In situ study on articular cartilage degeneration in simulated microgravity by HOF-ATR-FTIR spectroscopy.

Fourier transform infrared spectroscopy (FTIRS) can provide rich information on the composition and content of samples, enabling the detection of subtle changes in tissue composition and structure. This study represents the first application of FTIRS to investigate cartilage under microgravity. Simulated microgravity cartilage model was firstly established by tail-suspension (TS) for 7, 14 and 21 days, which would be compared to control samples. A self-developed hollow optical fiber attenuated total reflection (HOF-ATR) probe coupled with a FTIR spectrometer was used for the spectral acquisition of cartilage samples in situ, and one-way analysis of variance (ANOVA) was employed to analyze the changes in the contents of cartilage matrix at different stages. The results indicate that cartilage degenerates in microgravity, the collagen content gradually decreases with the TS time, and the structure of collagen fibers changes. The trends of proteoglycan content and collagen integrity show an initial decrease followed by an increase, ultimately significantly decreasing. The findings provide the basis for the cartilage degeneration in microgravity with TS time, which must be of real significance for space science and health detection.

The role of mice tails in response to external and self-generated balance perturbations on the roll plane.

Chordate tails exhibit considerable morphological and functional diversity, with variations in length, diameter and texture adapted to various ecological roles. While some animals, including humans, have lost or reduced their tails, many vertebrates retain and use their tails for activities such as balancing, climbing, and escaping predators. This study investigates how laboratory mice (Mus musculus) use their tails to maintain balance when dealing with external and self-generated perturbations. Mice crossed platforms of different widths, while responding to roll-plane tilts. Our findings show that mice swing their tails to counteract external roll perturbations, generating angular momentum to stabilize themselves. Mice were also found to use active (dynamic stabilizer) and passive (counterweight) tail movement strategies when locomoting on narrow platforms. The results suggest that the tail is a core component of mouse locomotion, especially in challenging balancing conditions.

Inferring the metabolic rate of zebrafish from ventilation frequency.

Fish schooling has attracted the interest of the scientific community for centuries. Energy savings have been long posited to be a key determinant for the emergence of schooling patterns. Yet, current methodologies do not allow the precise quantification of the metabolic rate of specific individuals within the school, typically leaving researchers with only a single, global measurement of metabolic rate for the collective. In this paper, we demonstrate the feasibility of inferring metabolic rate of swimming fish using the mouth-opening frequency, a simple proxy that can be scored utilizing video recordings in the laboratory or in the field, even for small fish. The mouth-opening frequency is independent of hydrodynamic interactions within the school, thereby mitigating potential confounding factors that arise when using locomotory measures associated with tail-beat motion. We assessed the reliability of mouth-opening frequency as a proxy for metabolic rate by conducting experiments on zebrafish (Danio rerio) using swimming respirometry. We varied the flow speed from 0.8 to 3.2 body lengths per second and extracted tail-beat motion and mouth opening from video recordings. Our results revealed a strong correlation between oxygen uptake and mouth-opening frequency for nonzero flow speeds but not in quiescent water. Contrary to our expectations, we did not find evidence in favor of the use of tail-beat frequency as a proxy for metabolic rate. Overall, our results open the door to the study of individual metabolic rates in fish schools without confounding factors related to hydrodynamic interactions.

Diverse Head-to-Tail Sequences in the Circular Genome of Human Bocavirus Genotype 1 among Children with Acute Respiratory Infections Implied the Switch of Template Chain in the Rolling-Circle Replication Model.

Head-to-tail sequences have been reported in human bocavirus (HBoV) 1-4. To reveal their features and functions, HBoV DNA was screened among respiratory specimens from pediatric patients with an acute respiratory infection (ARI) between April 2020 and December 2022, followed by HBoV genotyping. Head-to-tail sequences were detected using nested PCR, TA cloning, and Sanger sequencing, and these findings were confirmed by mNGS and amplicon sequencing. The secondary structure was predicted using the Mfold web server. The results indicated that head-to-tail sequences were detected in 42 specimens through TA cloning from 351 specimens positive for HBoV1 DNA, yielding 92 sequences into 32 types and 2 categories. Additionally, head-to-tail sequences were detected in 16 specimens by amplicon sequencing, yielding 60 sequences categorized into 23 types. The 374nt type, detected in 13 specimens, contains variants 374a and 374b, which differ in the unpaired loop regions of the palindrome or complementary reverse sequences, implying a switch of template chains during the replication process. The mNGS results in three specimens confirmed the presence of circular genome in copies below 1%. In conclusion, head-to-tail sequences of HBoV1 were common in children with ARI and were highly diverse in length and sequences. The variants may be generated by the switch of the template chain in the rolling-circle replication model.

Rearing conditions (isolated versus group rearing) affect rotenone-induced changes in the behavior of zebrafish (Danio rerio) embryos in the coiling assay.

Under regulations such as REACH, testing of novel and established compounds for their (neuro)toxic potential is a legal requirement in many countries. These are largely based on animal-, cost-, and time-intensive in vivo models, not in line with the 3 Rs' principle of animal experimentation. Thus, the development of alternative test methods has also received increasing attention in neurotoxicology. Such methods focus either on physiological alterations in brain development and neuronal pathways or on behavioral changes. An example of a behavioral developmental neurotoxicity (DNT) assay is the zebrafish (Danio rerio) embryo coiling assay, which quantifies effects of compounds on the development of spontaneous movement of zebrafish embryos. While the importance of embryo-to-embryo contact prior to hatching in response to environmental contaminants or natural threats has been documented for many other clutch-laying fish species, little is known about the relevance of intra-clutch contacts for zebrafish. Here, the model neurotoxin rotenone was used to assess the effect of grouped versus separate rearing of the embryos on the expression of the coiling behavior. Some group-reared embryos reacted with hyperactivity to the exposure, to an extent that could not be recorded effectively with the utilized software. Separately reared embryos showed reduced activity, compared with group-reared individuals when assessing. However, even the control group embryos of the separately reared cohort showed reduced activity, compared with group-reared controls. Rotenone could thus be confirmed to induce neurotoxic effects in zebrafish embryos, yet modifying one parameter in an otherwise well-established neurotoxicity assay such as the coiling assay may lead to changes in behavior influenced by the proximity between individual embryos. This indicates a complex dependence of the outcome of behavior assays on a multitude of environmental parameters.

Aggression to Biomembranes by Hydrophobic Tail Chains under the Stimulus of Reductant.

Stimulus-responsive materials hold significant promise for antitumor applications due to their variable structures and physical properties. In this paper, a series of peptides with a responsive viologen derivative, Pep-CnV (n = 1, 2, 3) were designed and synthesized. The process and mechanism of the interaction were studied and discussed. An ultraviolet-visible (UV) spectrophotometer and fluorescence spectrophotometer were used to study their redox responsiveness. Additionally, their secondary structures were measured by Circular Dichroism (CD) in the presence or absence of the reductant, Na2SO3. DPPC and DPPG liposomes were prepared to mimic normal and tumor cell membranes. The interaction between Pep-CnV and biomembranes was investigated by the measurements of surface tension and cargo leakage. Results proved Pep-CnV was more likely to interact with the DPPG liposome and destroy its biomembrane under the stimulus of the reductant. And the destruction increased with the length of the hydrophobic tail chain. Pep-CnV showed its potential as an intelligent antitumor agent.

Oncogenic plasmid DNA and liver injury agent dictates liver cancer development in a mouse model.

Primary liver cancer is an increasing problem worldwide and is associated with significant mortality. A popular method of modeling liver cancer in mice is plasmid hydrodynamic tail vein injection (HTVI). However, plasmid-HTVI models rarely recapitulate the chronic liver injury which precedes the development of most human liver cancer. We sought to investigate how liver injury using thioacetamide contributes to the pathogenesis and progression of liver cancer in two oncogenic plasmid-HTVI-induced mouse liver cancer models. Fourteen-week-old male mice received double-oncogene plasmid-HTVI (SB/AKT/c-Met and SB/AKT/NRas) and then twice-weekly intraperitoneal injections of thioacetamide for 6 weeks. Liver tissue was examined for histopathological changes, including fibrosis and steatosis. Further characterization of fibrosis and inflammation was performed with immunostaining and real-time quantitative PCR. RNA sequencing with pathway analysis was used to explore novel pathways altered in the cancer models. Hepatocellular and cholangiocellular tumors were observed in mice injected with double-oncogene plasmid-HTVI models (SB/AKT/c-Met and SB/AKT/NRas). Thioacetamide induced mild fibrosis and increased alpha smooth muscle actin-expressing cells. However, the combination of plasmids and thioacetamide did not significantly increase tumor size, but increased multiplicity of small neoplastic lesions. Cancer and/or liver injury up-regulated profibrotic and proinflammatory genes while metabolic pathway genes were mostly down-regulated. We conclude that the liver injury microenvironment can interact with liver cancer and alter its presentation. However, the effects on cancer development vary depending on the genetic drivers with differing active oncogenic pathways. Therefore, the choice of plasmid-HTVI model and injury agent may influence the extent to which injury promotes liver cancer development.

Detection of Trypanosoma cruzi DNA in lizards: Using non-lethal sampling techniques in a sylvatic species with zoonotic reservoir potential in Chile.

Several reptile species have been described as hosts of Trypanosoma cruzi, the causative agent of Chagas disease, and therefore, they have become vertebrates of epidemiological interest. In recent decades, there has been a growing interest in animal welfare, especially in populations with small numbers where lethal sampling could have catastrophic consequences, and non-lethal methodologies have been developed for detecting zoonotic parasites. In this study, we compared three non-lethal sampling methodologies for detecting T. cruzi DNA in 21 captured specimens of the native lizard Liolaemus monticola, collected from the semiarid Mediterranean ecosystem of Chile. Specimens were subjected to xenodiagnosis (XD), tail clipping, and living syringe sampling procedures to evaluate whether lizards could serve as sentinel species for T. cruzi in endemic regions. To detect the protozoan, real-time PCR (qPCR) was performed on the DNA extracted from the samples (intestinal contents, tail tissues, and blood from living syringes). Trypanosoma cruzi DNA was detected in 12 of 21 lizards, considering all three methodologies. By XD, 12 specimens showed infection (57.1 %), and both living syringe and tail sampling methodologies detected only one infected lizard (4.8 %). Therefore, T. cruzi can be detected in lizards by qPCR using the three methodologies but XD is by far the most effective non-lethal detection methodology. The use of tail and living syringe methodologies showed a large underestimation; however, they might be options for monitoring the presence of T. cruzi in lizard populations when large sample sizes are available.

Genetic analysis of key agronomic traits of local sheep breeds in Xinjiang, China.

The formation of sheep (Ovis aries) breeds is influenced by different ecological environments and populations with different living habits, resulting in the development of germplasm resources with stable genetic key agronomic traits. Thus, investigating the genetic mechanisms behind various agronomic traits can enhance the conservation and utilization of diverse sheep breeds. Here, we explored the sheep variome and selection signatures using the Ovine Infinium HD SNP BeadChip (600 K SNPs) from 23 sheep breeds, comprising a total of 1215 sheep. The genetic mechanisms of wool quality and tail morphology were analyzed by selective sweep and genome-wide association study. Based on the results of within-population selective sweep analysis, we performed gene network analysis and divided them into 6 gene communities. We identified genetic regions containing genes linked to sheep wool and tail, which have been and may continue to be important targets for breeding and selection. Furthermore, our results revealed the expression profiles of genes in these regions across different biological systems. Our study provides insights into categorizing sheep breeds into distinct gene communities, as well as references for constructing genetic network pathways related to key agronomic traits in sheep and other domestic animals.

Tuberculosis masquerading as a pancreatic cyst in a patient with cirrhosis.

Cystic lesions of the pancreas are being increasingly diagnosed incidentally in recent decades due to easy availability of abdominal imaging. They may be neoplastic or benign. Tuberculosis, the great masquerader, may rarely involve the pancreas mimicking pancreatic malignancy, cyst, or abscess. Identification of this rare entity is crucial to avoid potentially risky surgery as anti-tubercular pharmacotherapy is highly effective. We report a case of a 40-year old cirrhotic man who presented with a cystic hypodense lesion in the pancreatic tail and was subsequently diagnosed with pancreatic tuberculosis. Apart from the rarity of diagnosis, our case also highlights the utility of endoscopic ultrasound for minimally invasive assessment of pancreatic lesions.

Expression pattern and functional analysis of kinesin-14 KIFC1 in spermatogenesis of Macaca mulatta.

C-terminal kinesin motor KIFC1 is increasingly concerned with an essential role in germ cell development. During the spermatogenesis of mice, rats, and crustaceans, KIFC1 functions in regulating meiotic chromosome separation, acrosome vesicle transportation, and nuclear morphology maintenance. The expression pattern of KIFC1 is conservatively concentrated at the acrosome and nucleus of haploid sperm cells. However, whether KIFC1 has similar functions in non-human primates remains unknown. In this study, we constructed the testis-specific cDNA library and cloned different transcripts of KIFC1 based on the genomic sequence. New variants of KIFC1 were identified, and showed different functional domains from the predicted isoforms. The spatio-temporal expression of KIFC1 proteins in seminiferous tubules of rhesus monkeys showed an obvious nuclear localization, specifically expressed in the spermatocytes and early haploid spermatids. The transcripts of KIFC1 also exhibited considerable expression in the nucleus of rhesus LLC-MK2 cells. Besides, we demonstrated that KIFC1 located at the acrosome and microtubule flagella of the mature sperm, and KIFC1 inhibition resulted in sperm tail deformation as well as increased the instability of head-to-tail connection. In summary, this study filled a gap in the reproductive research of the KIFC1 gene in non-human primates.

Co-composting of tail vegetable with flue-cured tobacco leaves: analysis of nitrogen transformation and estimation as a seed germination agent for halophyte.

Resource utilization of tail vegetables has raised increasing concerns in the modern agriculture. However, the effect and related mechanisms of flue-cured tobacco leaves on the product quality, phytotoxicity and bacterially-mediated nitrogen (N) transformation process of tail vegetable composting were poorly understood. Amendments of high-dosed (5% and 10% w/w) tobacco leaves into the compost accelerated the heating process, prolonged the time of thermophilic stage, increased the peak temperature, thereby improving maturity and shortening composting duration. The tobacco leaf amendments at the 10% (w/w) increased the N conservation (TN and NH4-N content) of compost, due to the supply of N-containing nutrient and promotion of organic matter degradation by tobacco leaves. Besides, tobacco leaf amendments promoted the seed germination and root development of wild soybean, exhibiting the feasibility of composting product for promoting the growth of salt-tolerant plants, but no dose-dependent effect was found for tobacco leaf amendments. Addition of high dosed (5% and 10% w/w) tobacco leaves shifted the bacterial community towards lignocellulosic and N-fixing bacteria, contributing to increasing the compost maturity and N retention. PICRUSt 2 functional prediction revealed that N-related bacterial metabolism (i.e., hydroxylamine oxidation and denitrifying process) was enhanced in the tobacco leaf treatments, which contributed to N retention and elevated nutrient quality of composting. To the best knowledge, this was the first study to explore the effect of tobacco waste additives on the nutrient transformation and halophyte growth promotion of organic waste composting. These findings will deepen the understanding of microbially-mediated N transformation and composting processes involving flue-cured tobacco leaves.

Simulation of murine retinal hemodynamics in response to tail suspension.

The etiology of spaceflight-associated neuro-ocular syndrome (SANS) remains unclear. Recent murine studies indicate there may be a link between the space environment and retinal endothelial dysfunction. Post-fixed control (N = 4) and 14-day tail-suspended (TS) (N = 4) mice eye samples were stained and imaged for the vessel plexus and co-located regions of endothelial cell death. A custom workflow combined whole-mounted and tear reconstructed three-dimensional (3D) spherical retinal plexus models with computational fluid dynamics (CFD) simulation that accounted for the Fåhræus-Lindqvist effect and boundary conditions that accommodated TS fluid pressure measurements and deeper capillary layer blood flow distribution. TS samples exhibited reduced surface area (4.6 ± 0.5 mm2 vs. 3.5 ± 0.3 mm2, P = 0.010) and shorter lengths between branches in small vessels (<10 µm, 69.5 ± 0.6 µm vs. 60.4 ± 1.1 µm, P < 0.001). Wall shear stress (WSS) and pressure were higher in TS mice compared to controls, particularly in smaller vessels (<10 µm, WSS: 6.57 ± 1.08 Pa vs. 4.72 ± 0.67 Pa, P = 0.034, Pressure: 72.04 ± 3.14 mmHg vs. 50.64 ± 6.74 mmHg, P = 0.004). Rates of retinal endothelial cell death were variable in TS mice compared to controls. WSS and pressure were generally higher in cell death regions, both within and between cohorts, but significance was variable and limited to small to medium-sized vessels (<20 µm). These findings suggest a link may exist between emulated microgravity and retinal endothelial dysfunction that may have implications for SANS development. Future work with increased sample sizes of larger species or spaceflight cohorts should be considered.

Integrative proteome and metabolome analyses reveal molecular basis of the tail resorption during the metamorphic climax of Nanorana pleskei.

During the metamorphosis of anuran amphibians, the tail resorption process is a necessary and crucial change. One subject that has received relatively little or no attention is the expression patterns of proteins and metabolites in the different tail portions during metamorphosis, especially in highland amphibians. The mechanisms of tail resorption in three portions (the tip, middle and root) of the tail were investigated in N. pleskei G43 tadpole based on two omics (proteomic and metabolomic). Integrin αVß3 was found to be high expressed in the distal portion of the tail, which could improve the sensitiveness to thyroid hormones in the distal portion of the tail. Muscle regression displayed a spatial pattern with stronger regression in distal and weaker one in proximal portion. Probably, this stronger regression was mainly performed by the proteases of proteasome from the active translation by ribosomes. The suicide model and murder model coexisted in the tail resorption. Meanwhile, fatty acids, amino acids, pyrimidine, and purine which derived from the breakdown of tissues can be used as building blocks or energy source for successful metamorphosis. Our data improved a better comprehension of the tail resorption mechanisms underlying the metamorphism of N. pleskei tadpole through identifying important participating proteins and metabolites.

Hypocrellin A against intrahepatic Cholangiocarcinoma via multi-target inhibition of the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is an aggressive and highly lethal cancer with an increasing incidence worldwide that lacks effective treatment regimens. Hypocrellin A (HA), a natural small compound isolated from S. bambusicola, has multiple biomedical activities, including antitumor activity. PURPOSE: We intended to investigate the therapeutic effects of HA on ICC and its potential mechanisms. METHODS: RBE and HuccT1 cell lines were utilized for in vitro experiments. CCK8 assay, colony formation analysis, RTCA, and immunofluorescence staining of ki67 were employed to evaluate the suppression effects of HA on proliferation. The inhibitory effects of HA on cell migration and invasion were evaluate through transwell and wound healing assays, and Hoechst 33,258 staining was performed to evaluate apoptosis. Additionally, we performed transcriptome sequencing and molecular docking for targeting identification, and immunoblotting and immunofluorescence of key molecules for validation. Two in vivo models, HuccT1 xenografts, and the primary ICC model (KRAS/P19/SB) established via hydrodynamic tail-vein injection were implemented. Multiplex immunohistochemistry (mIHC) was used to illustrate the multi-target inhibitory effects of HA. RESULTS: The IC50 values of HA against RBE and HuccT1 cells were 4.612 µM and 10.01 µM for 24 h, as determined through the CCK8 assay. Our results confirmed that HA significantly repressed the proliferation, migration, invasion, and promoted the apoptosis of ICC cells at low concentrations. Moreover, HA exerted its anti-cancer effects through multi-target inhibition of the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways. This inhibitory effect was rescued by Recilisib, an activator of the PI3K-AKT-mTOR pathway. Bioinformatics analysis of a multi-center RNA-Seq cohort (n = 90) demonstrated significant associations between these target pathways and the occurrence and poor prognosis of ICC. Animal studies suggested that HA strongly inhibited tumor growth in xenograft ICC models, and repressed the tumor number and size in the liver of primary ICC models by suppressing these three crucial pathways. CONCLUSION: HA, a novel natural small molecule, demonstrated promising therapeutic efficacy against ICC through its multi-target inhibitory effects on the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways. Moreover, it exhibited notable therapeutic benefits in a primary ICC model (KRAS/P19/SB), positioning it as a novel therapeutic agent for ICC.

Analyzing the expression of the transcriptome in adipose tissue of fat- and thin-tailed sheep.

Significant efforts have been made to understand how fat deposition in sheep tail is regulated in genetic, transcriptomic, physiologic, biochemical, and metabolic levels in order to elucidate the complex mechanisms underlying the energy storage, lipid metabolism in adipose tissue, adaptability to harsh environments, and evolutionary domestication. Through RNA-seq data analysis, we are able to compare the gene expression of fat-tailed sheep versus thin-tailed sheep breeds in an acceptable resolution at transcriptome level. The purpose of this study was to compare the transcriptomes of Ghezel (fat-tailed) and Zel (thin-tailed) sheep. Total RNA from subcutaneous and tail tissue samples from healthy lambs was sequenced (150b PE) to identify differentially expressed genes (DEGs) between the two mentioned tissues and between the Ghezel and Zel sheep breeds. Further downstream pathway and network analyses were conducted afterwards. The results uncovered the association of the most important DEGs such as CAV1, ALB, and SOCS3 with cellular signaling pathways of lipids metabolism. It seems that the SOCS3 gene plays an important role in the differential deposition of lipid in the tails of two phenotypically different sheep breeds. Although the detail of gene expression in the tail and subcutaneous tissues of two morphologically different breeds was decoded here, to fully understand how differential expression of the SOCS3 gene affects the fat synthesis, further studies are needed.

From sequence to function: Exploring biophysical properties of bacteriophage BFK20 lytic transglycosylase domain from the minor tail protein gp15.

Bacteriophages have evolved different mechanisms of infection and penetration of bacterial cell walls. In Siphoviridae-like viruses, the inner tail proteins have a pivotal role in these processes and often encode lytic protein domains which increase infection efficiency. A soluble lytic transglycosylase (SLT) domain was identified in the minor tail protein gp15 from the BFK20 bacteriophage. Six fragments containing this SLT domain with adjacent regions of different lengths were cloned, expressed and purified. The biophysical properties of the two best expressing fragments were characterized by nanoDSF and CD spectroscopy, which showed that both fragments had a high refolding ability of 90 %. 3D modeling indicated that the bacteriophage BFK20 SLT domain is structurally similar to lysozyme. The degradation activity of these SLT proteins was evaluated using a lysozyme activity assay. BFK20 might use its transglycosylase activity to allow efficient phage DNA entry into the host cell by degrading bacterial peptidoglycan.

Prognostic implication of extra-pancreatic organ invasion in resectable pancreas ductal adenocarcinoma in the pancreas tail.

OBJECTIVES: To assess the prognostic significance of extra-pancreatic organ invasion in patients with resectable pancreatic ductal adenocarcinoma (PDAC) in the pancreas tail. MATERIALS & METHODS: This retrospective study included patients with resectable PDAC in the pancreas tail who received upfront surgery between 2014 and 2020 at a tertiary institution. Preoperative pancreas protocol computed tomography (CT) scans evaluated tumor size, peripancreatic tumor infiltration, suspicious metastatic lymph nodes, and extra-pancreatic organ invasion. The influence of extra-pancreatic organ invasion, detected by CT or postoperative pathology, on pathologic resection margin status was evaluated using logistic regression. The impact on recurrence-free survival (RFS) was analyzed using multivariable Cox proportional hazard models (clinical-CT and clinical-pathologic). RESULTS: The study included 158 patients (mean age, 65 years ± 8.8 standard deviation; 93 men). Extra-pancreatic organ invasion identified by either CT (p = 0.92) or pathology (p = 0.99) was not associated with a positive resection margin. Neither CT (p = 0.42) nor pathological (p = 0.64) extra-pancreatic organ invasion independently correlated with RFS. Independent predictors for RFS included suspicious metastatic lymph node (hazard ratio [HR], 2.05; 95 % confidence interval [CI], 1.08-3.9; p = 0.03) on CT in the clinical-CT model, pathological T stage (HR, 2.97; 95 % confidence interval [CI], 1.39-6.35; p = 0.005 for T2 and HR, 3.78; 95 % CI, 1.64-8.76; p = 0.002 for T3) and adjuvant therapy (HR, 0.62; 95 % confidence interval [CI], 0.42-0.92; p = 0.02) in the clinical-pathologic model. CONCLUSION: Extra-pancreatic organ invasion does not independently influence pathologic resection margin status and RFS in patients with resectable PDAC in the pancreas tail after curative-intent resection; therefore, it should not be considered a high-risk factor.

Transgenic poplar (Populus davidiana×P. bolleana Loucne) expressing dsRNA of insect chitinase gene: lines identification and resistance assay.

Poplar is a valuable tree species that is distributed all over the world. However, many insect pests infest poplar trees and have caused significant damage. To control poplar pests, we transformed a poplar species, Populus davidiana × P. bolleana Loucne, with the dsRNA of the chitinase gene of a poplar defoliator, Clostera anastomosis (Linnaeus) (Lepidoptera: Notodontidae), employing an Agrobaterium-mediated approach. The transgenic plant has been identified by cloning the T-DNA flanking sequences using TAIL-PCR and quantifying the expression of the dsRNA using qPCR. The toxicity assay of the transgenic poplar lines was carried out by feeding the target insect species (C. anastomosis). The results showed that, in C. anastomosis, the activity of chitinase was significantly decreased, consistent with the expression on mRNA levels, and the larval mortality was significantly increased. These results suggested that the transgenic poplar of dsRNA could be used for pest control.

Intracellular Construction of Organelle-like Compartments Facilitates Metabolic Flux in Escherichia coli.

The formation of well-designed synthetic compartments or membraneless organelles for applications in synthetic biology and cellular engineering has aroused enormous interest. However, establishing stable and robust intracellular compartments in bacteria remains a challenge. Here, we use the structured DIX domains derived from Wnt signaling pathway components, more specifically, Dvl2 and Axin1, as building blocks to generate intracellular synthetic compartments in Escherichia coli. Moreover, the aggregation behaviors and physical properties of the DIX-based compartments can be tailored by genetically embedding a specific dimeric domain into the DIX domains. Then, a pair of interacting motifs, consisting of the aforementioned dimeric domain and its corresponding binding ligand, was incorporated to modify the client recruitment pattern of the synthetic compartments. As a proof of concept, the human milk oligosaccharide lacto-N-tetraose (LNT) biosynthesis pathway was selected as a model metabolic pathway. The fermentation results demonstrated that the co-compartmentalization of sequential pathway enzymes into intracellular compartments created by DIX domain, or by the DIX domain in conjunction with interacting motifs, prominently enhanced the metabolic flux and increased LNT production. These synthetic protein compartments may provide a feasible and effective tool to develop versatile organelle-like compartments in bacteria for applications in cellular engineering and synthetic biology.