[Epidemiological characteristics of novel coronavirus pneumonia in Shijiazhuang, China].

Objective: To analyze the epidemiological characteristics of an outbreak of novel coronavirus pneumonia (COVID-19) in Shijiazhuang, Hebei Province in 2021 and to provide scientific basis for developing improved strategies to prevent and control the outbreak of COVID-19. Methods: Descriptive analysis of the outbreak of COVID-19 in Shijiazhuang, Hebei Province was performed with SPSS 21.0 and Excel software. The statistical analysis of the incubation period was performed using the rstan package in R4.0.4. Results: As of February 14th 2021, a total of 942 local confirmed cases were reported in Hebei Province, 869 cases in Shijiazhuang, of which 847 cases were available for case information. This outbreak was mainly in rural areas, with the largest number of confirmed cases in Xiaoguozhuang village, 249 (29.4%); followed by Nanqiaozhai village, 128 (15.1%); and Liujiazuo village, 85 (10.0%). The outbreak lasted from January 2nd, 2021 to February 14th, 2021, and was mainly transmitted among the farmers as well as the students through dining parties, public gatherings and family contacts, showing an obvious time and occupation concentration trend. An analysis of 116 local confirmed cases in this outbreak with specific exposure time and onset time indicated that the median incubation period was 6 [interquartile range(IQR): 3.3, 10.0] days; whereas another report including 264 local confirmed cases with specific exposure time window showed that a median incubation period was 8.5 [95% confidence interval (CI): 1.8-18.8] days. Conclusions: This outbreak was mainly related to rural areas, and was associated with parties, public gatherings and family gatherings. Self-protection and isolation of key areas and populations at risk should be effectively implemented to avoid close contact and other measures to reduce the occurrence of COVID-19 aggregation. Based on the results of the incubation period of this outbreak, the isolation period could be recommended to be extended to three weeks.

Predicting the survival rate of mouse embryonic stem cells cryopreserved in alginate beads.

Stem cell cryopreservation in three-dimensional (3D) scaffolds may offer better protection to cells leading to higher survival rates. However, it introduces heterogeneity in cryoprotective agent (CPA) concentrations, durations of exposure to CPA, and freezing and thawing rate within constructs. This paper applies a mathematical model which couples the mass transport of dimethyl sulphoxide (DMSO) in a cell-seeded spherical construct and cell membrane transport into mouse embryonic stem cells (mESCs) to predict overall cell survival rate (CSR) based on CPA equilibrium exposure times (t(E)) and concentrations. The effect of freeze-concentration is also considered. To enable such a prediction, a contour plot was constructed using experimental data obtained in cryopreservation of cell suspensions with DMSO at a cooling rate of 1 degrees C/min. Thereafter, the diffusion in the alginate bead and the membrane transport of CPA was numerically simulated. Results were mapped onto the survival rate contours yielding 'predicted' CSR. The effects of loading time, hindrance, construct radius, and CPA concentration on predicted CSR were examined. From these results, an operation window with upper and lower t(E) of 12-19 min (for 0.6 mm radius beads and 1.4 M DMSO) yielded an overall viability of 60 per cent. The model predictions and the best experimental cryopreservation results with encapsulated mESCs were in agreement. Hence, optimization based on post-thaw CSR can accelerate the identification of cryopreservation protocols and parameters for maximizing cell survival.

RGDS-fuctionalized alginates improve the survival rate of encapsulated embryonic stem cells during cryopreservation.

Cryopreservation of stem cells, especially embryonic stem cells, is problematic because of low post-thaw cell survival rates and spontaneous differentiation following recovery. In this investigation, mouse embryonic stem cells (mESCs) were encapsulated in arginine-glycine-aspartic acid-serine (RGDS)-coupled calcium alginates (1.2 percent, w/v), allowed to attach to the substratum and then cryopreserved in 10 percent (v/v) dimethyl sulfoxide (DMSO) solution at a slow cooling rate of 1 C per min. RGDS coupling to alginate was confirmed by Transmission Fourier Transform Infra-Red spectroscopy (T-FTIR) and quantified by using ninhydrin-Ultraviolet/Visible light (ninhydrin-UV/VIS) assay. Flow cytometry data showed that mESCs cryopreserved in RGDS-alginate beads had a higher expression of stem cell markers compared with cells cryopreserved in suspension or cells cryopreserved in unmodified alginates. Cell viability after thawing was assessed using trypan blue exclusion assay and monitored using Alamar blue assay for 6 hours. It was shown that post-thaw cell survival rate was significantly higher for cells encapsulated in RGDS-modified alginate (93 ± 2 percent, mean and standard error) than those in suspension (52 ± 2 percent) or in unmodified alginates (62 ± 3 percent). These results showed that cells encapsulated and attached to a substratum have better survival rate and stem cell marker expression 24 hours after cryopreservation than those in suspension. Encapsulation in RGDS-alginate was optimized for peptide concentration, cryoprotective agent loading time and cooling rate. The best result was obtained when using 12.5 mg peptide per g alginate, 30 minutes loading time and 1 C per min cooling rate.

Effect of various freezing solutions on cryopreservation of mesenchymal stem cells from different animal species.

The objective of this study is to compare the effects of different well defined freezing solutions with a reduced concentration of dimethylsulfoxide (DMSO) combined with polyethylene glycol (PEG) and/or trehalose on cryopreservation of mesenchymal stem cells (MSCs) from mice, rats and calves. Post-thaw cell viability, proliferation capacity and differentiation potential of MSCs from different species were assessed after cryopreservation with the conventional slow freezing method. Although the post-thaw viabilities and metabolic activities varied among the different species, satisfactory results were obtained with 5 percent (v/v) DMSO, 2 percent (w/v) PEG, 3 percent (w/v) trehalose and 2 percent (w/v) bovine serum albumin (BSA) as the freezing solution. Our results showed that mouse MSCs were more robust to cryopreservation compared with rat and bovine MSCs.

Application of multiple parallel perfused microbioreactors and three-dimensional stem cell culture for toxicity testing.

In this study, a multiple parallel perfused microbioreactor platform, TissueFlex, was developed which can be used to perform cell and tissue culture under almost uniform and precisely controlled environment in a mid-throughput and parallel manner. These microbioreactors were used to culture human bone marrow cells (hBMCs) in three-dimensional (3D) scaffolds and also in two-dimensional (2D) monolayer for comparison for upto 7 days. Several scaffolding materials were evaluated for this purpose in terms of easiness in handling, ability to support the hBMC growth, and feasibility for non-destructive optical assays. The feasibility and efficacy of using the developed 3D-hBMCs-based model tissue-constructs cultured in TissueFlex microbioreactors for drug evaluation and toxicity testing was then studied. As a demonstration case study, the cultured cells were challenged with two chemicals, trimethoprim and pyrimethamine, both known to be harmful to cellular activities, with different protocols. Cytotoxicity in terms of cell viability and growth was determined using the AlamarBlue assay. The 3D spatial variations in cell morphology and cell survival were also monitored using 3D optical imaging using non-linear multiphoton microscopy. The results show that (i) the data obtained from 3D hBMCs culture and from (2D) monolayer cultures on the effect of the tested chemicals on cell growth are significantly different, and that (ii) the perfused microbioreactor technology could provide a highly controlled and prolonged cell culture environment for testing of various drugs and chemicals. The outcome of this study demonstrated the feasibility and potentials of the using 3D stem cell based model tissues in TissueFlex microbioreactors for drug evaluation and toxicity testing of chemicals as an efficient and standardized alternative testing method.

3D bone tissue growth in hollow fibre membrane bioreactor: implications of various process parameters on tissue nutrition.

New experimental evidence shows that hollow fibre membrane bioreactor (HFMB) may be applied to grow bulky bone tissues which may then be implanted into patients to repair skeletal defects. To design effective bone tissue engineering protocols, it is necessary to determine the quantitative relationships between the cell environment and tissue behaviour in HFMBs and their relationship with nutrient supply. It is also necessary to determine under what conditions nutritional limitations may occur and, hence, may cause cell death. These require that the appropriate bioreactor conditions for generating neotissues, and the nutrient transfer behaviour and chemical reaction during cell growth and extracellular matrix formation are studied thoroughly. In this paper, we aim to use an existing mathematical framework to analyse the influence of various relevant parameters on nutrient supply for bone tissue growth in HFMB. We adopt the well-known Krogh cylinder approximation of the HFMB. The model parameters (e.g., cell metabolic rates) and operating conditions for the mathematical model have been obtained from, or correspond to, in-house experiments with the exception of a few variables which have been taken from the literature. The framework is then used to study oxygen and glucose transport behaviour in the HFMB. Influence of a number of important process parameters, e.g., reaction kinetics, cell density, inlet concentration of nutrients, etc, on the nutrient distributions have been systematically analysed. The work presented in this paper provides insights on unfavourable system designs and specifications which may be avoided to prevent mass transfer limitations for growing bone tissues in HFMB.

Microdialysis for monitoring the process of functional tissue culture.

Continuous monitoring is important during tissue culture. However, there are still technical difficulties in monitoring the internal status of cells or tissues. In this paper, microdialysis is adopted to monitor functional tissue growth in a bioreactor. Explanted bovine caudal intervertebral disc (IVD) was used as the test tissue. A microdialysis membrane probe of 100 kDa molecular weight cut-off was employed and in situ calibration methods with phenol red and fluorescent 40 kDa dextran were developed to measure the relative recovery of the solute of interest, and membrane fouling, respectively. Tissue metabolism was monitored successfully. At the same time soluble macromolecules were picked up by the probe and were detected and quantified by Fast Protein Liquid Chromatography (FPLC) and/or Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). These proteins were believed to be associated with biofunction of engineered tissue. Monitoring of phenol red content in the dialysate indicated that there was no significant fouling of the membrane probe during a 7-day culture period and the Relative Recovery of macromolecules of interests remained roughly 9%. We concluded that microdialysis could be used to sample a wide range of molecular species released during cell metabolism and extracellular matrix turnover, which were direct or indirect indications of cell and tissue functions. The application of the developed system could be extended to monitor tissue repair in vivo, and the development of the engineered tissue.

Using an improved 1D boundary layer model with CFD for flux prediction in gas-sparged tubular membrane ultrafiltration.

Tubular membrane ultrafiltration and microfiltration are important industrial separation and concentration processes. Process optimisation requires reduction of membrane build-up. Gas slug introduction has been shown to be a useful approach for flux enhancement. However, process quantification is required for design and optimisation. In this work we employ a non-porous wall CFD model to quantify hydrodynamics in the two-phase slug flow process. Mass transfer is subsequently quantified from wall shear stress, which was determined from the CFD. The mass transfer model is an improved one-dimensional boundary layer model, which empirically incorporates effects of wall suction and analytically includes edge effects for circular conduits. Predicted shear stress profiles are in agreement with experimental results and flux estimates prove more reliable than that from previous models. Previous models ignored suction effects and employed less rigorous fluid property inclusion, which ultimately led to under-predictive flux estimates. The presented model offers reliable process design and optimisation criteria for gas-sparged tubular membrane ultrafiltration.

Potent antitumoral efficacy of a novel replicative adenovirus CNHK300 targeting telomerase-positive cancer cells.

PURPOSE: Telomerase reverse transcriptase (hTERT) is the key determinant of telomerase activity and plays a crucial role in cellular immortalization and oncogenesis. It will be a promising target for cancer gene therapy. We constructed a novel replicative adenovirus CNHK300 in which hTERT promoter with three extra E-boxes downstream of the promoter was introduced and used to regulate adenoviral E1a gene, and studied its properties of selective replication in cancer cells and antitumoral activity. METHODS: Luciferase assay was used to detect hTERT promoter activity. The selective replication of CNHK300 in cancer cells was investigated by E1a Western blot and green fluorescent protein (GFP) reporter gene assay. The antitumoral activity of CNHK300 and its toxicity were measured on animal models. RESULTS: Luciferase assay showed that introducing extra E-boxes downstream of hTERT promoter is beneficial to decreasing the promoter activity in normal cells without affecting its strong activity in cancer cells. Experiments in vitro and in vivo demonstrated that CNHK300 can selectively target to hTERT-positive cancer cells and replicate in them, resulting in oncolytic or antitumoral effect. CNHK300 is superior to ONYX-015 in terms of selective replication and oncolytic or antitumoral effect. The toxicity assay showed no signs of toxicity to liver cells even at the higher dosage of CNHK300 in vivo. CONCLUSION: The hTERT promoter-controlled, replication-competent adenovirus CNHK300 is a promising system for targeted cancer gene therapy.

Modeling of the co-transport of cryoprotective agents in a porous medium as a model tissue.

Cryopreservation is likely the choice for long-term preservation of natural and engineered tissues, and high concentration multiple cryoprotective agents (CPAs) are usually used in such a process. To achieve high cell viability after cryopreservation, cells at all locations within the tissue must be protected properly by the CPAs during freezing. It is hence essential to know the distribution and concentration of CPAs within the tissue during multiple-CPA addition, to maximize cell survival and minimize tissue damage. In this work, a model to describe the CPA transport during multiple-CPA addition in a one-dimensional porous medium, as a simplified model of living tissue, was developed on the basis of the Maxwell-Stefan (M-S) equations. The UNIFAC and UNIQUAC models were used to evaluate the activity coefficients, and the Siddiqi-Lucas correlation was used for estimation of Maxwell-Stefan diffusivities. Simulations were carried out to examine the effect of temperature, tissue property, CPA type and the interactions between solutes on the CPA transport within construct during the CPA addition. It was found that these parameters, especially the interactions between the different CPA molecules, which was neglected before, significantly affect the transport of each individual CPA component. It is hence concluded that the traditional single-component analysis on the CPA diffusion is not adequate to quantify the multiple-CPA distribution in the tissue, particularly when the CPA concentrations are relatively high.

Modeling of cryopreservation of engineered tissues with one-dimensional geometry.

Long-term storage of engineered bio-artificial tissues is required to ensure the off-the-shelf availability to clinicians due to their long production cycle. Cryopreservation is likely the choice for long-term preservation. Although the cryopreservation of cells is well established for many cell types, cryopreservation of tissues is far more complicated. Cells at different locations in the tissue could experience very different local environmental changes, i.e., the change of concentration of cryoprotecting chemicals (CPA) and temperature, during the addition/removal of CPA and cooling/warming, which leads to nonuniformity in cell survival in the tissue. This is due to the limitation of mass and heat transfer within the tissue. A specific aim of cryopreservation of tissue is to ensure a maximum recovery of cells and their functionality throughout a tissue. Cells at all locations should be protected adequately by the CPA and frozen at rates conducive to survival. It is hence highly desirable to know the cell transient and final states during cryopreservation within the whole tissue, which can be best studied by mathematical modeling. In this work, a model framework for cryopreservation of one-dimensional artificial tissues is developed on the basis of solving the coupled equations to describe the mass and heat transfer within the tissue and osmotic transport through the cell membrane. Using an artificial pancreas as an example, we carried out a simulation to examine the temperature history, cell volume, solute redistribution, and other state parameters during the freezing of the spherical heterogeneous construct (a single bead). It is found that the parameters affecting the mass transfer of CPA in tissue and through the cell membrane and the freezing rate play dominant roles in affecting the cell volume transient and extracellular ice formation. Thermal conductivity and extracellular ice formation kinetics, on the other hand, have little effect on cell transient and final states, as the heat transfer rate is much faster than mass diffusion. The outcome of such a model study can be used to evaluate the construct design on its survivability during cryopreservation and to select a cryopreservation protocol to achieve maximum cell survival.

The influence of model parameter values on the prediction of skin surface temperature: II. Contact problems.

A model of heat transfer and temperature distribution in the skin and superficial tissues which is based on a finite difference numerical solution of the one-dimensional multilayer coupled bioheat equation is presented. The model is used to investigate the influence of the values chosen to represent the physiological and thermal properties of the tissues on the skin surface temperature after contact with an external medium. It was found that the skin blood flow and dermal conductivity were the main cutaneous parameters which influence the contact response, but in terms of normalized temperature the response was little influenced by cutaneous metabolic heat generation and deep dermal temperature. For contact with a good conductor, the transient behaviour was sensitive to the heat transfer coefficient on the outer surface and the thickness of the contact material, but insensitive to the conductivity of the material.

The influence of model parameter values on the prediction of skin surface temperature: I. Resting and surface insulation.

A model is presented of heat transfer and temperature distributions in the skin and superficial tissues. It is based on a finite difference numerical solution of the one-dimensional multilayer coupled bioheat equation. In this paper, the model is used to investigate the influence of the values of parameters chosen to represent the physiological and heat transfer processes on the temperature of the skin under resting conditions and after insulation of the skin surface. Equilibrium resting temperatures were strongly influenced by deep body temperature especially at lower heat transfer coefficients on the skin surface, but slightly affected by the values chosen for skin blood flow and metabolic heat generation; both the heat transfer coefficients and environmental temperature strongly influenced the surface temperature. After surface insulation the temperature elevation was strongly influenced by the thermal conductivities of tissues, skin blood flow and deep boundary temperature; metabolic heat generation was only significantly at unphysiologically high values.

Abnormal thermal hyperaemia in the skin in paraplegia.

Surface insulation, together with laser Doppler flowmetry, was used to assess the skin microcirculation of paraplegic patients. Two control groups of five male and five female subjects were used to establish the response of normals with which to compare the results obtained from six paraplegic subjects. No significant sex related difference was revealed from this study. It was found that in normal subjects, surface insulation resulted in a significant increase in both skin temperature and skin blood flow. In paraplegic patients, the temperature increase was significantly less than in the normal subjects and there was no significant thermally induced hyperaemia after surface insulation.

Alpha-fetoprotein reaction to Pisum sativum agglutinin in differentiation of benign liver diseases from hepatocellular carcinoma.

Alpha-fetoprotein reactive to Pisum sativum agglutinin levels (AFP-R-PSA) was measured in sera from 124 patients with hepatocellular carcinoma (HCC) and 54 patients with benign liver diseases (BLD). The level of AFP-R-PSA in the HCC group (42% +/- 22%) was significantly higher than that in the BLD group (10% +/- 8%). When an AFP-R-PSA level above 25% was used as a value highly suggestive of HCC, the sensitivity of the test was 82%, the specificity was 96%, the accuracy was 86%, and the positive prediction value was 98%. The positive rates of AFP-R-PSA in HCC patients with a serum AFP level below 100 micrograms/L and with a serum AFP level below 400 micrograms/L were 78% and 84%, respectively. Corresponding value was 74% for 31 patients with a tumor size less than 5cm. If AFP was combined with AFP-R-PSA, the detection rate of small HCC in this study would be increased from 32% to 87%. These data indicate that measurement of AFP-R-PSA is useful for the differentiation of BLD and for the early diagnosis of HCC.

Phenacetin O-deethylation in extrahepatic tissues of rats.

Phenacetin O-deethylation is a marker reaction of CYP450 1A2 activity. The drug-metabolizing enzyme is constitutively expressed in liver. In this study, an in vivo rat model for assessment of extrahepatic metabolism was used to investigate phenacetin O-deethylation and the alterations in the disposition of phenacetin due to the loss of liver function. Rats were divided into the model and normal control groups. The model was established according to our previously described method. The concentrations of phenacetin and its major metabolites acetaminophen, glucuronate-acetaminophen and sulfate-acetaminophen in plasma and urine were determined by HPLC. 30 min after intravenous administration of 0.16% phenacetin 10 mg x kg(-1), plasma acetaminophen in the model group was only 3.6% of that in the control group (0.09+/-0.04 microg x mL(-1) vs 2.49+/-0.85 microg x mL(-1), n = 8). 30 min after intragastric injection of 0.4% phenacetin 30 mg x kg(-1), plasma acetaminophen formation was very slight, about 8.6% of plasma phenacetin in the model group (0.74+/-0.43 microg x mL(-1) acetaminophen vs 8.57+/-8.42 microg x mL(-1) phenacetin) and 6.8% in the control group (1.06+/-0.59 microg x mL(-1) acetaminophen vs 15.47+/-7.21 microg x mL(-1) phenacetin, n = 8); no significant differences were observed in plasma phenacetin, total acetaminophen and the ratio of acetaminophen to phenacetin between control and model groups. In the urine collected for 3 h after intravenous administration of 0.16% phenacetin 10 mg x kg(-1), the total recovery of acetaminophen (as free, glucuronate- and sulfate-acetaminophen ) in the model group was 4.6% of that in the control group (4.47+/-4.27 microg vs 96.63+/-8.50 microg, n = 6), but phenacetin recovery in the model group was 9 times higher than that in the control group (15.03+/-17.72 microg vs 1.66+/-0.50 microg). The results indicate that phenacetin O-deethylation in the extrahepatic tissues and the first-pass metabolism of the probe compound seem to be negligible in rats, but the renal excretion of phenacetin, as a compensation, dramatically increases in model rats.

[Alpha-fetoprotein (AFP) variant in the diagnosis of small liver cancer].

From May 1983 to June 1986, 40 patients with primary liver cancer, less than 5 cm in diameter, were treated by operation. The smallest lesion was 1.4 x 0.9 x 0.4 cm in size. Fifteen patients came to the hospital for treatment due to the finding of AFP greater than or equal to 31 ng/ml by public survey and 8 with space occupying lesion (SOL) in the liver by ultrasonography (US). The serum AFP levels ranged from 0 to 6800 ng/ml in this group with AFP negative in 6 cases, 31-400 ng/ml in 19 and over 400 ng/ml in 15. The positive rate of AFP was 37.5%. US displayed SOL in 25 cases, suspicious SOL in 5 and negative in 10. The positive rate was 62.5% by US. The overall positive rate was 77.5% by AFP and US combined. In addition, AFP variant was determined by LCA affino-crossed-immunoelectrophoresis autoradiography. LCA-nonreactive-AFP varied from 0 to 100% with a mean value of 57.0 +/- 26.7%. Taking less than 75% as diagnostic criterion for liver cancer, the positive rate of LCA-nonreactive-AFP was 65.0%. 12 patients who were AFP variant positive but SOL suspicious or negative were regularly followed by US for 1-20 months. They all showed distinct SOL. Composite results of AFP, AFP variant and US gave a diagnostic rate of 97.5% for small liver cancer. The determination of AFP variant is helpful in early detection of small liver cancer.

Mechanisms of structure generation during plastic compression of nanofibrillar collagen hydrogel scaffolds: towards engineering of collagen.

Operator control of cell/matrix density of plastically compressed collagen hydrogel scaffolds critically depends on reproducibly limiting the extent of scaffold compaction, as fluid expulsion. A functional model of the compression process is presented, based on the idea that the main fluid-leaving surface (FLS) behaves as an ultrafiltration membrane, allowing fluid (water) out but retaining collagen fibrils to form a cake. We hypothesize that accumulation of collagen at the FLS produces anisotropic structuring but also increases FLS hydraulic resistance (R(FLS) ), in turn limiting the flux. Our findings show that while compressive load is the primary determinant of flux at the beginning of compression (load-dependent phase), increasing FLS collagen density (measured by X-ray attenuation) and increasing R(FLS) become the key determinants of flux as the process proceeds (flow-dependent phase). The model integrates these two phases and can closely predict fluid loss over time for a range of compressive loads. This model provides a useful tool for engineering cell and matrix density to tissue-specific levels, as well as generating localized 3D nano micro-scale structures and zonal heterogeneity within scaffolds. Such structure generation is important for complex tissue engineering and forms the basis for process automation and up-scaling.