- Invited Review - Reproductive technologies needed for the generation of precise gene-edited pigs in the pathways from laboratory to farm.

Gene editing (GE) offers a new breeding technique (NBT) of sustainable value to animal agriculture. There are 3 GE working sites covering 5 feasible pathways to generate GE pigs along with the crucial intervals of GE/genotyping, microinjection/electroporation, induced pluripotent stem cells, somatic cell nuclear transfer, cryopreservation, and nonsurgical embryo transfer. The extension of NBT in the new era of pig breeding depends on the synergistic effect of GE and reproductive biotechnologies; the outcome relies not only on scientific due diligence and operational excellence but also on the feasibility of application on farms to improve sustainability.

Activated macrophages of CD 163 gene edited pigs generated by direct cytoplasmic microinjection with CRISPR gRNA/Cas9 mRNA are resistant to PRRS virus assault.

Porcine reproductive and respiratory syndrome virus (PRRSV) infects placental and lung macrophages, causing a global epidemic with economic loss. Attempts to develop an effective vaccine to control the disease have not been effective. Currently, developing PRRSV disease-resistant pigs via a gene editing (GE) strategy to mutate the PRRSV receptor or to delete the binding domain on the macrophage appears promising. In this study, we used the strategy of Edinburg University to construct two guide RNAs (gRNAs) located on the proximal front and post sites of exon 7. Directive microinjection of two gRNAs and Cas9 mRNA into the cytoplasm of pronuclear zygotes efficiently generated four piglets confirmed as CD163 knockout (KO) and/or CD163 exon 7 deleted (CD163ΔE7). In four GE piglets, three pigs carried two chromosome CD163 KO or ΔE7. Peripheral blood mononuclear cells (PBMCs) from three GE and wild-type (WT) pigs were activated into macrophages for in vitro transfection. The results showed that the activated macrophages from all GE pigs were significantly more viable than those from WT pig. Current results suggest that we have successfully generated PRRSV-resistant pigs, although in vivo challenge is needed to validate that the pigs are PRRSV resistant.

Orthotopic Versus Allotopic Implantation: Comparison of Radiological and Pathological Characteristics.

BACKGROUND: In experimental animal models, implantation location might influence the heterogeneity and overall development of the tumor, leading to an interpretation bias. PURPOSE: To investigate the effects of implantation location in experimental tumor model using magnetic resonance imaging (MRI) and pathological findings. STUDY TYPE: Prospective. SUBJECTS: Forty-five breast cancer-bearing mice underwent orthotopic (N = 15) and heterotopic (intrahepatic [N = 15] and subcutaneous [N = 15]) implantation. FIELD STRENGTH/SEQUENCE: Sequences including: T1-weighted turbo spin echo sequence, T2-weighted blade sequence, diffusion-weighted imaging, pre- and post-contrast T1 mapping, multi-echo T2 mapping at 3.0 T. ASSESSMENT: MRI was performed at 7, 14, and 21 days after implantation. Native T1, post-contrast T1, T2, and apparent diffusion coefficient (ADC) of tumors, the tumor volume and necrosis volume within tumor were obtained. Lymphocyte cells from H&E staining, Ki67-positive, and CD31-positive cells from immunohistochemistry were determined. STATISTICAL TESTS: One-way analysis of variance and Spearman's rank correlation were performed. P value <0.05 was considered statistically significant. RESULTS: The tumor volume (intrahepatic vs. orthotopic vs. subcutaneous: 587.50 ± 77.62 mm3 vs. 814.00 ± 43.85 mm3 vs. 956.13 ± 119.22 mm3 ), necrosis volume within tumor (89.10 ± 26.60 mm3 vs. 292.41 ± 57.92 mm3 vs. 179.91 ± 31.73 mm3 , respectively), ADC at day 21 (543.41 ± 42.28 vs. 542.92 ± 99.67 vs. 369.83 ± 42.90, respectively), and post-contrast T1 at all timepoints (day 7: 442.00 ± 11.52 vs. 435.00 ± 22.90 vs. 394.33 ± 29.95; day 14: 459.00 ± 26.11 vs. 436.83 ± 26.01 vs. 377.00 ± 27.83; day 21: 463.50 ± 23.49 vs. 458.00 ± 34.28 vs. 375.00 ± 30.55) were significantly different between three groups. Necrosis volumes of subcutaneous and intrahepatic tumors were significantly lower than those of orthotopic tumors. The CD31-positive rate in the intrahepatic implantation was significantly higher than in orthotopic and subcutaneous groups. Necrosis volume (r = -0.71), ADC (r = -0.85), and post-contrast T1 (r = -0.75) were strongly correlated with vascular invasion index. DATA CONCLUSION: Orthotopic and heterotopic tumors have their unique growth kinetics, necrosis volume, and vascular invasion. Non-invasive MR quantitative parameters, including ADC and post-contrast T1, may reflect vascular invasion in mice. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 3.

The Preschool-Aged and School-Aged Children Present Different Odds of Mortality than Adults in Southern Taiwan: A Cross-Sectional Retrospective Analysis.

Background: This study aimed to profile the epidemiology of injury among preschool-aged and school-aged children in comparison to those in adults. Methods: According to the Trauma Registry System of a level I trauma center, the medical data were retrieved from 938 preschool-aged children (aged less than seven years), 670 school-aged children (aged 7⁻12 years), and 16,800 adults (aged 20⁻64 years) between 1 January 2009 and 31 December 2016. Two-sided Pearson’s, chi-squared, and Fisher’s exact tests were used to compare categorical data. A one-way analysis of variance (ANOVA) with the Games-Howell post-hoc test was used to assess the differences in continuous variables among different groups of patients. The mortality outcomes of different subgroups were assessed by a multivariable regression model under the adjustment of sex, injury mechanisms, and injury severity. Results: InFsupppjury mechanisms in preschool-aged and school-aged children were remarkably different from that in adults; in preschool-aged children, burns were the most common cause of injury requiring hospitalization (37.4%), followed by falls (35.1%) and being struck by/against objects (11.6%). In school-aged children, injuries were most commonly sustained from falls (47.8%), followed by bicycle accidents (14%) and being struck by/against objects (12.5%). Compared to adults, there was no significant difference of the adjusted mortality of the preschool-aged children (AOR = 0.9; 95% CI 0.38⁻2.12; p = 0.792) but there were lower adjusted odds of mortality of the school-aged children (AOR = 0.4; 95% CI 0.10⁻0.85; p = 0.039). The school-aged children had lower odds of mortality than adults (OR, 0.2; 95% CI, 0.06⁻0.74; p = 0.012), but such lower odds of risk of mortality were not found in preschool-aged children (OR, 0.7; 95% CI, 0.29⁻1.81; p = 0.646). Conclusions: This study suggests that specific types of injuries from different injury mechanisms are predominant among preschool-aged and school-aged children. The school-aged children had lower odds of mortality than adults; nonetheless there was no difference in mortality rates of preschool-aged children than adults, with or without controlling for sex, injury mechanisms and ISS. These results highlight the importance of injury prevention, particularly for preschool-aged children in Southern Taiwan.

Artificial neural network approach to predict surgical site infection after free-flap reconstruction in patients receiving surgery for head and neck cancer.

BACKGROUND: The aim of this study was to develop an effective surgical site infection (SSI) prediction model in patients receiving free-flap reconstruction after surgery for head and neck cancer using artificial neural network (ANN), and to compare its predictive power with that of conventional logistic regression (LR). MATERIALS AND METHODS: There were 1,836 patients with 1,854 free-flap reconstructions and 438 postoperative SSIs in the dataset for analysis. They were randomly assigned tin ratio of 7:3 into a training set and a test set. Based on comprehensive characteristics of patients and diseases in the absence or presence of operative data, prediction of SSI was performed at two time points (pre-operatively and post-operatively) with a feed-forward ANN and the LR models. In addition to the calculated accuracy, sensitivity, and specificity, the predictive performance of ANN and LR were assessed based on area under the curve (AUC) measures of receiver operator characteristic curves and Brier score. RESULTS: ANN had a significantly higher AUC (0.892) of post-operative prediction and AUC (0.808) of pre-operative prediction than LR (both P<0.0001). In addition, there was significant higher AUC of post-operative prediction than pre-operative prediction by ANN (p<0.0001). With the highest AUC and the lowest Brier score (0.090), the post-operative prediction by ANN had the highest overall predictive performance. CONCLUSION: The post-operative prediction by ANN had the highest overall performance in predicting SSI after free-flap reconstruction in patients receiving surgery for head and neck cancer.

Patterns Associated with Adult Mandibular Fractures in Southern Taiwan-A Cross-Sectional Retrospective Study.

Purpose: This study aimed to determine the patterns associated with adult mandibular fractures from a Level-I trauma center in southern Taiwan. Methods: The data of adult trauma patients admitted between 1 January 2009 and 31 December 2014 were retrieved from the Trauma Registry System and retrospectively reviewed. Fracture site and cause of injury were categorized into groups for comparison, and corresponding odds ratios (ORs) and 95% confidence intervals (CIs) were obtained by multivariate logistic regression. Results: Motorcycle accidents were the most common cause of mandibular fractures (76.3%), followed by falls (10.9%), motor vehicle accidents (4.8%), and being struck by/against objects (4.5%). Of the 503 cases of mandibular fractures, the condylar neck and head were the most common sites (32.0%), followed by the parasymphysis (21.7%), symphysis (19.5%), angle and ramus (17.5%), and body (9.3%). The location of mandibular fractures in patients who had motorcycle accidents was similar to that in all patients. Motor vehicle accidents resulted in a significantly higher number of body fractures (OR 3.3, 95% CI 1.24-8.76, p = 0.017) and struck injury in a significantly higher number of angle and ramus fractures (OR 3.9, 95% CI 1.48-10.26, p = 0.006) compared to motorcycle accidents. The helmet-wearing status and body weight were not associated with the location of mandibular fractures in motorcycle accidents. Conclusions: Our study revealed that the anatomic fracture sites of mandible were specifically related to different etiologies. In southern Taiwan, motorcycle accidents accounted for the major cause of mandibular fractures and were associated with the condylar neck and head as the most frequent fracture sites. In contrast, motor vehicle accidents and struck injuries tended to cause more body fracture as well as angle and ramus fracture compared to motorcycle accidents. Furthermore, the status of helmet-wearing and body weight were not associated with the location of mandible fractures caused by motorcycle accidents.

Generation of GGTA1 Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors.

This study was conducted to confirm that 1-site and 4-site ppU6-GGTA1-gRNA CRISPR vectors together with the pCX-Flag2-NLS1-Cas9-NLS2 plasmid can both generate KO pigs by direct pronuclear microinjection. In total, 41 and 53 fertilized eggs were microinjected on 1-site and 4-site strategies, respectively. The 1-site construction generated a litter of 8 piglets, and 2 were mono-allelic mutant (mMt). The injection of 4-site constructions resulted in one biallelic mutant (bMt) and one mMt piglet in a litter of 7. Those 3 mMt pigs had a 4 bp deletion, 5 bp insertion, or 7 bp insertion at site I, and the bMt pig had 5 types of mutations at cleavage sites I and III. The expression of alpha-Gal on the bMt peripheral blood mononuclear cells (PBMCs) was reduced, and survival rate of bMt PBMCs was maintained as indicated by results of cultivation with sera of humans or Formosan Macaques. We concluded that mutant pigs could be generated by direct pronuclear microinjection of ppU6-GGTA1-gRNA CRISPR vectors with the pCX-Flag2-NLS1-Cas9-NLS2 plasmid and that the 4-site strategy has a better mutant efficiency. Porcine U6 promoter was firstly used to express KO vectors and effectively generate mutant pigs, worthily to adopt for future KO studies.

Influence of alcohol use on mortality and expenditure during hospital admission: a cross-sectional study.

OBJECTIVES: This study was designed to investigate the effect of alcohol intoxication on clinical presentation of hospitalised adult trauma patients at a Level I trauma centre using propensity score matching. DESIGN: Cross-sectional study. SETTING: Taiwan. PARTICIPANTS: Detailed data of 929 hospitalised adult trauma patients with alcohol intoxication, aged 20-65 years, and 10 104 corresponding patients without alcohol intoxication were retrieved from the Trauma Registry System between 1 January 2009 and 31 December 2014. Alcohol intoxication was defined as a blood alcohol concentration (BAC) ≥50 mg/dL. MAIN OUTCOME MEASURES: In-hospital mortality and expenditure. RESULTS: Patients with alcohol intoxication presented with significantly higher short-term mortality (OR: 3.0, 95% CI 2.0 to 4.4; p<0.001) than patients without alcohol intoxication. However, on comparison with propensity score-matched patients with respect to sex, age, comorbidity, Glasgow Coma Scale (GCS), injury region based on Abbreviated Injury Scale (AIS) and Injury Severity Score (ISS), alcohol intoxication did not significantly influence mortality (OR: 0.8, 95% CI 0.5 to 1.4; p=0.563). This implied that the higher mortality of alcohol-intoxicated patients was attributable to patient characteristics such as a higher injury severity rather than alcohol intoxication. Even on comparison with sex-matched, age-matched and comorbidity-matched patients without alcohol intoxication, patients with alcohol intoxication still had significantly higher total expenditure (17.4% higher), cost of operation (40.3% higher), cost of examination (52.8% higher) and cost of pharmaceuticals (38.3% higher). CONCLUSIONS: The associated higher mortality of adult trauma patients with alcohol intoxication was completely attributable to other patient characteristics and associated injury severity rather than the effects of alcohol. However, patients with alcohol intoxication incurred significantly higher expenditure than patients without alcohol intoxication, even on comparison with sex-matched, age-matched and comorbidity-matched patients without alcohol intoxication.

Image quality improvement in three-dimensional time-of-flight magnetic resonance angiography using the subtraction method for brain and temporal bone diseases.

BACKGROUND: Time-of-flight (TOF) magnetic resonance (MR) angiography is based on flow-related enhancement using the T1-weighted spoiled gradient echo, or the fast low-angle shot gradient echo sequence. However, materials with short T1 relaxation times may show hyperintensity signals and contaminate the TOF images. The objective of our study was to determine whether subtraction three-dimensional (3D) TOF MR angiography improves image quality in brain and temporal bone diseases with unwanted contaminations with short T1 relaxation times. METHODS: During the 12-month study period, patients who had masses with short T1 relaxation times noted on precontrast T1-weighted brain MR images and 24 healthy volunteers were scanned using conventional and subtraction 3D TOF MR angiography. The qualitative evaluation of each MR angiogram was based on signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR) and scores in three categories, namely, (1) presence of misregistration artifacts, (2) ability to display arterial anatomy selectively (without contamination by materials with short T1 relaxation times), and (3) arterial flow-related enhancement. RESULTS: We included 12 patients with intracranial hematomas, brain tumors, or middle-ear cholesterol granulomas. Subtraction 3D TOF MR angiography yielded higher CNRs between the area of the basilar artery (BA) and normal-appearing parenchyma of the brain and lower SNRs in the area of the BA compared with the conventional technique (147.7 ± 77.6 vs. 130.6 ± 54.2, p < 0.003 and 162.5 ± 79.9 vs. 194.3 ± 62.3, p < 0.001, respectively) in all 36 cases. The 3D subtraction angiography did not deteriorate image quality with misregistration artifacts and showed a better selective display of arteries (p < 0.0001) and arterial flow-related enhancement (p < 0.044) than the conventional method. CONCLUSION: Subtraction 3D TOF MR angiography is more appropriate than the conventional method in improving the image quality in brain and temporal bone diseases with unwanted contaminations with short T1 relaxation times.

Synergetic degradation of konjac glucomannan by γ-ray irradiation and hydrogen peroxide.

Konjac glucomannan (KGM) samples were irradiated with (60)Co γ-rays with a radiation dose of 50kGy in the presence and absence of hydrogen peroxide. The average molecular weight (Mw) and polydispersity index (PDI) of untreated and degraded samples were measured by gel permeation chromatography (GPC), revealing that γ-rays and hydrogen peroxide had a synergetic effect on degradation. Structures of untreated and degraded products were characterized with ultraviolet-visible (UV) and Fourier-transform infrared (FT-IR) spectroscopies, and X-ray diffraction (XRD). Results showed that there was no significant change in the main chain of KGM following degradation, although the crystallinity of KGM decreased. A synergetic effect of γ-rays and hydrogen peroxide was also found in the structural characterization of KGM. The physical properties of KGM changed markedly following degradation, which may increase its application potential. The mechanism of degradation of KGM in the presence and absence of hydrogen peroxide was also investigated.

Study on preparation and separation of Konjac oligosaccharides.

To study the preparation and separation of Konjac oligosaccharides, Konjac Glucomannan was degraded by the combination of γ-irradiation and ß-mannanase, and then the degradation product was separated by ultrafiltration. To our interest, for most of Konjac oligosaccharides obtained by this method, the molecular mass was lower than 2200 Da. In addition, the 1000 Da molecular weight cut off membrane could effectively separate the Konjac oligosaccharides. In conclusion, the combination of γ-irradiation and ß-mannanase was an efficient method to obtain Konjac oligosaccharides, and the oligosaccharides of molecular mass lower than 1000 Da could be effectively separated by ultrafiltration.

Sonographic diagnosis of a subclinical wandering spleen: role of the decubitus position.

A wandering spleen is a rare condition. It is usually diagnosed when abdominal pain develops secondary to splenic torsion. Although splenic hypermobility is the pathognomonic feature of a wandering spleen, it is rarely revealed by imaging in the subclinical stage. We report 3 patients with a subclinical wandering spleen who had incidental sonographic findings of splenomegaly. Gray scale and color Doppler sonography in the right decubitus position can easily show the migratory nature and perfusion status of a wandering spleen in real time.

Structural characterization and properties of konjac glucomannan/curdlan blend films.

A series of novel edible blend films of konjac glucomannan (KGM) and curdlan were prepared by a solvent-casting technique with different blending ratios of the two polymers. The Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), etc. were used to characterize the change of structure and properties of blend films. The results showed that the strong intermolecular hydrogen bonds took place between KGM and curdlan. And the interaction of the blend film was much greater than that of the others when the KGM content in the blend films was around 70 wt% (KC7), resulting in excellent miscibility. The conclusion of the electron tensile testing analysis indicated that the blend film KC7 showed the maximum tensile strength (42.93±1.92 MPa). In addition, the blend films displayed excellent moisture barrier properties, which had a potential application in the food field.