Principal factors that determine the extension of detection range in molecular beacon aptamer/conjugated polyelectrolyte bioassays.

A strategy to extend the detection range of weakly-binding targets is reported that takes advantage of fluorescence resonance energy transfer (FRET)-based bioassays based on molecular beacon aptamers (MBAs) and cationic conjugated polyelectrolytes (CPEs). In comparison to other aptamer-target pairs, the aptamer-based adenosine triphosphate (ATP) detection assays are limited by the relatively weak binding between the two partners. In response, a series of MBAs were designed that have different stem stabilities while keeping the constant ATP-specific aptamer sequence in the loop part. The MBAs are labeled with a fluorophore and a quencher at both termini. In the absence of ATP, the hairpin MBAs can be opened by CPEs via a combination of electrostatic and hydrophobic interactions, showing a FRET-sensitized fluorophore signal. In the presence of ATP, the aptamer forms a G-quadruplex and the FRET signal decreases due to tighter contact between the fluorophore and quencher in the ATP/MBA/CPE triplex structure. The FRET-sensitized signal is inversely proportional to [ATP]. The extension of the detection range is determined by the competition between opening of the ATP/MBA G-quadruplex by CPEs and the composite influence by ATP/aptamer binding and the stem interactions. With increasing stem stability, the weak binding of ATP and its aptamer is successfully compensated to show the resistance to disruption by CPEs, resulting in a substantially broadened detection range (from millimolar up to nanomolar concentrations) and a remarkably improved limit of detection. From a general perspective, this strategy has the potential to be extended to other chemical- and biological-assays with low target binding affinity

Production of transgenic first filial puppies expressing mutated human amyloid precursor protein gene

Propagation of transgenic animals by germline transmission using assisted reproductive technologies such as in vitro fertilization (IVF) is the most efficient way to produce transgenic colonies for biomedical research. The objective of this study was to generate transgenic puppies from a founder dog expressing the mutated human amyloid precursor protein (mhAPP) gene. Experiment I assessed the characteristics of the semen prepared by freshly diluted, swim-up, and Percoll gradient methods using a computer-assisted semen analyzer (CASA). Motile and progressively motile sperm counts were higher in the Percoll gradient samples (p < 0.05) than in the swim-up and freshly diluted samples. In Experiment II, a total of 59, 70, and 65 presumptive zygotes produced by fresh, Percoll gradient, and swim-up methods, respectively, were transferred to surrogates (5 for each group); the Percoll gradient (27.27%) and swim-up samples (14.29%) showed the highest blastocyst formation rates, while fresh diluted semen did not produce any blastocyst. Experiment III examined the full-term developmental ability of embryos. Among the 5 surrogates in the Percoll gradient group, one (20.0%) became pregnant; it had 4 (6.15%) sacs and delivered 4 (6.15%; 2 males and 2 females) live puppies. Among the 4 puppies, 2 (50.0%) were found to transmit the transgene on their nail and toe under GFP fluorescence. Furthermore, the integration and expression of the mhAPP transgene were examined in the umbilical cords of all the IVF-derived puppies, and the presence of the transgene was only observed in the GFP-positive puppies. Thus, semen prepared by the Percoll method could generate transgenic puppies by male germline transmission using the IVF technique. Our result will help propagate transgenic dogs efficiently, which will foster human biomedical research

Prediction of tannin content and quality parameters in astringent persimmons from visible and near-infrared spectroscopy

IntroductionTannin content and postharvest quality characteristics of persimmon fruit are often determined by the destructive analysis that consumes time, does not allow the acquisition of data from the same fruit continuously, and requires expensive high-performance equipment. This research was done to investigate the potential for non-destructive estimation of astringency and quality parameters in persimmon fruit based on visible/near-infrared (VNIR) spectra.MethodsVNIR spectra readings, the reference tannin content, and quality parameters were measured from fruits of “Cheongdo-Bansi” and “Daebong” persimmon cultivars at harvest and throughout the ripening/deastringency period. The spectra readings from half of the total fruit were utilized for the calibration set, while the other half readings were used for the prediction set. To develop models correlating the spectra data to the measured reference parameters data, the partial least square regression (PLSR) method was utilized.Results and discussionIn the case of ‘Daebong’, the coefficients of determination (R2) between VNIR spectra and the actual measured values of TSS, firmness, simple sugars, and tannin content were (0.95, 0.94, 0.96, and 0.96) and (0.93, 0.89, 0.96, and 0.93), for the calibration and prediction sets, respectively. Similarly, the R2-values of (0.86, 0.93, 0.79, and 0.81) and (0.83, 0.91, 0.75, and 0.75) were recorded in ‘Cheongdo-Bansi’ for the calibration and prediction sets, respectively. Additionally, the acquired data were divided into two sets in a 3:1 ratio to develop predictive models and to validate the models in multiple regressions. PLSR models were developed in multiple regression to estimate the tannin content of both cultivars from firmness and simple sugars with R2-values of 0.83 and 0.79 in ‘Cheongdo-Bansi’ for the calibration and prediction sets, respectively, whereas, R2-values of 0.80 and 0.84 were recorded in ‘Daebong’ for the calibration and prediction sets, respectively. The overall findings of this study showed the possibility of using VNIR spectra for the prediction of postharvest quality and tannin contents from intact persimmon fruit with quick, chemical-free, and low-cost assessment methods. Also, the multiple regression using physicochemical parameters could fairly predict the tannin content in persimmon fruit though destructively but save time and low-cost

Autoimmune Hypoglycemia in a Patient with Characterization of Insulin Receptor Autoantibodies

BackgroundType B insulin resistance syndrome is a manifestation of autoantibodies to the insulin receptor that results in severe hyperglycemia and acanthosis nigricans. However, the mechanisms by which these autoantibodies induce hypoglycemia are largely unknown. In this paper, we report the case of patient with type B insulin resistance syndrome who presented with frequent severe fasting hypoglycemia and acanthosis nigricans.MethodsTo evaluate the mechanism of hypoglycemia, we measured the inhibition of insulin binding to erythrocytes and IM9 lymphocytes in a sample of the patient's dialyzed serum before and after immunosuppressive therapy.ResultsIn the patient's pre-treatment serum IgG, the binding of 125I-insulin to erythrocytes was markedly inhibited in a dose-dependent manner until the cold insulin level reached 10-9 mol/L. We also observed dose-dependent inhibition of insulin binding to IM9 lymphocytes, which reached approximately 82% inhibition and persisted even when diluted 1:20. After treatment with glucocorticoids, insulin-erythrocyte binding activity returned to between 70% and 80% of normal, while the inhibition of insulin-lymphocyte binding was reduced by 17%.ConclusionWe treated a patient with type B insulin resistance syndrome showing recurrent fasting hypoglycemia with steroids and azathioprine. We characterized the patient's insulin receptor antibodies by measuring the inhibition of insulin binding

Single- and repeated-dose toxicities of aloe fermentation products in rats

In this study, aloe fermentation products were derived from mycelia from 3 mushrooms: Ganoderma lucidum (AG), Hericium erinaceum (AH), and Phellinus linteus (AP). Levels of aloin A and B increased with fermentation time. The highest levels were measured on the fifth day of fermentation. β-Glucan levels decreased with fermentation time. The safety of aloe fermentation products were examined in male and female Sprague-Dawley rats. Rats were orally administered the three aloe fermentation products at dose levels of 1, 2 or 5 g/kg for single-dose toxicity test and 0.5, 1, or 2 g/kg for repeated-dose toxicity test. There were no significant differences in body weight gain between vehicle control and AG-, AH- or AP-treated rats. Also, significant changes in daily feed intake and water consumption were not observed. In hematological analysis, none of the parameters were affected by aloe fermentation products with mushroom mycelia. This suggests that there are no negative effects on homeostasis and immunity. In blood biochemistry analysis, none of the markers were affected by feeding rats with AG, AH or AP. Similarly, there were no significant effects on markers for liver, kidney, skeletal and heart muscle functions. No remarkable lesions were observed in these organs at histopathology. Since there were no adverse effects of AG, AH and AP in single- or repeated-dose toxicity tests, even at higher doses than normal, we conclude that the aloe fermentation products with mushroom mycelia possess long-term safety and could be candidates as multifunctional nutrients for the improvement of intestinal function and immunity

Surgical outcomes of strabismus after iatrogenic ophthalmic artery occlusion caused by cosmetic filler injections

Background To investigate the surgical outcomes of strabismus related to iatrogenic occlusion of the ophthalmic artery and its branches from cosmetic facial filler injection. Methods A retrospective study was performed on 6 patients who underwent strabismus surgery among 23 patients who had suffered occlusion of the ophthalmic artery and its branches after cosmetic facial filler injection. Initial, preoperative and final ocular motility examinations, the type of surgery and surgical outcomes were evaluated. Results At initial presentation, visual acuity was no light perception in 5 patients and hand motion in one patient. Five out of 6 patients showed initial ophthalmoplegia. Among these 5 patients, eye motility fully recovered in 3 patients although sensory strabismus developed during follow-up, while the remaining 2 patients had persistent ocular motility limitations. Strabismus surgery was performed at 2.2 ± 1.5 years after iatrogenic ophthalmic artery occlusion. Preoperatively, 5 of the 6 patients showed exotropia, and one patient had esotropia. Vertical deviation was found in 3 out of 6 patients in addition to the horizontal deviation. Successful outcome was achieved only in the 4 patients without persistent ophthalmoplegia after 1.4 ± 1.0 years from surgery. The other two patients with persistent ocular motility limitations failed to achieve successful alignment after surgery, and one patient eventually underwent evisceration due to phthisis bulbi. Conclusions In our study, surgical outcomes of strabismus caused by cosmetic facial filler injection were successful only in patients without persistent ophthalmoplegia at the time of surgery.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No.2017R1A2B4011450). The funding organization had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Reconstruction of a Severely Crushed Leg with Interpositional Vessel Grafts and Latissimus Dorsi Flap

We present a case of a near total amputation at the distal tibial level, in which the patient emphatically wanted to save the leg. The anterior and posterior tibial nerves were intact, indicating a high possibility of sensory recovery after revascularization. The patient had open fractures at the tibia and fibula, but no bone shortening was performed. The posterior tibial vessels were reconstructed with an interposition saphenous vein graft from the contralateral side and a usable anterior tibial artery graft from the undamaged ipsilateral distal portions. The skin and soft tissue defects were covered using a subatmospheric pressure system for demarcating the wound, and a latissimus dorsi myocutaneous free flap for definite coverage of the wound. At 6 months after surgery, the patient was ambulatory without requiring additional procedures. Replantation without bone shortening, with use of vessel grafts and temporary coverage of the wound with subatmospheric pressure dressings before definite coverage, can shorten recovery time