL) of single-walled carbon nanotubes (SWNTs). Biosensing techniques using sensitive responses of SWNTs have been intensively studied. When a small amount of an oxidant or reductant solution was injected into the SWNT suspensions, the PL intensity of the SWNTs is significantly changed. However, distinguishing between different molecules remains challenging. In this study, we comparably injected saponin and banana solutions, which are known antioxidant chemicals, into an SWNT suspension. The SWNTs were solubilized by wrapping them with DNA molecules. The results show that 69.1 and 155.2% increases of PL intensities of SWNTs were observed after injection of 20 and 59 µg/mL saponin solutions, respectively. Subsequently, the increase in PL was saturated. With the banana solution, 18.1 and 175.4% increases in PL intensities were observed with 20 and 59 µg/mL banana solutions, respectively. Based on these results, the two antioxidant molecules could be distinguished based on the different PL responses of the SWNTs. In addition, the much higher saturated PL intensities observed with the banana solution suggests that the banana solution increased the capacity of the PL increase for the same SWNT suspension. These results provide helpful information for establishing biosensing applications of SWNTs, particularly for distinguishing chemicals.

Stable Near-Infrared Photoluminescence of Single-Walled Carbon Nanotubes Dispersed Using a Coconut-Based Natural Detergent.

We prepared single-walled carbon nanotube (SWNT) suspensions in phosphate buffer solutions containing 1% of a coconut-based natural detergent (COCO) or 1% of sodium dodecyl sulfate (SDS). The suspensions exhibited strong photoluminescence (PL) in the near-infrared region, suggesting that the SWNTs, such as those with (9, 4) and (7, 6) chiralities, were monodispersed. Upon diluting the suspensions with a detergent-free phosphate buffer solution, the PL intensity of the SDS-containing SWNT suspension was significantly lower than that of the COCO-containing SWNT suspension. The COCO-containing SWNT suspension was more stable than the SDS-containing SWNT suspension. The SWNT concentration of the suspensions prepared via bath-type sonication was lower than that of the suspensions prepared via probe-type sonication. However, near-infrared (NIR) PL intensity of the SWNT suspensions prepared via bath-type sonication was much higher than that of the SWNT suspensions prepared via probe-type sonication regardless of the detergent. This suggested that the fraction of monodispersed SWNTs of the suspensions prepared via bath-type sonication was larger than that of the suspensions prepared via probe-type sonication, although the SWNT concentration was low. Our results indicated that COCO favored the fabrication of SWNT suspensions with stable and strong NIR PL, which are useful for various biological applications.

Prediction of Severe Cisplatin-Induced Neutropenia Using Serum Albumin Concentration-A Retrospective Study.

BACKGROUND: Cisplatin that is used in the treatment of gastric cancer not only has gastrointestinal side effects but also has a high serum protein-bound fraction. Reduction of serum albumin concentration may cause increase the risk of cisplatin- induced neutropenia. Hence, alteration of serum albumin concentration poses a major safety issue during anticancer therapy. METHODS: For gastric cancer patients who received cisplatin plus S-1 therapy, we investigated the relationship between the serum albumin concentration before cisplatin administration in the treatment course during which the neutrophil count reached nadir and the neutrophil count fluctuation after cisplatin administration. RESULTS: In the grade 3-4 neutropenia and grade 0-2 neutropenia groups, the mean serum albumin concentration before cisplatin administration was 3.39±0.60 and 3.85±0.59 g/dL, respectively; in the former group were significantly lower than in the latter group(p=0.006). Lower serum albumin concentrations before cisplatin administration were significantly correlated with a decrease in neutrophil count after cisplatin administration(r=0.463, p<0.001). According to the receiver operating characteristic curve analysis, patients with serum albumin concentrations below 3.25 g/dL before cisplatin administration exhibited a significantly higher incidence of grade 3-4 neutropenia(odds ratio: 4.33). CONCLUSIONS: Decreased serum albumin levels were found to be strongly associated with the prediction of the development of severe neutropenia. Our findings emphasize serum albumin concentration needs to be evaluated before each administration of cisplatin.

Evaluation of risk factors for lymph node metastasis in T2 lower rectal cancer to perform chemoradiotherapy after local resection.

The oncological outcome of chemoradiotherapy (CRT) after local excision (LE) for T2 lower rectal cancer has demonstrated a high local recurrence (LR) rate. The aim of the present study was to determine the risk factors for lymph node metastasis (LNM) in order to reduce LR in T2 lower rectal cancer after LE and CRT. Specimens were collected from 95 consecutive patients with T2 lower rectal adenocarcinoma who underwent R0 resection by total mesenteric excision or tumor-specific mesenteric excision between January 2008 and December 2018 at Osaka International Cancer Institute. All specimens were checked and evaluated to determine the risk factors for LNM. LNM was observed in 26 patients (27%), including 2 patients (2%) with lateral pelvic lymph node metastasis. Univariate analysis indicated lymphovascular invasion (LVI; P=0.008), tumor budding (P=0.012) and histology other than well-differentiated adenocarcinoma (P=0.08) were associated with LNM; multivariate analysis revealed that LVI (P=0.03) was the only independent risk factor for LNM. LNM was confirmed in 0% (0/8) of patients without LVI, tumor budding and histological type. LVI, tumor budding and histological type can be risk factors for LNM in lower rectal cancer. The present study may be helpful to select patients for performing LE and CRT with good oncological outcome.

[Effect of Hypoalbuminemia on Severe Neutropenia Induced by Paclitaxel Monotherapy and Paclitaxel plus Ramucirumab Combination Therapy in Patients with Gastric Cancer].

Hypoalbuminemia is often observed in patients receiving chemotherapy for gastric cancer. The risk of hematologic toxicity is increased by the pharmacokinetic alteration of paclitaxel(PTX)owing to high serum protein binding in patients with hypoalbuminemia. Here, we examined the relationshipbetween the frequency of GradeB3 neutropenia and serum albumin concentration in 30 patients receiving PTX monotherapy, and 29 patients receiving PTX plus ramucirumab(RAM)combination therapy-a second-line treatment for advanced/recurrent gastric cancer. The number of patients who developed GradeB3 neutropenia was 8(27%)and 14(48%)of those who received monotherapy and combination therapy, respectively, with mean serum albumin concentrations of 3.31 g/dL and 3.15 g/dL, respectively. Serum albumin concentrations were significantly lower in the GradeB3 neutropenia group than in the non-GradeB3 neutropenia group in both regimens. When the serum albumin concentration of patients receiving PTX or PTX plus RAM was below the cut-off values of 3.75 g/dL and 3.45 g/dL, respectively, the odds ratio of GradeB3 neutropenia was 12.25 and 7.33, respectively. Hypoalbuminemia was associated with the development of chemotherapy-induced GradeB3 neutropenia, in patients with gastric cancer treated with either PTX monotherapy or PTX plus RAM combination therapy. Therefore, not only neutrophils but also serum albumin concentration should be monitored during chemotherapy.

Risk stratification for lymph node metastasis using Epstein-Barr virus status in submucosal invasive (pT1) gastric cancer without lymphovascular invasion: a multicenter observational study.

BACKGROUND: Lymphovascular invasion (LVI) is a strong predictive factor for lymph node metastasis (LNM) in early gastric cancer (GC). This study investigated the risk for LNM in pT1b GC without LVI based on Epstein-Barr virus (EBV) status in addition to conventional clinicopathological parameters. METHODS: In total, 847 consecutive patients of pT1b GC without LVI who underwent surgery at three high-volume centers between 2005 and 2014 were retrospectively analyzed. Clinicopathological parameters and EBV status were evaluated, and univariate and multivariate analyses were performed to estimate LNM risk. With regard to the presence of those three parameters, risk stratification for LNM was performed and compared with a previously proposed risk classification that included low-risk (LNM < 3.0%), intermediate-risk (LNM ≥ 3.0 and < 19.6%), and high-risk (LNM ≥ 19.6%) groups. RESULTS: EBV-positive GC (EBVGC) accounted for 11.3% (96 of 847) of cases; LNM was lower in EBVGC than in non-EBVGC (1 of 96, 1.0% vs. 71/751, 9.5%). In the multivariate analysis, non-EBVGC [odds ratio (OR) 10.8, 95% confidence interval (CI) 1.48-78.9], age < 65 years (OR 2.13, 95% CI 1.30-3.48), and tumor diameter > 3 cm (OR 2.26, 95% CI 1.36-3.74) were independent risk factors for LNM. Patients with EBVGC were at low risk for LNM whereas those with all of three independent risk factors were at high risk (36 of 168, 21.4%, 95% CI 15.5-28.4). CONCLUSION: LNM risk stratification that includes EBV status is useful for clinical decision-making in pT1b GC cases without LVI.

[Identification of the Risk Factors for Cabazitaxel-Induced Neutropenia with Preventive Administration of Pegfilgrastim].

Prophylaxis using pegfilgrastim is recommended to prevent cabazitaxel-induced neutropenia. We observed GradeB3 neutropenia in a patient after administration of cabazitaxel, despite prophylaxis using pegfilgrastim. To identify the risk factors associated with GradeB3 neutropenia, we retrospectively investigated the records of 10 patients who received prophylaxis with pegfilgrastim after administration of cabazitaxel. They were divided into GradeB3 neutropenia and non-GradeB3 neutropenia groups, and we compared the background data and laboratory values between the 2 groups. A univariate analysis revealed that hypoalbuminemia was significantly observed in patients with cabazitaxel-induced GradeB3 neutropenia. The incidence of GradeB3 neutropenia was significantly high in patients with serum albumin levels<3.6 g/dL. Cabazitaxel has a high rate of protein binding; moreover, serum albumin levels<3.6 g/dL might be associated with high concentrations of unbound cabazitaxel, and thus an increase in the incidence of GradeB3 neutropenia. Therefore, hypoalbuminemia at the time of administration of cabazitaxel may be a risk factor related to the development of GradeB3 neutropenia.

Relatively high rates of cefotaxime- and ceftriaxone-non-susceptible isolates among group B streptococci with reduced penicillin susceptibility (PRGBS) in Japan.

OBJECTIVES: We have previously identified group B Streptococcus (GBS) clinical isolates with reduced penicillin susceptibility (PRGBS) that were non-susceptible to cefotaxime; however, the rates of cefotaxime and ceftriaxone non-susceptibility among PRGBS isolates have never been reported. Therefore, we first determined the MICs of 22 antibacterial drugs/compounds for 74 PRGBS isolates and then determined the rates of cefotaxime and ceftriaxone non-susceptibility among these isolates. METHODS: We used 74 clinical PRGBS isolates, previously collected in Japan and confirmed to harbour relevant amino acid substitutions in PBP2X. We also used 80 penicillin-susceptible GBS (PSGBS) clinical isolates as controls. The MICs of 22 antibacterial drugs/compounds for all 154 GBS isolates were determined via microdilution and/or agar dilution methods, as recommended by the CLSI. RESULTS: The rates of non-susceptibility/resistance to ampicillin, cefotaxime, ceftriaxone and levofloxacin for the 80 PSGBS isolates were 0%, 0%, 0% and 30%, respectively, but were 15% (P = 0.0003), 28% (P < 0.0001), 36% (P < 0.0001) and 93% (P < 0.0001) for the 74 PRGBS isolates, respectively. No PRGBS isolates were identified to be non-susceptible to meropenem, doripenem, vancomycin, quinupristin/dalfopristin, daptomycin or linezolid. CONCLUSIONS: We found that cefotaxime- and ceftriaxone-non-susceptible PRGBS isolates occur at relatively high rates in Japan. Importantly, this finding suggests that the range of drugs likely to be effective in treating PRGBS infections may be limited compared with those available for PSGBS infections; therefore, clinicians should exercise care when considering drug choice and efficacy for PRGBS infections.

[Retrospective Investigation of the Risk Factors for Severity in Panitumumab-Induced Hypomagnesemia].

Hypomagnesemia caused by panitumumab can often lead to severe adverse effects, such as arrhythmia. However, the risk factors are still controversial. To clarify the risk factors and time to onset of panitumumab-induced hypomagnesemia, we retrospectively investigated the records of 30 patients who had received panitumumab. They were divided into Grade B2 hypomagnesemia and non-Grade B2 hypomagnesemia groups, according to their serum magnesium levels, and we compared patients' backgrounds, laboratory values, and concomitant drugs between the 2 groups. Univariate analysis revealed that hypocalcemia and non-administration of an oral magnesium preparation were significantly associated with Grade B2 hypomagnesemia induced by panitumumab. Incipient grade 1 hypocalcemia was observed after incipient Grade 1 hypomagnesemia in both groups. The occurrence of these complications was significant in the Grade B2 hypomagnesemia group. Thereafter, in all patients of the Grade B2 hypomagnesemia group that exhibited both complications, hypomagnesemia developed to Grade 2 or higher. The development of Grade 3 and Grade 4 hypomagnesemia without the development of Grade 2 hypomagnesemia was observed in 2 patients each. Thus, aggravation of hypomagnesemia can be expected upon the administration of panitumumab, and therefore, not only serum magnesium levels, but also serum calcium levels need to be monitored.

Case Reports of Severe Congenital Neutropenia Treated With Unrelated Cord Blood Transplantation With Reduced-intensity Conditioning.

Allogeneic stem cell transplantation is a curative treatment for severe congenital neutropenia (SCN). However, a standard conditioning regimen and donor source have not been established. We report 3 consecutive cases of SCN who were successfully treated by cord blood transplantation (CBT) with reduced-intensity conditioning consisting of fludarabine, melphalan, and low-dose total body irradiation. All cases achieved complete donor chimerism without severe infectious complications and have maintained normal neutrophil counts for between 3 and 9 years after CBT. These results suggest that CBT with reduced-intensity conditioning can be an alternative therapy for SCN when human leukocyte antigen-matched bone marrow donor is unavailable.

A prospective stratified case-cohort study on statins and multiple adverse events in Japan.

PURPOSE: To assess the association between statins and diverse adverse events in Japanese population. METHODS: New users of statin who started statin after 6-month period of non-use were identified in 68 hospitals between January 2008 and July 2010. In addition to the random sample subcohort, we selected additional subcohort members to make the stratified sample subcohort have at least one patient in all subgroups stratified by each combination of statin and hospital. By abstraction from medical records, detailed information was obtained for all potential cases and pre-selected subcohort members. The event review committee consisting of 3 specialists judged whether possible cases met the definition of one of the adverse events of interest, and for adjudicated cases the committee further judged whether statin was a certain, probable or possible cause of the occurrence of the event. Adjusted for covariates including age, gender, status of "switcher", use of high daily dose and comorbidities at baseline, hazard ratio (HR) was estimated by the Cox proportional hazards model with Barlow's weighting method. Data were also analyzed by the method proposed by Breslow in 2009. RESULTS: A total of 6,877 new users of a statin were identified (median age: 66 years; males: 52%). The hazard ratios of increase in serum creatinine for atorvastatin and fluvastatin have wide confidence intervals, but both of the point estimates were around 2.5. Estimates of hazard ratios by the method of Barlow (1999) were similar to those by the method of Breslow (2009). CONCLUSIONS: Use of statin was not associated with a significant increased risk for renal, liver and muscle events. However, the hazard ratio of increase in serum creatinine tended to be high with atorvastatin and fluvastatin to require further studies.