Enhanced Calvarial Bone Repair Using ASCs Engineered with RNA-Guided Split dCas12a System that Co-Activates Sox 5, Sox6, and Long Non-Coding RNA H19.

Healing of large calvarial bone defects remains challenging. An RNA-guided Split dCas12a system is previously harnessed to activate long non-coding RNA H19 (lncRNA H19, referred to as H19 thereafter) in bone marrow-derived mesenchymal stem cells (BMSCs). H19 activation in BMSCs induces chondrogenic differentiation, switches bone healing pathways, and improves calvarial bone repair. Since adipose-derived stem cells (ASCs) can be harvested more easily in large quantity, here it is aimed to use ASCs as an alternative cell source. However, H19 activation alone using the Split dCas12a system in ASCs failed to elicit evident chondrogenesis. Therefore, split dCas12a activators are designed more to co-activate other chondroinductive transcription factors (Sox5, Sox6, and Sox9) to synergistically potentiate differentiation. It is found that co-activation of H19/Sox5/Sox6 in ASCs elicited more potent chondrogenic differentiation than activation of Sox5/Sox6/Sox9 or H19 alone. Co-activating H19/Sox5/Sox6 in ASCs significantly augmented in vitro cartilage formation and in vivo calvarial bone healing. These data altogether implicated the potentials of the Split dCas12a system to trigger multiplexed gene activation in ASCs for differentiation pathway reprogramming and tissue regeneration.

Source-apportionment and spatial distribution analysis of VOCs and their role in ozone formation using machine learning in central-west Taiwan.

This study assessed the machine learning based sensitivity analysis coupled with source-apportionment of volatile organic carbons (VOCs) to look into new insights of O3 pollution in Yunlin County located in central-west region of Taiwan. One-year (Jan 1 to Dec 31, 2021) hourly mass concentrations data of 54 VOCs, NOX, and O3 from 10 photochemical assessment monitoring stations (PAMs) in and around the Yunlin County were analyzed. The novelty of the study lies in the utilization of artificial neural network (ANN) to evaluate the contribution of VOCs sources in O3 pollution in the region. Firstly, the station specific source-apportionment of VOCs were carried out using positive matrix factorization (PMF)-resolving six sources viz. AAM: aged air mass, CM: chemical manufacturing, IC: Industrial combustion, PP: petrochemical plants, SU: solvent use and VE: vehicular emissions. AAM, SU, and VE constituted cumulatively more than 65% of the total emission of VOCs across all 10 PAMs. Diurnal and spatial variability of source-segregated VOCs showed large variations across 10 PAMs, suggesting for distinctly different impact of contributing sources, photo-chemical reactivity, and/or dispersion due to land-sea breezes at the monitoring stations. Secondly, to understand the contribution of controllable factors governing the O3 pollution, the output of VOCs source-contributions from PMF model along with mass concentrations of NOX were standardized and first time used as input variables to ANN, a supervised machine learning algorithm. ANN analysis revealed following order of sensitivity in factors governing the O3 pollution: VOCs from IC > AAM > VE ≈ CM ≈ SU > PP ≈ NOX. The results indicated that VOCs associated with IC (VOCs-IC) being the most sensitive factor which need to be regulated more efficiently to quickly mitigate the O3 pollution across the Yunlin County.

Neuroprotective effects of intraoperative dexmedetomidine versus saline infusion combined with goal-directed haemodynamic therapy for patients undergoing cranial surgery: A randomised controlled trial.

BACKGROUND: By inhibiting neuroinflammation dexmedetomidine may be neuroprotective in patients undergoing cranial surgery, but it reduces cardiac output and cerebral blood flow. OBJECTIVE: To investigate whether intra-operative dexmedetomidine combined with goal-directed haemodynamic therapy (GDHT) has neuroprotective effects in cranial surgery. DESIGN: A double-blind, single-institution, randomised controlled trial. SETTING: A single university hospital, from April 2017 to April 2020. PATIENTS: A total of 160 adults undergoing elective cranial surgery. INTERVENTION: Infusion of dexmedetomidine (0.5 µg kg-1 h-1) or saline combined with GDHT to optimise stroke volume during surgery. MAIN OUTCOME MEASURES: The proportion who developed postoperative neurological complications was compared. Postoperative disability was assessed using the Barthel Index at time points between admission and discharge, and also the 30-day modified Rankin Scale (mRS). Postoperative delirium was assessed. The concentration of a peri-operative serum neuroinflammatory mediator, high-mobility group box 1 protein (HMGB1), was compared. RESULTS: Fewer patients in the dexmedetomidine group developed new postoperative neurological complications (26.3% vs. 43.8%; P = 0.031), but the number of patients developing severe neurological complications was comparable between the two groups (11.3% vs. 20.0%; P = 0.191). In the dexmedetomidine group the Barthel Index reduction [0 (-10 to 0)] was less than that in the control group [-5 (-15 to 0)]; P = 0.023, and there was a more favourable 30-day mRS (P = 0.013) with more patients without postoperative delirium (84.6% vs. 64.2%; P = 0.012). Furthermore, dexmedetomidine induced a significant reduction in peri-operative serum HMGB1 level from the baseline (222.5 ±â€Š408.3 pg ml-1) to the first postoperative day (152.2 ±â€Š280.0 pg ml-1) P = 0.0033. There was no significant change in the control group. The dexmedetomidine group had a lower cardiac index than did the control group (3.0 ±â€Š0.8 vs. 3.4 ±â€Š1.8 l min-1 m-2; P = 0.0482) without lactate accumulation. CONCLUSIONS: Dexmedetomidine infusion combined with GDHT may mitigate neuroinflammation without undesirable haemodynamic effects during cranial surgery and therefore be neuroprotective. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT02878707.

Accuracy of dynamic preload variables for predicting fluid responsiveness in patients with pediatric liver cirrhosis: A prospective study.

BACKGROUND: We have previously reported that dynamic preload variables predicted fluid responsiveness in adult patients with liver cirrhosis. However, pediatric patients with cirrhosis may present with unique hemodynamic characteristics, and therefore, the predictive accuracy of these variables in such patients must be clarified. AIMS: To investigate the accuracy of dynamic preload variables for predicting fluid responsiveness in pediatric patients with cirrhosis. METHODS: A total of 27 pediatric patients with cirrhosis undergoing orthotopic liver transplantation were enrolled in this study. Patients' pulse pressure variation, stroke volume variation, stroke volume index, and central venous pressure were measured using the calibrated pulse contour cardiac output system. The plethysmographic variability index was measured using a Masimo Radical 7 co-oximeter. During the hepatic dissection phase of the surgery, repeated intraoperative fluid challenges with 10 mL kg-1 of crystalloid within 15 minutes were administered. Fluid responsiveness was defined as an increase in stroke volume index of ≥15% after fluid challenge. RESULTS: A total of 61 fluid challenges were administered resulting in 15 fluid responders and 46 fluid nonresponders. Fluid challenge induced significant decreases in all three dynamic preload variables but not in the fluid nonresponders. However, the area under the receiver operating characteristic curves for pulse pressure variation, stroke volume variation, plethysmographic variability index, and central venous pressure for predicting fluid responsiveness were 0.67 (95% confidence interval: 0.52-0.82; P = .0255), 0.68 (95% confidence interval: 0.54-0.83; P = .0140), 0.56 (95% confidence interval: 0.40-0.71; P = .4724), and 0.57 (95% confidence interval: 0.40-0.74; P = .4192), respectively. CONCLUSIONS: Dynamic preload variables do not predict fluid responsiveness in pediatric patients with liver cirrhosis.

Photochemical oxidation of VOCs and their source impact assessment on ozone under de-weather conditions in Western Taiwan.

The application of statistical models has excellent potential to provide crucial information for mitigating the challenging issue of ozone (O3) pollution by capturing its associations with explanatory variables, including reactive precursors (VOCs and NOX) and meteorology. Considering the large contribution of O3 in degrading the air quality of western Taiwan, three-year (2019-2021) hourly concentration data of VOC, NOX and O3 from 4 monitoring stations of western Taiwan: Tucheng (TC), Zhongming (ZM), Taixi (TX) and Xiaogang (XG), was evaluated to identify the effect of anthropogenic emissions on O3 formation. Owing to the high-ambient reactivity of VOCs on the underestimation of sources, photochemical oxidation was assessed to calculate the consumed VOC (VOCcons) which was followed by the source identification of their initial concentrations. VOCcons was observed to be highest in the summer season (16.7 and 22.7 ppbC) at north (TC and ZM) and in the autumn season (17.8 and 11.4 ppbC) in southward-located stations (TX and XG, respectively). Results showed that VOCs from solvents (25-27%) were the major source at northward stations whereas VOCs-industrial emissions (30%) dominated in south. Furthermore, machine learning (ML): eXtreme Gradient Boost (XGBoost) model based de-weather analysis identified that meteorological factors favor to reduce ambient O3 levels at TC, ZM and XG stations (-67%, -47% and -21%, respectively) but they have a major role in accumulating the O3 (+38%) at the TX station which is primarily transported from the upwind region of south-central Taiwan. Crucial insights using ML outputs showed that the finding of the study can be utilized for region-specific data-driven control of emission from VOCs-sources and prioritized to limit the O3-pollution at the study location-ns as well as their accumulation in distant regions.

Split dCas12a activator for lncRNA H19 activation to enhance BMSC differentiation and promote calvarial bone healing.

Healing of large calvarial bone defects in adults is challenging. We previously showed that inducing chondrogenic differentiation of mesenchymal stem cells from bone marrow (BMSC) or adipose tissue (ASC) before implantation can switch the repair pathway and improve calvarial bone healing. Split dCas12a activator is a new CRISPR activation system comprising the amino (N) and carboxyl (C) fragments of dCas12a protein, each being fused with synthetic transcription activators at both termini. The split dCas12a activator was shown to induce programmable gene expression in cell lines. Here we exploited the split dCas12a activator to activate the expression of chondroinductive long non-coding RNA H19. We showed that co-expression of the split N- and C-fragments resulted in spontaneous dimerization, which elicited stronger activation of H19 than full-length dCas12a activator in rat BMSC and ASC. We further packaged the entire split dCas12a activator system (13.2 kb) into a hybrid baculovirus vector, which enhanced and prolonged H19 activation for at least 14 days in BMSC and ASC. The extended H19 activation elicited potent chondrogenic differentiation and inhibited adipogenesis. Consequently, the engineered BMSC promoted in vitro cartilage formation and augmented calvarial bone healing in rats. These data implicated the potentials of the split dCas12a activator for stem cell engineering and regenerative medicine.

The correlation of the presence and expression levels of cry genes with the insecticidal activities against Plutella xylostella for Bacillus thuringiensis strains.

The use of Bacillus thuringiensis (Bt) strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the expression levels of cry1 genes, with their insecticidal activities against Plutella xylostella, were investigated. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) results revealed that the genotypes of these Bt strains are highly diversified. Also, a considerable number of the Bt strains isolated in Taiwan were found to have high insecticidal activities. Since strains that showed individual combined patterns of PFGE and RAPD exhibited distinct insecticidal activities against P. xylostella, thus, these genotypes may be useful for the identification of the new Bt strains and those which have been used in bioinsecticides. In addition, although the presence of cry2Aa1 may have a greater effect on the insecticidal activity of Bt strains in bioassay than other cry genes, only high expression level of cry1 genes plays a key role to determine the insecticidal activity of Bt strains. In conclusion, both RAPD and PFGE are effective in the differentiation of Bt strains. The presence of cry2Aa1 and, especially, the expression level of cry1 genes are useful for the prediction of the insecticidal activities of Bt strains against P. xylostella.

Transplantation of human Wharton's Jelly-derived stem cells alleviates chemically induced liver fibrosis in rats.

There is currently no effective treatment method available for liver fibrosis. We therefore evaluated the use of Wharton's jelly stem cells (WJSCs; the major umbilical cord stem cell population) to treat chemically induced liver fibrosis via intraperitoneal injection of thioacetamide. WJSCs were transplanted into liver-damaged rats via the portal vein and the treatment was evaluated by assessing serum biochemistry and histopathology. Transplanted WJSCs were distributed in the fibrotic area and around blood vessels, and hepatic recovery was accelerated. Serum prothrombin time significantly recovered, and serum albumin also improved at 21 days posttransplantation; collagen accumulation also decreased at 14 days. Thus, human WJSCs promoted recovery after chronic liver damage. Using immunohistochemical analyses, we determined that transplanted WJSCs produce albumin, hepatocyte growth factor (HGF), and metalloproteinase (MMP) after transplantation to chemically injured liver, indicating that WJSC may help to decrease liver collagen and thus may be useful for treating liver fibrosis.