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Calvarial bone healing is challenging, especially for individuals with osteoporosis because stem cells from osteoporotic patients are highly prone to adipogenic differentiation. Based on previous findings that chondrogenic induction of adipose-derived stem cells (ASCs) can augment calvarial bone healing, we hypothesized that activating chondroinductive Sox Trio genes (Sox5, Sox6, Sox9) and repressing adipoinductive genes (C/ebp-α, Ppar-γ) in osteoporotic ASCs can reprogram cell differentiation and improve calvarial bone healing after implantation. However, simultaneous gene activation and repression in ASCs is difficult. To tackle this problem, we built a CRISPR-BiD system for bi-directional gene regulation. Specifically, we built a CRISPR-AceTran system that exploited both histone acetylation and transcription activation for synergistic Sox Trio activation. We also developed a CRISPR interference (CRISPRi) system that exploited DNA methylation for repression of adipoinductive genes. We combined CRISPR-AceTran and CRISPRi to form the CRISPR-BiD system, which harnessed three mechanisms (transcription activation, histone acetylation, and DNA methylation). After delivery into osteoporotic rat ASCs, CRISPR-BiD significantly enhanced chondrogenesis and in vitro cartilage formation. Implantation of the engineered osteoporotic ASCs into critical-sized calvarial bone defects significantly improved bone healing in osteoporotic rats. These results implicated the potential of the CRISPR-BiD system for bi-directional regulation of cell fate and regenerative medicine.

Chinese hamster ovary (CHO) cell is the predominant cell factory to produce biopharmaceuticals such as immunoglobulin G (IgG), but in CHO cells lactate accumulation and cell death compromise IgG production while fucosylation mitigates the antibody-dependent cellular cytotoxicity (ADCC) of IgG. To tackle these problems, we harnessed a burgeoning gene silencing system, CRISPR-Cas13d, to effectively suppress the endogenous genes governing lactate production (LDHA), fucosylation (GFT) and cell death (DDIT3), hence repressing lactate accumulation and core fucosylation. We further exploited the Sleeping Beauty system to integrate the CRISPR-Cas13d module co-targeting these 3 genes and generated a CHO cell platform for IgG production. The new platform exhibited simultaneous knockdown of LDHA, GFT and DDIT3, accumulated less lactate, had prolonged longevity, produced 59 ± 6% more IgG with less fucosylation and stronger ADCC efficacy. These data collectively warrant the potentials of CRISPR-Cas13d for CHO cell engineering and improving antibody production with regard to quantity and quality.

Chinese hamster ovary (CHO) cell is the predominant cell factory to produce biopharmaceuticals such as immunoglobulin G (IgG), but in CHO cells lactate accumulation and cell death compromise IgG production while fucosylation mitigates the antibody-dependent cellular cytotoxicity (ADCC) of IgG. To tackle these problems, we harnessed a burgeoning gene silencing system, CRISPR-Cas13d, to effectively suppress the endogenous genes governing lactate production (LDHA), fucosylation (GFT) and cell death (DDIT3), hence repressing lactate accumulation and core fucosylation. We further exploited the Sleeping Beauty system to integrate the CRISPR-Cas13d module co-targeting these 3 genes and generated a CHO cell platform for IgG production. The new platform exhibited simultaneous knockdown of LDHA, GFT and DDIT3, accumulated less lactate, had prolonged longevity, produced more IgG with less fucosylation and stronger ADCC efficacy. These data collectively warrant the potentials of CRISPR-Cas13d for CHO cell engineering and improving antibody production with regard to quantity and quality.

Healing of large calvarial bone defects in adults adopts intramembranous pathway and is difficult. Implantation of adipose-derived stem cells (ASC) that differentiate towards chondrogenic lineage can switch the bone repair pathway and improve calvarial bone healing. Long non-coding RNA DANCR was recently uncovered to promote chondrogenesis, but its roles in rat ASC (rASC) chondrogenesis and bone healing stimulation have yet to be explored. Here we first verified that DANCR expression promoted rASC chondrogenesis, thus we harnessed CRISPR activation (CRISPRa) technology to upregulate endogenous DANCR, stimulate rASC chondrogenesis and improve calvarial bone healing in rats. We generated 4 different dCas9-VPR orthologues by fusing a tripartite transcription activator domain VPR to catalytically dead Cas9 (dCas9) derived from 4 different bacteria, and compared the degree of activation using the 4 different dCas9-VPR. We unveiled surprisingly that the most commonly used dCas9-VPR derived from Streptococcus pyogenes barely activated DANCR. Nonetheless dCas9-VPR from Staphylococcus aureus (SadCas9-VPR) triggered efficient activation of DANCR in rASC. Delivery of SadCas9-VPR and the associated guide RNA into rASC substantially enhanced chondrogenic differentiation of rASC and augmented cartilage formation in vitro. Implantation of the engineered rASC remarkably potentiated the calvarial bone healing in rats. Furthermore, we identified that DANCR improved the rASC chondrogenesis through inhibition of miR-203a and miR-214. These results collectively proved that DANCR activation by SadCas9-VPR-based CRISPRa provides a novel therapeutic approach to improving calvarial bone healing.

Chinese hamster ovary (CHO) cells are the predominant cell chassis for biopharmaceutical production. Engineering cellular pathways related to cell death, metabolism, and glycosylation in CHO cells is desired but challenging. Here, we present a novel approach that exploits CRISPR-Cas13d for gene silencing and CHO cell engineering. CRISPR-Cas13d is a burgeoning system that exploits Cas13d nuclease and guide RNA (gRNA) for RNA cleavage and gene knockdown. We first showed that CRISPR-Cas13d effectively knocked down exogenous genes in CHO cell lines (K1, DG44, and DUXB11) commonly used for recombinant protein production. We next demonstrated that CRISPR-Cas13d robustly suppressed the expression of exogenous genes and various endogenous genes involved in gene amplification, apoptosis, metabolism, and glycosylation (e.g., GS, BAK, BAX, PDK1, and FUT8) in CHO cells with efficiencies ranging from 60% to 80%, simply by transient transfection. By integrating the entire CRISPR-Cas13d system with the Sleeping Beauty system and optimal gRNA design, we further improved the knockdown efficiency and rapidly generated stable cells with ≈80%-90% knockdown. With this approach, we knocked down FUT8 expression for >90% and significantly attenuated the IgG fucosylation. These data altogether implicated the potentials of CRISPR-Cas13d for gene regulation, glycoengineering, and cell engineering of CHO cells.

The decellularization of long segments of tubular tissues such as blood vessels may be improved by perfusing decellularization solution into their lumen. Particularly, transmural flow that may be introduced by the perfusion, if any, is beneficial to removing immunogenic cellular components in the vessel wall. When human umbilical arteries (HUAs) were perfused at a transmural pressure, however, very little transmural flow was observed. We hypothesized that a watertight lining at the abluminal surface of HUAs hampered the transmural flow and tested the hypothesis by subjecting the abluminal surface to enzyme digestion. Specifically, a highly viscous collagenase solution was applied onto the surface, thereby restricting the digestion to the surface. The localized digestion resulted in a water-permeable vessel without damaging the vessel wall. The presence of the abluminal lining and its successful removal were also supported by evidence from SEM, TEM, and mechanical testing. The collagenase-treated HUAs were decellularized with 1% sodium dodecyl sulfate (SDS) solution under either rotary agitation, simple perfusion, or pressurized perfusion. Regardless of decellularization conditions, the decellularization of HUAs was significantly enhanced after the abluminal lining removal. Particularly, complete removal of DNA was accomplished in 24 h by pressurized perfusion of the SDS solution. We conclude that the removal of the abluminal lining can improve the perfusion-assisted decellularization.

PurposeMore attention should be paid to project managers’ (PMs) job satisfaction as they play an important role in ensuring projects are completed successfully. The purpose of this paper is to identify human resource management (HRM) policies and practices that lead to higher PMs’ job satisfaction.Design/methodology/approachA questionnaire survey was conducted on PMs who are working in construction firms and project management consultancy firms. Data were collected via random, convenience and snowball sampling. The data collected were analysed using partial least square-structural equation modelling, independent samplest-test and Pearson’s correlation.FindingsThe findings show that PMs who are satisfied with their firms’ HRM practices and job rewards also have higher job satisfaction. Several HRM strategies that give rise to higher job satisfaction are identified, e.g. a system to recognise and develop talent, and taking active steps to identify and develop backups in case of emergency. Unfortunately, some practices are not implemented to a significant extent, and these include: systematically recruiting and retaining talented PMs, encouraging PMs to plan for their careers, offering performance and development coaching, and appraising employees.Research limitations/implicationsThe limitations include the low response rate and the relatively small sample size of 81. The profile of respondents is largely from construction companies with more than 150 staff, and, therefore, the findings are more applicable to medium- to large-sized construction firms.Practical implicationsThe study identified many HRM practices and policies that are significantly associated with PMs’ job satisfaction, yet many of these are not implemented to a significant extent by the employers. The practical implication is that employers of PMs should systematically implement these in order that their PMs have higher job satisfaction which is important for a project’s success.Originality/valueThe originality of this research is that the HRM practices and policies that are associated with job satisfaction of PMs are uncovered. Its value is in showing that PMs derive greater job satisfaction when HRM policies encompass talent development, career coaching and a personalised management style. Among these important practices, those that have been neglected were also identified. The study offers recommendations on the HRM practices that firms should be put in place for their PMs to experience higher job satisfaction.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer as the overall five-year survival rate was less than 5%. More than 90% of PDAC patients harbor Kras mutation, but only mutant Kras is insufficient to give rise to PDAC. Inflammation is essential to cooperate with mutant Kras for the initiation of pancreatic precancerous lesion, and attracted M1-polarized macrophages-induced inflammation are known to contribute to the process. Among TRIM family proteins, TRIM37 is the only protein containing MATH domain that endows it being able to interact with proteins containing TRAF domain, indicating its possible role in regulating pattern recognition receptor-, TNFR- and cytokine-mediated signaling pathways. We have observed that, when TRIM37 was knocked-down, macrophage cytokines production, such as IL-1β, IFN-β, IL-12, TNF-a, IL-6 and CCL-2 in response to LPS was reduced, and the regulation was both epigenetically and non-epigenetically. Interestingly, pro-inflammatory cytokines also upregulated TRIM37 expression in macrophages. Thus our data implicated that upregulated TRIM37 that positively regulate macrophage cytokine production might contribute to the initiation of PDAC. However, when macrophages were co-cultured with pancreatic cancer cells, the expression level of TRIM37 in macrophages were downregulated, this led to the loss of its ability in maintaining macrophages in a stable M1 phenotype, and instead, skewing the macrophages into a M2 phenotype, which promotes cancer progression. In conclusion, our data demonstrate that during pancreatic cancer development, the expression of TRIM37 in macrophages are differential regulated to favor cancer development and progression.

The operation strategy for a lab-scale grid-connected photovoltaic generation system integrated with battery energy storage is proposed in this paper. The photovoltaic generation system is composed of a full-bridge inverter, a DC–DC boost converter, an isolated bidirectional DC–DC converter, a solar cell array and a battery set. Since the battery set acts as an energy buffer to adjust the power generation of the solar cell array, the negative impact on power quality caused by the intermittent and unstable output power from a solar cell array is alleviated, so the penetration rate of the grid-connected photovoltaic generation system is increased. A lab-scale prototype is developed to verify the performance of the system. The experimental results show that it achieves the expected performance.

Titania nanofibers with fiber diameters ranging from 194 to 441 nm were electrospun as light-scattering photoactive materials. Transmittance UV–vis spectroscopy revealed that the scattering behavio...

Skeletal muscle neoplasms, in contrast to other groups of tumors, are almost malignant. The benign variant, rhabdomyoma, is distinctly rare. Rhabdomyomas can be classified generally into two types: cardiac and extracardiac. Extracardiac rhabdomyoma can be further divided into three subtypes: adult, fetal, and genital type. Adult rhabdomyoma is the most common subtype of rhabdomyoma even though it remains relatively rare. Fetal rhabdomyomas are less common than the adult type. In this paper we report a rare case of a fetal rhabdomyoma with polyp-like appearance originating from right tonsil. Punch biopsy and then right tonsillectomy were performed for complete excision. There was no obvious recurrence.

This paper presents an efficient, simple, hierarchical, and sparse three-dimensional capacitance extraction algorithm, i.e., ICCAP. Most previous capacitance extraction algorithms, such as FastCap and HiCap, introduce intermediate variables to facilitate the hierarchical potential calculation, but still preserve the basic panels as basis. In this paper, we discover that those intermediate variables are a fundamentally much better basis than leaf panels. As a result, we are able to explicitly construct the sparse potential coefficient matrix and solve it with linear memory and linear run time in comparison with the most recent hierarchical O(nlogn) approach in PHiCap. Furthermore, the explicit sparse formulation of a potential matrix not only enables the usage of preconditioned Krylov subspace iterative methods, but also the reordering technique. A new reordering technique, i.e., level-oriented reordering (LOR), is proposed to further reduce over 20% of memory consumption and run time compared with no reordering techniques applied. In fact, LOR is even better than the state-of-the-art minimum degree reordering and more efficient. Without complicated orthonormalization matrix computation, ICCAP is very simple, efficient, and accurate. Experimental results demonstrate the superior run time and memory consumption over previous approaches while achieving similar accuracy.

To evaluate the behavior of TCP on a path with a wireless link, we build a wireline-wireless network model, and assume the wireless link is the bottleneck which causes packets to be buffered at the wireline-wireless interface buffer. A reliable link layer protocol is assumed on the wireless link which hides the fluctuations of the wireless medium from TCP. We first build the link layer model and the wireless channel Markov chain model, and use the matrix geometric method to evaluate the packet loss probability. We then analyze the long-term steady-state send rate and throughput of a TCP connection.

Poly(methyl methacrylate) nanofibers with desired fiber diameters that ranged from 336 to 896 nm were electrospun as light scattering and propagation materials. The light scattering behavior of these samples as a function of the fiber diameter and fiber deposition thickness was examined by UV-vis spectrophotometry, which revealed the scattering bands in the absorption spectra. The scattering bands of these nanofibers were linearly proportional to the fiber diameter, which shows good agreement with a scattering model based on the Mie theory. The light scattering and prolonged light path lengths in the nanofiber scaffolds were monitored and quantified by the photoluminescence of a fluorescent dye, Coumarin 6, which was preloaded into the polymer nanofibers. The photoluminescence after proper normalization showed a second-order dependence on the dye loading per unit area, which is significantly different from the spin-coated thin-film samples following a first-order relationship. Nonlinear photoluminescence enhancements indicated prolonged light path lengths and multiple light absorptions within the fiber scaffolds as a result of light scattering. Even with relatively broad scattering band widths, the light scattering and photoluminescence of the electrospun nanofibers exhibited considerable wavelength selectivity, especially as the scattering bands overlapped with the excitation wavelengths of the fluorescence reagent.