The triangular prism approximation method for volume estimation of deep inferior epigastric artery perforator flap in breast reconstruction.

BACKGROUND: Many methods to predict the amount of tissue needed for breast reconstruction have been reported, but some require complicated software and special systems. The purpose of this report was to present a simpler method for predicting the volume required for deep inferior epigastric artery perforator (DIEP) flaps. The accuracy of this method was evaluated based on both actual flap design and computed tomography. METHODS: The weight and horizontal (x cm) and vertical (y cm) lengths of the DIEP flap were recorded, and the maximum thickness of subcutaneous tissue (z cm) was measured from computed tomography in 36 cases of breast reconstruction using DIEP flap in our hospital performed between January 2019 and December 2020. Flap volume was calculated using three methods of approximation: triangular prisms using physical and CT measurements (1/2xyz cm3 ); quadrangular and triangular prisms using physical and CT measurements (3/4xyz cm3 ); and a previously reported method using measurements from CT angiography alone and calculation with a standard mathematical formula. These three groups were compared using Bland-Altman plots and intraclass correlation coefficients (ICCs) to assess consistency between predicted and measured values. RESULTS: On Bland-Altman plots, values were distributed almost randomly around the average value of the difference, and no proportional error was evident in the methods. The ICC between predicted and actual values of triangular prisms using physical and CT measurements was largest: ICC (1, 2) = 0.978 (0.825-0.981; 95% confidence interval for ICC). A sufficient flap volume was able to be transplanted in all cases. CONCLUSION: The methods presented appear useful to calculate flap volume closer to the measured value without complicated software systems. These results suggest that the method using two symmetric triangular prisms could predict volume more easily than previously reported methods and may facilitate good breast reconstruction.

ANESTHETIC MANAGEMENT OF AN INDO-PACIFIC BOTTLENOSE DOLPHIN (TURSIOPS ADUNCUS) REQUIRING SURGICAL DEBRIDEMENT OF A TAIL ABSCESS.

This report describes the anesthetic management of a 14-yr-old, 160-kg, female Indo-Pacific bottlenose dolphin ( Tursiops aduncus ) that underwent surgical debridement for a refractory subcutaneous abscess twice within a 6-mo interval. The animal was otherwise in good physical condition at each anesthetic procedure. Following premedication with intramuscular midazolam and butorphanol, anesthesia was induced with propofol and maintained with sevoflurane by intubation. During surgery ventilation was controlled. Blood pressure was indirectly estimated using either oscillometric or pulse oximetry. Presumed hypotension was managed by adjusting the sevoflurane concentration and infusion of dopamine. During recovery, the dolphin regained adequate spontaneous respiration following intravenous administration of flumazenil and doxapram. The dolphin was extubated at 85 min and 53 min after the first and second surgeries, respectively. Successful weaning from the ventilator and initiation of spontaneous respiration was the most important complication encountered. Establishment of a reliable blood pressure measurement technique is critical to success for anesthesia in this species.

Fluorescent nanoparticles consisting of lipopeptides and fluorescein-modified polyanions for monitoring of protein kinase activity.

Protein kinase (PK)-responsive nanoparticles (NPs) comprising a hydrophobically modified peptide substrate for PKs and a fluorescein-labeled polyanion (pA-F) were reported for monitoring PK activity via fluorescence intensity measurements. In this system, the formation of NPs by mixing lipopeptides and pA-Fs results in fluorescence quenching, while the quenched fluorescence recovered following dissociation of the NPs owing to the phosphorylation reaction of PKs. Eleven lipopeptides with different hydrophobic moieties (hydrocarbon and lithocholic acid) and four pA-Fs having main chains with differing flexibilities and fluorescein contents were synthesized and used to fabricate a series of twenty-four PK-responsive NP probes. The responses of the PK-responsive NP probes to PKs were evaluated to screen the most suitable NP probes. The assay system was then used to determine the IC(50) values for five inhibitors, the results of which were very similar to those previously reported. Thus, PK-responsive NPs are useful tools for high-throughput screening (HTS) of PK inhibitors.

Morphological and functional behaviors of rat hepatocytes cultured on single-walled carbon nanotubes.

This study describes the morphological and functional behaviors of rat hepatocytes on single-walled carbon nanotube (CNT)-coated surfaces. Although the hydrophobic characteristics of CNT-coated surfaces increased with increasing CNT density, hepatocyte adhesion decreased, indicating that the interaction between hepatocytes and CNTs is weak. We found that hepatocytes on a CNT-coated surface gradually gather together and form spheroids (spherical multicellular aggregates). These spheroids exhibit compact spherical morphology with a smooth surface and express connexin-32, an intracellular communication molecule. In contrast, collagen treatment in conjunction with the CNT-coated surface improved hepatocyte adhesion, and the cells maintained a monolayer configuration throughout the culture period. The albumin secretion and ammonia removal activities of hepatocyte spheroids were maintained at elevated levels for at least 15 days of culturing as compared with hepatocyte monolayers. These results indicate that CNTs can be used for the formation and long-term culture of hepatocyte spheroids.

Objective assessment of reconstructed breast hardness using a durometer.

BACKGROUND: Whether a durometer was suitable for objectively measuring reconstructed breast hardness was evaluated. METHODS: Subjects were 81 women who underwent expander-implant reconstructions following breast cancer ablation. Capsular contracture was evaluated with Baker grading. Capsular thickness was measured with T1-weighted MRI at the upper areola area. The durometer was placed on the upper areola. Multiple logistic regression analysis was performed to compare variables. RESULTS: On Baker grading, 17 breasts were Baker grade I, 52 breasts were Baker grade II, 11 breasts were Baker grade III, and 1 breast was Baker grade IV. Mean capsular thickness on MRI was 1.1 (SD 0.4) mm with Baker grade I, 1.2 (SD 0.3) mm with Baker grade II, 1.4 (SD 0.4) mm with Baker grade III, and 1.9 mm with Baker grade IV. Mean durometer value was 0 with Baker grade I, 0.2 (SD 0.5) with Baker grade II, 2.0 (SD 1.7), with Baker grade III, and 8 with Baker grade IV. Baker grade IV was excluded from analysis because there was only one case. When Baker grade III was defined as positive for hardness, multiple logistic regression analysis showed that durometer value was associated with Baker grade III (p = 0.0005), but capsular thickness was not. On receiver operating characteristic curve analysis of the durometer value for Baker grade III, the optimal cutoff value was 0.5 (sensitivity 0.92, 1-specificity 0.17, area under the curve 0.92). CONCLUSIONS: The durometer offers an objective index of hardness that might replace the subjective Baker grading. Further studies are needed to confirm the utility of this index.

Long-term monitoring of circulating progesterone and its relationship to peripheral white blood cells in female false killer whales Pseudorca crassidens.

Long-term monitoring of circulating progesterone levels in three captive female false killer whales, Pseudorca crassidens, was conducted to characterize their reproductive events and to reveal the relationship between their estrous cycles or pregnancies and peripheral white blood cell (WBC) counts. Blood samples were collected at 2-3-day intervals or on a weekly-to-monthly basis for up to 10 years, from 2006 to 2017. In two mature females (initial body lengths of 4.22 and 4.07 m), some cyclic progesterone elevations were detected during the study period; the estimated mean (± SE) estrous cycle length was 40.5 ± 0.7 days (n=12). The seasonality of ovulation, estimated from the elevation of progesterone levels, varied among individuals or years, and ovulation did not occur every year. The third female (3.26 m) showed progesterone elevations, despite irregular cycles after sexual maturity, and became pregnant. The progesterone levels during pregnancy ranged from 7.3 to 42.2 ng/ml, and the gestation period lasted for 14 months until parturition. The mean WBC counts during estrous cycles were the lowest before the progesterone levels began to increase and then gradually increased toward the luteal phase. The WBC counts were significantly higher during pregnancy than before and were particularly high in early pregnancy. To the best of our knowledge, this is the first report of the relationship between the estrous cycle or pregnancy and WBC counts in cetaceans.

Effect of separation distance on the growth and differentiation of mouse embryoid bodies in micropatterned cultures.

Embryoid body (EB) culture has been widely used for in vitro differentiation of embryonic stem (ES) cells. Micropatterning of cultures is a promising technique for regulating EB development, because it allows for controlling the EB size and the distance between neighboring EBs. In this study, we examined the relationship of EB separation distance to their growth and differentiation using a micropatterned chip. The basic chip design consisted of 91 gelatin spots (300 µm in diameter) in a hexagonal arrangement on a glass substrate that served as the cell adhesion area; the region without gelatin spots was modified with polyethylene glycol to create the non-adhesion area. Two similar chips were fabricated with distances between gelatin spots of 500 and 1500 µm. Mouse ES cells adhered on the gelatin spots and then proliferated to form EBs. When the EB-EB distance was at 1500 µm, their size and the expression of developmental gene markers were almost the same for all EBs on the chip. This indicated that interference between neighboring EBs was avoided. In contrast, when the EB-EB distance was at 500 µm, the size of EBs located in the inside region of the chip was smaller than that in the outside region. Additionally, in the inside region, hepatic differentiation of EB cells was increased over cardiac and vascular differentiation. These results indicate that the distance between EBs is an important factor in the regulation of their growth and differentiation.

Sperm granuloma and sperm agglutination in a dog with asthenozoospermia.

A mongrel dog, aged 2 years, was found to have only a small number of sperm, immobilization of all sperm, and many sperm agglutinations in its ejaculates, and scrotal palpation revealed a small nodule in the left cauda epididymis. Addition of the dog's seminal plasma or serum to the semen of 2 normal dogs caused immobilization and agglutination of their sperm. Histological examination showed that the nodule was a sperm granuloma. Many lymphocytes were seen in the stroma around the sperm granuloma. Anti-sperm antibodies are presumed to be present in the semen and serum of the asthenozoospermic dog.

Tailor-made cell patterning using a near-infrared-responsive composite gel composed of agarose and carbon nanotubes.

Micropatterning is useful for regulating culture environments. We developed a highly efficient near-infrared-(NIR)-responsive gel and established a new technique that enables cell patterning by NIR irradiation. As a new culture substratum, we designed a tissue culture plate that was coated with a composite gel composed of agarose and carbon nanotubes (CNTs). A culture plate coated with agarose only showed no response to NIR irradiation. In contrast, NIR laser irradiation induced heat generation by CNTs; this permitted local solation of the CNT/agarose gel, and consequently, selective cell-adhesive regions were exposed on the tissue culture plate. The solation area was controlled by the NIR intensity, magnification of the object lens and CNT concentration in the gel. Furthermore, we formed circular patterns of HeLa cells and linear patterns of 3T3 cells on the same culture plate through selective and stepwise NIR irradiation of the CNT/agarose gel, and we also demonstrated that individual 3T3 cells migrated along a linear path formed on the CNT/agarose gel by NIR irradiation. These results indicate that our technique is useful for tailor-made cell patterning of stepwise and/or complex cell patterns, which has various biological applications such as stepwise co-culture and the study of cell migration.

Characterization of mouse embryoid bodies cultured on microwell chips with different well sizes.

Microwell chip culture is a promising technique for the generation of homogenous embryoid bodies (EBs). In this study, we focused on the relationship between microwell size and mouse EB properties. The basic chip design was 195 microwells in a triangular arrangement on a polymethylmethacrylate plate with a surface modified by polyethylene glycol to render it nonadhesive, and 4 similar chips were fabricated with microwell diameters of 400, 600, 800, and 1000 µm. The cell proliferation rate of EBs in larger microwells was higher than that of EBs in smaller microwells. The decrease in the expression levels of undifferentiated marker genes (Oct3/4 and Nanog) in larger microwells was faster than that in smaller microwells. The expression of hepatic (transthyretin and alpha-fetoprotein), cardiac (Nkx2.5 and alpha-myosin heavy chain), and vascular (fetal liver kinase-1; Flk1) markers in larger microwells was higher than that in smaller microwells. The expression levels of differentiation markers except Flk1 in the chip with a diameter of 1000 µm were similar to those in hanging drop culture. However, Flk1 expression in microwell chip was markedly lower than that in hanging drop culture, suggesting that microwell chip culture promotes differentiation of hepatic and cardiac lineages. Furthermore, glucose consumption and lactate production were higher in smaller microwells, suggesting that the culture proceeds under anaerobic conditions in smaller microwells. These results indicate that the difference in microwell size affects the proliferation and differentiation of embryonic stem cells, and that microwell culture is a promising technique to control EB properties.

Effect of introduction of chondroitin sulfate into polymer-peptide conjugate responding to intracellular signals.

We recently developed a novel tumor-targeted gene delivery system responding to hyperactivated intracellular signals. Polymeric carrier for gene delivery consists of hydrophilic neutral polymer as main chains and cationic peptide substrate for target enzyme as side chains, and was named polymer-peptide conjugate (PPC). Introduction of chondroitin sulfate (CS), which induces receptor-medicated endocytosis, into polymers mainly with a high cationic charge density such as polyethylenimine can increase tumor-targeted gene delivery. In the present study, we examined whether introduction of CS into PPC containing five cationic amino acids can increase gene expression in tumor cells. Size and zeta potential of plasmid DNA (pDNA)/PPC/CS complex were <200 nm and between -10 and -15 mV, respectively. In tumor cell experiments, pDNA/PPC/CS complex showed lower stability and gene regulation, compared with that of pDNA/PPC. Moreover, no difference in gene expression was identified between positive and negative polymer. These results were caused by fast disintegration of pDNA/PPC/CS complexes in the presence of serum. Thus, we suggest that introduction of negatively charged CS into polymers with a low charge density may lead to low stability and gene regulation of complexes.