Identification of novel mammalian viruses in tree shrews ( Tupaia belangeri chinensis).

The Chinese tree shrew ( Tupaia belangeri chinensis), a member of the mammalian order Scandentia, exhibits considerable similarities with primates, including humans, in aspects of its nervous, immune, and metabolic systems. These similarities have established the tree shrew as a promising experimental model for biomedical research on cancer, infectious diseases, metabolic disorders, and mental health conditions. Herein, we used meta-transcriptomic sequencing to analyze plasma, as well as oral and anal swab samples, from 105 healthy asymptomatic tree shrews to identify the presence of potential zoonotic viruses. In total, eight mammalian viruses with complete genomes were identified, belonging to six viral families, including Flaviviridae, Hepeviridae, Parvovirinae, Picornaviridae, Sedoreoviridae, and Spinareoviridae. Notably, the presence of rotavirus was recorded in tree shrews for the first time. Three viruses - hepacivirus 1, parvovirus, and picornavirus - exhibited low genetic similarity (<70%) with previously reported viruses at the whole-genome scale, indicating novelty. Conversely, three other viruses - hepacivirus 2, hepatovirus A and hepevirus - exhibited high similarity (>94%) to known viral strains. Phylogenetic analyses also revealed that the rotavirus and mammalian orthoreovirus identified in this study may be novel reassortants. These findings provide insights into the diverse viral spectrum present in captive Chinese tree shrews, highlighting the necessity for further research into their potential for cross-species transmission.

Characterization of long-term ex vivo expansion of tree shrew spermatogonial stem cells.

Tree shrews ( Tupaia belangeri chinensis) share a close relationship to primates and have been widely used in biomedical research. We previously established a spermatogonial stem cell (SSC)-based gene editing platform to generate transgenic tree shrews. However, the influences of long-term expansion on tree shrew SSC spermatogenesis potential remain unclear. Here, we examined the in vivo spermatogenesis potential of tree shrew SSCs cultured across different passages. We found that SSCs lost spermatogenesis ability after long-term expansion (>50 passages), as indicated by the failure to colonize the seminiferous epithelium and generate donor spermatogonia (SPG)-derived spermatocytes or spermatids marking spermatogenesis. RNA sequencing (RNA-seq) analysis of undifferentiated SPGs across different passages revealed significant gene expression changes after sub-culturing primary SPG lines for more than 40 passages on feeder layers. Specifically, DNA damage response and repair genes (e.g., MRE11, SMC3, BLM, and GEN1) were down-regulated, whereas genes associated with mitochondrial function (e.g., NDUFA9, NDUFA8, NDUFA13, and NDUFB8) were up-regulated after expansion. The DNA damage accumulation and mitochondrial dysfunction were experimentally validated in high-passage cells. Supplementation with nicotinamide adenine dinucleotide (NAD +) precursor nicotinamide riboside (NR) exhibited beneficial effects by reducing DNA damage accumulation and mitochondrial dysfunction in SPG elicited by long-term culture. Our research presents a comprehensive analysis of the genetic and physiological attributes critical for the sustained expansion of undifferentiated SSCs in tree shrews and proposes an effective strategy for extended in vitro maintenance.

Cognitive Deficits and Alzheimer's Disease-Like Pathologies in the Aged Chinese Tree Shrew.

Alzheimer's disease (AD) is the most common chronic progressive neurodegenerative disease in the elderly. It has an increasing prevalence and a growing health burden. One of the limitations in studying AD is the lack of animal models that show features of Alzheimer's pathogenesis. The tree shrew has a much closer genetic affinity to primates than to rodents and has great potential to be used for research into aging and AD. In this study, we aimed to investigate whether tree shrews naturally develop cognitive impairment and major AD-like pathologies with increasing age. Pole-board and novel object recognition tests were used to assess the cognitive performance of adult (about 1 year old) and aged (6 years old or older) tree shrews. The main AD-like pathologies were assessed by Western blotting, immunohistochemical staining, immunofluorescence staining, and Nissl staining. Our results showed that the aged tree shrews developed an impaired cognitive performance compared to the adult tree shrews. Moreover, the aged tree shrews exhibited several age-related phenotypes that are associated with AD, including increased levels of amyloid-ß (Aß) accumulation and phosphorylated tau protein, synaptic and neuronal loss, and reactive gliosis in the cortex and the hippocampal tissues. Our study provides further evidence that the tree shrew is a promising model for the study of aging and AD.

Rethinking neutrophil extracellular traps.

Neutrophils are a major subset of leukocytes in human circulating blood. In some circumstances, neutrophils release neutrophil extracellular traps (NETs). lnitially, NETs were considered to have a strong antibacterial capacity. However, currently, NETs have been shown to have a pivotal impact on various diseases. Different stimulators induce the production of different types of NETs, and their biological functions and modes of clearance do not appear to be the same. In this review, we will discuss several important issues related to NETs in order to better understand the relationship between NETs and diseases, as well as how to utilize the characteristics of NETs for disease treatment.

Pro-inflammatory microenvironment and systemic accumulation of CXCR3+ cell exacerbate lung pathology of old rhesus macaques infected with SARS-CoV-2.

Understanding the pathological features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in an animal model is crucial for the treatment of coronavirus disease 2019 (COVID-19). Here, we compared immunopathological changes in young and old rhesus macaques (RMs) before and after SARS-CoV-2 infection at the tissue level. Quantitative analysis of multiplex immunofluorescence staining images of formalin-fixed paraffin-embedded (FFPE) sections showed that SARS-CoV-2 infection specifically induced elevated levels of apoptosis, autophagy, and nuclear factor kappa-B (NF-κB) activation of angiotensin-converting enzyme 2 (ACE2)+ cells, and increased interferon α (IFN-α)- and interleukin 6 (IL-6)-secreting cells and C-X-C motif chemokine receptor 3 (CXCR3)+ cells in lung tissue of old RMs. This pathological pattern, which may be related to the age-related pro-inflammatory microenvironment in both lungs and spleens, was significantly correlated with the systemic accumulation of CXCR3+ cells in lungs, spleens, and peripheral blood. Furthermore, the ratio of CXCR3+ to T-box protein expression in T cell (T-bet)+ (CXCR3+/T-bet+ ratio) in CD8+ cells may be used as a predictor of severe COVID-19. These findings uncovered the impact of aging on the immunopathology of early SARS-CoV-2 infection and demonstrated the potential application of CXCR3+ cells in predicting severe COVID-19.

COVID-19-like symptoms observed in Chinese tree shrews infected with SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues to pose a global threat to the human population. Identifying animal species susceptible to infection with the SARS-CoV-2/ HCoV-19 pathogen is essential for controlling the outbreak and for testing valid prophylactics or therapeutics based on animal model studies. Here, different aged Chinese tree shrews (adult group, 1 year old; old group, 5-6 years old), which are close relatives to primates, were infected with SARS-CoV-2. X-ray, viral shedding, laboratory, and histological analyses were performed on different days post-inoculation (dpi). Results showed that Chinese tree shrews could be infected by SARS-CoV-2. Lung infiltrates were visible in X-ray radiographs in most infected animals. Viral RNA was consistently detected in lung tissues from infected animals at 3, 5, and 7 dpi, along with alterations in related parameters from routine blood tests and serum biochemistry, including increased levels of aspartate aminotransferase (AST) and blood urea nitrogen (BUN). Histological analysis of lung tissues from animals at 3 dpi (adult group) and 7 dpi (old group) showed thickened alveolar septa and interstitial hemorrhage. Several differences were found between the two different aged groups in regard to viral shedding peak. Our results indicate that Chinese tree shrews have the potential to be used as animal models for SARS-CoV-2 infection.

Studies of particle size distribution of Non-Exchangeable Organically Bound Tritium activities in the soil around Qinshan Nuclear Power Plant.

The NE-OBT (Non-Exchangeable Organically Bound Tritium) in the soil plays a significant role in tritium migration and transformation. In order to further understand the NE-OBT activity in the soil, the particle size, vertical profile and spatial distribution of the NE-OBT activities in the soil were determined around the Qinshan Nuclear Power Plant (NPP) in China. The experimental results indicated that the NE-OBT preferred to concentrate in the soil particle sizes of 53-250 µm within the soil depth of 5 cm-25 cm. The NE-OBT activity showed significantly vertical variations, however, its largest activity did not appear at the surface soil (0-5 cm). Meanwhile, the NE-OBT had a significant spatial distribution, its activity decreased with the increasing distance from the NPP, especially from the HWRs. In this study, the NE-OBT activities have no significant relationship to the organic matter content in the soil. But the vertical profile distribution of the NE-OBT activity has a strong correlation with the NE-OBT/HTO ratio in the soil, which reflect the capability of living organisms converting HTO into NE-OBT. According to these analyses, we supposed that the NE-OBT in the soil may be derived from the microbial transformation of HTO.

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis.

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Long-term propagation of tree shrew spermatogonial stem cells in culture and successful generation of transgenic offspring.

Tree shrews have a close relationship to primates and have many advantages over rodents in biomedical research. However, the lack of gene manipulation methods has hindered the wider use of this animal. Spermatogonial stem cells (SSCs) have been successfully expanded in culture to permit sophisticated gene editing in the mouse and rat. Here, we describe a culture system for the long-term expansion of tree shrew SSCs without the loss of stem cell properties. In our study, thymus cell antigen 1 was used to enrich tree shrew SSCs. RNA-sequencing analysis revealed that the Wnt/ß-catenin signaling pathway was active in undifferentiated SSCs, but was downregulated upon the initiation of SSC differentiation. Exposure of tree shrew primary SSCs to recombinant Wnt3a protein during the initial passages of culture enhanced the survival of SSCs. Use of tree shrew Sertoli cells, but not mouse embryonic fibroblasts, as feeder was found to be necessary for tree shrew SSC proliferation, leading to a robust cell expansion and long-term culture. The expanded tree shrew SSCs were transfected with enhanced green fluorescent protein (EGFP)-expressing lentiviral vectors. After transplantation into sterilized adult male tree shrew's testes, the EGFP-tagged SSCs were able to restore spermatogenesis and successfully generate transgenic offspring. Moreover, these SSCs were suitable for the CRISPR/Cas9-mediated gene modification. The development of a culture system to expand tree shrew SSCs in combination with a gene editing approach paves the way for precise genome manipulation using the tree shrew.

Early embryonic development and transplantation in tree shrews.

As a novel experimental animal model, tree shrews have received increasing attention in recent years. Despite this, little is known in regards to the time phases of their embryonic development. In this study, surveillance systems were used to record the behavior and timing of copulations; embryos at different post-copulation stages were collected and cultured in vitro; and the developmental characteristics of both early-stage and in vitro cultured embryos were determined. A total of 163 females were collected following effective copulation, and 150 were used in either unilateral or bilateral oviduct embryo collections, with 307 embryos from 111 females obtained (conception rate=74%). Among them, 237 embryos were collected from 78 females, bilaterally, i.e., the average embryo number per female was 3.04; 172 fertilized eggs collected from 55 females, bilaterally, were cultured for 24-108 h in vitro for developmental observations; finally, 65 embryos from 23 bilateral cases and 70 embryos from 33 unilateral cases were used in embryo transplantation.

An improved combustion apparatus for the determination of organically bound tritium in environmental samples.

This paper reports an improved combustion apparatus for the determination of organically bound tritium in environmental samples. The performance of this apparatus including the recovery rate and reproducibility was investigated by combusting lettuce and pork samples. To determine the factors for the different recovery rates of lettuce and pork and investigate whether the samples were completely oxidized, the ashes and exhaust gases produced by the combustion were analyzed. The results indicate that the apparatus showed an excellent performance in the combustion of environmental samples. Thus, the improvements conducted in this study were effective.

Preparation of lysine-coated magnetic Fe2O3 nanoparticles and influence on viability of A549 lung cancer cells.

OBJECTIVE: To explore the effect of lysine-coated oxide magnetic nanoparticles (Lys@MNPs) on viability and apoptosis of A549 lung cancer cells. METHODS: Transmission electron microscopy (TEM), vibrating sample magnetometer (VSM) and Zeta potentiometric analyzer were employed to characterize Lys@MNPs. Then Lys@MNPs and lung cancer A549 cells were co-cultured to study the effect of Lys@MNPs on cell viability and apoptosis. The pathway of Lys@MNPs entering A549 cells was detected by TEM and cell imaging by 1.5 T MRI. RESULTS: Lys@MNPs were 10.2 nm in grain diameter, characterized by small size, positive charge, and superparamagnetism. Under low-dose concentration of Lys@MNPs (<40 µg/mL), the survival rate of A549 cells was decreased but remained higher than 95% while under high-dose concentration (100 µg/mL), the survival ratewas still higher than 80%, which suggested Lys@MNPs had limited influence on the viability of A549 cells, with good biocompatibility and and no induction of apoptosis. Moreover, high affinity for cytomembranes, was demonstrated presenting good imaging effects. CONCLUSION: Lys@MNPs can be regarded as a good MRI negative contrast agents, with promising prospects in biomedicine.

[Phylogenetic relationships of seabuckthorn based on ITS sequences].

Sequences of the internal transcribed spacer (ITS) was compared in three seabuckthrons (Hippophae rhamnoides subsp. sinensis, H. tibetana and H. neurocarpa) distributed in Qinghai Province, then the systematic positions of 15 seabuckthron samples were analyzed with Elaeagnaceae angustifolia being outgroup. The results showed that ITS sequences in three seabuckthron species varied in length from 600 to 605 bp. ITS-1, 5.8S and ITS-2 varied from 201 to 203 bp, 166 to 167 bp and 232 to 236 bp, respectively. The sequence divergence among three seabuckthorn species was also remarkably high; Cluster analysis based on ITS indicated that H. gonicocarpa subsp. litangensis and H. gonicocarpa subsp. goniocarpa were distinct and could be classified as H. gonicocarpa and H. litangensis. H. gyantsensis had the closest genetic relationship with H. salicifolia and the distant relationship with H. rhamnoides subsp. sinensis. The positions of nine subspecies of H. rhamnoides based on morphological classification were different from the results of ITS.

A highly selective polymeric membrane barium(II) electrode based on a macrocyclic tetrabasic ligand as neutral carrier.

A novel barium(II) ion-selective polymeric membrane electrode based on a macrocyclic tetrabasic ligand that derived from the reaction of 4,6-diacetylresorcinol and ethylenediamine in the molar ratio 1:1 as neutral carrier was described. The electrode, with optimum membrane composition, exhibited an excellent near-Nernstian response for Ba(2+) ion ranging from 3.6 x 10(-6) to 1.0 x 10(-1) mol/L with a detection limit of 1.9 x 10(-6) mol/L and a slope of 29.7 +/- 0.2 mV/dec in pH 4.0 nitrate buffer solution at 25 degrees C. It had a relatively fast response time (10 s), good selectivity towards Ba(2+) ion, and could be used over a wide pH range of 2.5-7.5. Then, the response mechanism was discussed in view of UV-visible spectroscopy and the A.C. impedance technique. Finally, the proposed electrode was successfully applied to the detection of Ba(2+) ions in real samples as well as in sulfate(II) ions in water.

[Simultaneous determination of six main constituents in Swertia of Qinghai Province and Sichuan Province by HPLC].

OBJECTIVE: To establish a quantitative method of simultaneously determination of swertiamarin, gentiopicroside, mangiferin, swertianolin, isoorientin, 1,8-drihydroxy-3-methoxy-xthanone in Swertia from Qinghai province and Sichuan province by HPLC. METHOD: The samples were separated on the column of Kromasil C18 (4. 6 mm x 250 mm, 5 microm) which eluted with methanol and water (content 0.02% phosphoric acid). The ratio of methanol increased from 20% to 80% during 20-50 min, and from 80% to 100% during 50-60 min, with detected wavelength 254 nm, flow rate at 1 mL x min(-1), column temperature 35 degrees C. RESULT: Six compounds were base-isolated, the linear ranges of swertiamarin, gentiopicroside, mangiferin, 4-swertianolin, 5-isoorientin, 1,8-drihydroxy-3-methoxy-xthanone were excellent. CONCLUSION: The method was rapid and precise, and can be use for controlling medicinal materials quality.