Characterization of porcine extraembryonic endoderm cells.

OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.

Twenty-three Years of Sea Turtle Stranding/ bycatch Research in Taiwan.

Coastal sea turtle stranding and bycatch are common phenomena worldwide and have received more attention in recent years. They are caused by both natural and anthropogenic factors. One thousand and seventy-two turtles were reported to be victims of these phenomena from March 1997 to November 2019 in Taiwan. Number of stranding/bycatch were variable and infrequent for the first 14 years, but increased each year after 2012 and peaked in 2019 with 217 cases. Most turtles were juveniles to subadults. All five of Taiwan's species were reported in stranding and bycatch records, and the green turtle was reported the most common. The main reported seasons lasted from winter to spring, when the weather changes dramatically. The sex ratio (female: male) ranged from 7 in the hawksbill turtle to 0.7 in the olive ridley, with an average of 2.4 for all species. Green turtles were the dominant stranded species, and more loggerhead turtles were by-caught. The hotspots were the towns of Dougou and Tochen in Yilan County, and Gongliao District in New Taipei City, located in NE coast of Taiwan respectively. Stranding was the more common of the two phenomena reported, and 80% of all stranded turtles were subadult green turtles. Eighty percent of all stranded/bycaught turtles were dead. Pond-nets were the fishing gear that accounted for the most bycatch, and captured mainly living young and subadult green turtles as well as subadult loggerhead turtles. The hotspots for bycatch were the towns of Dongou and Tochen in Yilan County. The Coast Guard and concerned citizen were the main sources of reports. This is the first study to analyze the long-term stranding/bycatch of sea turtles in Taiwan.

Risk factors associated with acute/subacute cerebral infarction in HIV-negative patients with cryptococcal meningitis.

OBJECTIVE: To explore the risk factors associated with acute/subacute cerebral infarction (ASCI) in HIV-negative patients with cryptococcal meningitis (CM). METHODS: This case-control study included 10 HIV-negative CM patients with ASCI and 30 age- and sex-matched HIV-negative control (1:3) CM patients without ASCI. The clinical manifestations and neuroimaging findings were collected. Risk factors for ASCI in the HIV-negative CM patients were confirmed by conditional logistic regression analysis. RESULTS: Among the 10 HIV-negative CM patients with ASCI, all cases had lacunar infarctions. Single infarctions were found in 6 patients, and multiple infarctions in 4. Hydrocephalus (p=0.020, OR=23.77, 95% CI, 1.67-339.33) and smoking (p=0.039, OR=11.63, 95% CI, 1.14-118.96) were found to be independently associated with the occurrence of ASCI. CONCLUSIONS: Hydrocephalus and smoking may increase the risk of ASCI in HIV-negative CM patients. In the clinical course, cerebral infarction should be strongly suspected in CM patients with hydrocephalus or smoking histories.

Population pharmacokinetics of valproic acid in adult Chinese epileptic patients and its application in an individualized dosage regimen.

BACKGROUND: There are several reports describing population pharmacokinetic (PPK) models of valproic acid (VPA). However, little was known in Chinese adult patients with epilepsy. The present study aimed to establish a PPK model for VPA in Chinese adult epileptic patients and to demonstrate its use for dose individualization. METHODS: Data were obtained from a prospective study of 199 adult epileptic patients at 5 hospitals. The trough concentrations at steady state were measured by fluorescence polarization immunoassay. Data were analyzed using the Nonlinear Mixed Effects Model software. The serum trough concentrations at steady state were also measured using samples (n = 20) collected prospectively from a different hospital from those providing the data for deriving the original model. These independent samples served as an evaluation group. RESULTS: The important determinants of apparent VPA clearance were daily dose, body weight, and combination with carbamazepine, phenytoin, or phenobarbital. The final model predicted the individualized doses accurately. A total of 85% of the trough concentrations in the evaluation group were accurately predicted by the final model, whereas the prediction errors of the other patients were all < ± 31%. CONCLUSIONS: A PPK model was developed to estimate the individual clearance for patients taking VPA and could be applied for individualizing doses in the target population.

[Activation of Stra 8 gene during the differentiation of spermatogonial stem cells].

Retinoic acid plays an important role in maintaining the structure and function in male testis. Recent studies showed that there is a group of genes that can be specially activated by retinoic acid during the development of male reproductive gland. The gene Stra 8 (Stimulated by Retinoic Acid) was one of the gene in this group. In mouse, Stra 8 is restrictively expressed in male germ line cells, and its function is related to the development of sperm. In order to investigate the feature of Stra 8 gene expression,the 1.4 kb (-1407 - +7) promoter region of Stra 8 gene was amplified from mouse genomic DNA. The DNA fragment was then cloned into a promoter less vector to form the construct that contained the 1.4 kb promoter region, and the reporter gene of EGFP that was regulated by 1.4kb Stra 8 promoter. To investigate the specificity of Stra 8 promoter,the vector pStro-EGFP was transfected into undifferentiated mouse stem cells such as ES-129, bone marrow mesenchymal stem cell (mMSC) and spermatogonial stem cell (mSSC). The results showed that the expression of GFP was only observed in the mSSC cells,which indicated that Stra 8 gene was specially regulated in testis tissue. As the gene marker,vector pStra8-EGFP was then transfected to undifferentiated mMSC cells. After being selected by G418 for 2 weeks,the mMSC cells were induced by retinoic acid. After 12 hours induction, some induced cells started to express GFP protein, which was observed under the fluorescence microscope. At the same time, several stem cell specificity biomarkers such as Oct4, and spermatogonial stem cell biomarkers such as CyclinA2 and Stra 8 were detected in the induced cells by RT-PCR method. These results showed that the mMSCs would differentiated to spermatognial stem cells after induced by Retinoic Acid.