Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.

Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.

Genomic characterization and related functional genes of γ- poly glutamic acid producing Bacillus subtilis.

γ- poly glutamic acid (γ-PGA), a high molecular weight polymer, is synthesized by microorganisms and secreted into the extracellular space. Due to its excellent performance, γ-PGA has been widely used in various fields, including food, biomedical and environmental fields. In this study, we screened natto samples for two strains of Bacillus subtilis N3378-2at and N3378-3At that produce γ-PGA. We then identified the γ-PGA synthetase gene cluster (PgsB, PgsC, PgsA, YwtC and PgdS), glutamate racemase RacE, phage-derived γ-PGA hydrolase (PghB and PghC) and exo-γ-glutamyl peptidase (GGT) from the genome of these strains. Based on these γ-PGA-related protein sequences from isolated Bacillus subtilis and 181 B. subtilis obtained from GenBank, we carried out genotyping analysis and classified them into types 1-5. Since we found B. amyloliquefaciens LL3 can produce γ-PGA, we obtained the B. velezensis and B. amyloliquefaciens strains from GenBank and classified them into types 6 and 7 based on LL3. Finally, we constructed evolutionary trees for these protein sequences. This study analyzed the distribution of γ-PGA-related protein sequences in the genomes of B. subtilis, B. velezensis and B. amyloliquefaciens strains, then the evolutionary diversity of these protein sequences was analyzed, which provided novel information for the development and utilization of γ-PGA-producing strains.

Chromatin accessibility uncovers KRAS-driven FOSL2 promoting pancreatic ductal adenocarcinoma progression through up-regulation of CCL28.

BACKGROUND: The epigenetic mechanisms involved in the progression of pancreatic ductal adenocarcinoma (PDAC) remain largely unexplored. This study aimed to identify key transcription factors (TFs) through multiomics sequencing to investigate the molecular mechanisms of TFs that play critical roles in PDAC. METHODS: To characterise the epigenetic landscape of genetically engineered mouse models (GEMMs) of PDAC with or without KRAS and/or TP53 mutations, we employed ATAC-seq, H3K27ac ChIP-seq, and RNA-seq. The effect of Fos-like antigen 2 (FOSL2) on survival was assessed using the Kaplan-Meier method and multivariate Cox regression analysis for PDAC patients. To study the potential targets of FOSL2, we performed Cleavage Under Targets and Tagmentation (CUT&Tag). To explore the functions and underlying mechanisms of FOSL2 in PDAC progression, we employed several assays, including CCK8, transwell migration and invasion, RT-qPCR, Western blotting analysis, IHC, ChIP-qPCR, dual-luciferase reporter, and xenograft models. RESULTS: Our findings indicated that epigenetic changes played a role in immunosuppressed signalling during PDAC progression. Moreover, we identified FOSL2 as a critical regulator that was up-regulated in PDAC and associated with poor prognosis in patients. FOSL2 promoted cell proliferation, migration, and invasion. Importantly, our research revealed that FOSL2 acted as a downstream target of the KRAS/MAPK pathway and recruited regulatory T (Treg) cells by transcriptionally activating C-C motif chemokine ligand 28 (CCL28). This discovery highlighted the role of an immunosuppressed regulatory axis involving KRAS/MAPK-FOSL2-CCL28-Treg cells in the development of PDAC. CONCLUSION: Our study uncovered that KRAS-driven FOSL2 promoted PDAC progression by transcriptionally activating CCL28, revealing an immunosuppressive role for FOSL2 in PDAC.

Frequency and distribution of H1N1 influenza A viruses with oseltamivir-resistant mutations worldwide before and after the 2009 pandemic.

H1N1 influenza has brought serious threats to people's health and a high socioeconomic burden to society. Oseltamivir, a kind of neuraminidase (NA) inhibitor, is the second-generation specific drug that is broadly used currently. However, H1N1 influenza viruses have exhibited oseltamivir resistance in the past decades, which might be a hidden danger. To understand the frequency and distribution laws of oseltamivir-resistant viruses, we conducted a thorough and deep analysis of the available NA protein sequences of H1N1 influenza viruses worldwide from 1918 to 2020. The differences and similarities before and after 2009 were also considered since the dominant viruses changed in this period. Results showed that 3.76% of H1N1 viruses harbored oseltamivir resistance currently. Among various significative mutations, H274Y had the highest frequency of 3.30%, while the frequencies of the other mutations were far below this whether before or after 2009. The oseltamivir resistance was mainly found in three hosts, humans, swine, and avian. Different mutation sites could exhibit different distributions in each host. Our results showed that the resistance level reached a peak during the 2007-2008 influenza season and then quickly decreased in 2009. The resistance also displayed a global distribution. The densely populated countries usually had a high resistance level. However, frequent significative mutations were also found in some small countries. Our findings indicated the necessity of monitoring oseltamivir resistance around the world. The study could provide a unique perspective toward the cognition of viruses and facilitate the future study of both pandemic and drug development.

Helicobacter pylori inhibits autophagic flux and promotes its intracellular survival and colonization by down-regulating SIRT1.

Helicobacter pylori (H. pylori) is the strong risk factor for a series of gastric pathological changes. Persistent colonization of H. pylori leading to chronic infection is responsible for gastritis and malignancy. Autophagy is an evolutionary conserved process which can protect cells and organisms from bacterial infection. Here, we demonstrated that H. pylori infection induced autophagosome formation but inhibited autophagic flux. SIRT1, a class III histone deacetylase, was down-regulated at both mRNA and protein levels by H. pylori infection in gastric cells. Further investigation showed that the transcriptional factor RUNX3 accounted for down-regulation of SIRT1 in H. pylori-infected gastric cells. SIRT1 promoted autophagic flux in gastric cells and activation of SIRT1 restored the autophagic flux inhibited by H. pylori infection. Furthermore, SIRT1 exerted inhibitory effects on intracellular survival and colonization of H. pylori. And activation of autophagic flux in SIRT1-inhibited gastric cells could significantly reduce intracellular load of H. pylori. Moreover, the relationship between H. pylori infection and SIRT1 expression was identified in clinical specimen. Our findings define the importance of SIRT1 in compromised autophagy induced by H. pylori infection and bacterial intracellular colonization. These results provide evidence that SIRT1 can serve as a therapeutic target to eradicate H. pylori infection.

Distribution and evolution of H1N1 influenza A viruses with adamantanes-resistant mutations worldwide from 1918 to 2019.

H1N1 influenza is a kind of acute respiratory infectious disease that has a high socioeconomic and medical burden each year around the world. In the past decades, H1N1 influenza viruses have exhibited high resistance to adamantanes, which has become a serious issue. To understand the up-to-date distribution and evolution of H1N1 influenza viruses with adamantanes-resistant mutations, we conducted a deep analysis of 15875 M2 protein and 8351 MP nucleotides sequences. Results of the distribution analyses showed that 77.32% of H1N1 influenza viruses harbored-resistance mutations of which 73.52% were S31N, And the mutant variants mainly appeared in North America and Europe and H1N1 influenza viruses with S31N mutation became the circulating strains since 2009 all over the world. In addition, 80.65% of human H1N1 influenza viruses and 74.61% of swine H1N1 influenza viruses exhibited adamantanes resistance, while the frequency was only 1.86% in avian H1N1 influenza viruses. Studies from evolutionary analyses indicated that the avian-origin swine H1N1 influenza viruses replaced the classical human H1N1 influenza viruses and became the circulating strains after 2009; The interspecies transmission among avian, swine, and human strains over the past 20 years contributed to the 2009 swine influenza pandemic. Results of our study clearly clarify the historical drug resistance level of H1N1 influenza viruses around the world and demonstrated the evolution of adamantanes-resistant mutations in H1N1 influenza viruses. Our findings emphasize the necessity for monitoring the adamantanes susceptibility of H1N1 influenza viruses and draw attention to analyses of the evolution of drug-resistant H1N1 influenza variants.

Development and application of a TaqMan-based real-time PCR assay for specifically detecting feline astrovirus.

Feline astrovirus (FeAstV), an enteric RNA virus of recent concern that is associated with diarrheal illness in cats, has been described in several countries throughout the world. However, no scientific and sensitive diagnostic method against FeAstV was reported up to now. Here, we developed a specific, sensitive and repeatable TaqMan fluorescence quantitative PCR (qPCR) assay to investigate the prevalence of FeAstV in domestic cats from China, especially low copy numbers in clinical sample. Specific assay showed that no cross-reactivity was observed with other non-FeAstV cat-derivied pathogens, suggesting this method was highly specific for FeAstV. The lowest detection limit of this assay was 3.52 copies/µl, and 1000-times more sensitive than conventional PCR. Intra- and inter-assay variability was less than 1.72%, means a high degree of repeatability. A total of 578 clinical fecal samples were collected from northeast China, and were tested for FeAstV using our developed qPCR assay. 105 samples were positive for FeAstV with an overall prevalence of 18.17%. Moreover, a higher positive rate was found in cats with diarrhea (32.26%, 80/248) than that in asymptomatic cats (7.58%, 25/330), further demonstrating that FeAstV infection was associated with diarrhea in cats. In brief, our developed assay showed high specificity, sensitivity, reproducibility for detecting FeAstV, and can be used for clinical diagnosis and epidemiological investigation of FeAstV.

Development of a cross-priming isothermal amplification assay based on the glycoprotein B gene for instant and rapid detection of feline herpesvirus type 1.

A cross-priming isothermal amplification (CPA) assay was developed for detection of feline herpesvirus type 1 (FHV-1). In this assay, the target fragment of the FHV-1 glycoprotein B gene is amplified rapidly by Bst DNA polymerase at a constant temperature (63 °C, 45 min), using a simple thermostat. The assay had no cross-reactions with four types of feline viruses, and the detection limit was 100 copies/µl. The positive rate of clinical samples from CPA was 100% consistent with qPCR but higher than ordinary PCR, indicating its superiority to ordinary PCR. Visualization was achieved using SYBR Green I dye.

The evolution and molecular characteristics of H9N2 avian influenza viruses in Jiangxi of China.

To understand the evolution and molecular characteristics of Jiangxi H9N2 viruses, we isolated 17 viruses in 2011 and analyzed their characteristics. Phylogenetic analyses revealed that their hemagglutinin genes originate from JS/1/00-like sublineage, neuraminidase genes originate from BJ/94-like sublineage, PB1, PA, NP, and NS genes all come from SH/F/98-like sublineage, PB2 genes originate from ST/163/04-like sublineage, while M genes come from G1-like sublineage. Genotype analysis showed that our isolates were classified as genotype 57. Molecular analyses indicated that our strains contained specific sites characteristic of low-pathogenic viruses. The current study once again highlights the necessity for continued surveillance of novel H9N2 viruses.

Development and application of a multiplex PCR method for the simultaneous detection and differentiation of feline panleukopenia virus, feline bocavirus, and feline astrovirus.

A multiplex polymerase chain reaction (mPCR) assay was developed to detect and distinguish feline panleukopenia virus (FPV), feline bocavirus (FBoV) and feline astrovirus (FeAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of the three viruses and were used to specifically amplify targeted fragments of 237 bp from the VP2 gene of FPV, 465 bp from the NP1 gene of FBoV and 645 bp from the RdRp gene of FeAstV. The results showed that this mPCR assay was effective, because it could detect at least 2.25-4.04 × 104 copies of genomic DNA of the three viruses per µl, was highly specific, and had a good broad-spectrum ability to detect different genotypes of the targeted viruses. A total of 197 faecal samples that had been screened previously for FeAstV and FBoV were collected from domestic cats in northeast China and were tested for the three viruses using the newly developed mPCR assay. The total positive rate for these three viruses was 59.89% (118/197). From these samples, DNA from FPV, FBoV and FeAstV was detected in 73, 51 and 46 faecal samples, respectively. The mPCR testing results agreed with the routine PCR results with a coincidence rate of 100%. The results of this study show that this mPCR assay can simultaneously detect and differentiate FPV, FBoV and FeAstV and can be used as an easy, specific and efficient detection tool for clinical diagnosis and epidemiological investigation of these three viruses.

Complete genome sequence analysis of canine bocavirus 1 identified for the first time in domestic cats.

In this study, we investigated the presence of canine bocaviruses (CBoVs) in fecal samples from 105 cats with diarrhea and 92 asymptomatic cats in northeast China. One fecal sample, 17CC0312, collected from an asymptomatic cat, was found to be positive for canine bocavirus 1 (CBoV1). The nearly complete genome of this virus was cloned and sequenced. The viral genome was 5,069 nucleotides (nt) in length and combined four open reading frames (ORFs) in the order 5'-NS1-ORF4-NP1-VP1/VP2-3'. The 17CC0312 virus shared more than 90.3% nucleotide sequence identity with CBoV1 reference sequences and was placed within the CBoV1 group in a phylogenetic tree based on complete genome sequences. Further phylogenetic analysis based on the deduced amino acid sequence of the VP2 gene showed that this feline CBoV1 strain belongs to CBoV1 lineage 3. These data provide the first molecular evidence of the presence of CBoV1 in a domestic cat and suggest that cats might be carriers of CBoV1.

A potential high risk for fatty liver disease was found in mice generated after assisted reproductive techniques.

Abnormal gametogenesis and embryonic development may lead to poor health status of the offspring. The operations involved in the assisted reproductive technologies (ARTs) occur during the key stage of gametogenesis and early embryonic development. To assess the potential risk of abnormal lipid metabolism in the liver of adult ARTs offspring, two ARTs mice models derived from preimplantation genetic diagnosis (PGD group) and in vitro cultured embryos without biopsy (IVEM group) were constructed. And control mice were from in vivo naturally conceived (Normal group). The results showed that ARTs offspring had increased body weight and body fat content comparing to normal group. An increasing volume and amount of lipid droplets as well as lipid droplet fusion were found in the hepatocytes of ARTs mice, and a significantly increased liver TG content was also shown in the ARTs mice, which due to the increased TG synthesis and decreased TG transport in the liver. All the results indicated that the manipulations involved in ARTs might play an important role in the lipid accumulation of adult offspring. By analyzing the DNA methylation profiles of 7.5dpc embryos, we proposed that methylation deregulation of the genes related to liver development in ARTs embryos might contribute to the abnormal phenotype in the offspring. The study demonstrated that ARTs procedures have adverse effect on liver development which resulted in abnormal lipid metabolism and induced the potential high risk of fatty liver in adulthood.

The role of S-nitrosylation of kainate-type of ionotropic glutamate receptor 2 in epilepsy induced by kainic acid.

Epilepsy is a chronic brain disease affecting millions of individuals. Kainate receptors, especially kainate-type of ionotropic glutamate receptor 2 (GluK2), play an important role in epileptogenesis. Recent data showed that GluK2 could undergo post-translational modifications in terms of S-nitrosylation (SNO), and affect the signaling pathway of cell death in cerebral ischemia-reperfusion. However, it is unclear whether S-nitrosylation of GluK2 (SNO-GluK2) contributes to cell death induced by epilepsy. Here, we report that kainic acid-induced SNO-GluK2 is mediated by GluK2 itself, regulated by neuronal nitric oxide synthase (nNOS) and the level of cytoplasmic calcium in vivo and in vitro hippocampus neurons. The whole-cell patch clamp recordings showed the influence of SNO-GluK2 on ion channel characterization of GluK2-Kainate receptors. Moreover, immunohistochemistry staining results showed that inhibition of SNO-GluK2 by blocking nNOS or GluK2 or by reducing the level of cytoplasmic calcium-protected hippocampal neurons from kainic acid-induced injury. Finally, immunoprecipitation and western blotting data revealed the involvement of assembly of a GluK2-PSD95-nNOS signaling complex in epilepsy. Taken together, our results showed that the SNO-GluK2 plays an important role in neuronal injury of epileptic rats by forming GluK2-PSD95-nNOS signaling module in a cytoplasmic calcium-dependent way, suggesting a potential therapeutic target site for epilepsy.

Detection and genetic characterization of feline bocavirus in Northeast China.

BACKGROUND: Bocaviruses have been reported to cause respiratory tract infection and gastroenteritis in most animal species. In cats, different genotype bocaviruses have been identified in USA, Japan, Hong Kong and Portugal. However, the clear relationship between the clinical symptoms and FBoV infection is unknown, and the prevalence of FBoV and the distribution of FBoV genotypes in China are still unclear. RESULTS: In this study, 197 fecal samples from cats with diarrhea (n = 105) and normal cats (n = 92) were collected in different regions between January 2016 and November 2017 and investigated using PCR targeting different FBoV genotypes. Screening results showed that 51 of 197 samples (25.9%) were positive for FBoV, and a higher positive rate was observed in cats with diarrhea (33.3%, 35/105) than in normal cats (17.4%, 16/92). Of these FBoV-positive samples, 35 were identified as FBoV-1, 12 as FBoV-2 and 4 as coinfection of FBoV-1 and FBoV-2. A phylogenetic analysis based on partial NS1 gene indicated that 24 sequences from randomly selected FBoV-positive samples were divided into 2 different FBoV groups: FBoV-1 and FBoV-2. Furthermore, 6 strains were randomly selected, and the complete genome was sequenced and analyzed. These strains exhibited the typical genome organization of bocavirus and were closely related to FBoV. Two FBoV-2 identified strains shared high homologies with FBoV-2 reference strains based on the complete genome and entire encoding gene, but lower identities were exhibited in the NP1 and VP1 regions for the other 4 FBoV-1 identified strains compared with FBoV-1 reference strains. CONCLUSION: These findings demonstrate that genetically diverse FBoV-1 and FBoV-2 widely circulate in cats in Northeast China and that FBoV-1 is more prevalent. The high prevalence of FBoV in cats with diarrhea symptoms suggests that FBoV infection may be associated with diarrhea in cats.

Long-term effects of repeated superovulation on the uterus and mammary gland in rhesus monkeys.

PURPOSE: The aim of this study is to evaluate the effect of repeated controlled ovarian hyperstimulation (COH) on the structure and function of the uterus and mammary gland. METHODS: Three adult female rhesus monkeys were superovulated up to four times, and three spontaneously ovulating monkeys were used as controls. After a 5-year period, the uterus and mammary gland tissue samples were collected for examination of their structure and function. Further, the expression of certain tumor markers was examined to assess the cancer risk for each organ. RESULTS: Expression of Wnt7a (associated with the functional/developmental status of the uterus) was significantly decreased in the uterus of superovulated monkeys, and decreased expression of proliferation marker PCNA was found in uterine cells. Meanwhile, abnormal Golgi-derived secretory vesicles with an irregular shape were observed in the mammary glands of the superovulated monkeys, and decreased PCNA expression together with increased expression of caspase-3 (an apoptosis marker) was indicated in the mammary cells. The expression of tumor molecular markers of the uterus and mammary gland was not significantly different between the two groups. CONCLUSIONS: Repeated COH affects the expression of the uterine development-related gene several years later, and uterine cells exhibited a low proliferation status. The ultrastructure of the mammary gland epithelial cells was abnormal, and the cells exhibited both low proliferation and high apoptosis status. Cancer risk for these organs was not observed. Given that primates are the closest relatives of humans, the results obtained from this study provide more intuitive information for optimization of clinical COH.

miR-543 promotes gastric cancer cell proliferation by targeting SIRT1.

SIRT1, a class III histone deacetylase, exerts inhibitory effects on tumorigenesis and is downregulated in gastric cancer. However, the role of microRNAs in the regulation of SIRT1 in gastric cancer is still largely unknown. Here, we identified miR-543 as a predicted upstream regulator of SIRT1 using 3 different bioinformatics databases. Mimics of miR-543 significantly inhibited the expression of SIRT1, whereas an inhibitor of miR-543 increased SIRT1 expression. MiR-543 directly targeted the 3'-UTR of SIRT1, and both of the two binding sites contributed to the inhibitory effects. In gastric epithelium-derived cell lines, miR-543 promoted cell proliferation and cell cycle progression, and overexpression of SIRT1 rescued the above effects of miR-543. The inhibitory effects of miR-543 on SIRT1 were also validated using clinical gastric cancer samples. Moreover, we found that miR-543 expression was positively associated with tumor size, clinical grade, TNM stage and lymph node metastasis in gastric cancer patients. Our results identify a new regulatory mechanism of miR-543 on SIRT1 expression in gastric cancer, and raise the possibility that the miR-543/SIRT1 pathway may serve as a potential target for the treatment of gastric cancer.

Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice.

Blastomere biopsy is used in preimplantation genetic diagnosis; however, the long-term implications on the offspring are poorly characterized. We previously reported a high risk of memory defects in adult biopsied mice. Here, we assessed nervous function of aged biopsied mice and further investigated the mechanism of neural impairment after biopsy. We found that aged biopsied mice had poorer spatial learning ability, increased neuron degeneration, and altered expression of proteins involved in neural degeneration or dysfunction in the brain compared to aged control mice. Furthermore, the MeDIP assay indicated a genome-wide low methylation in the brains of adult biopsied mice when compared to the controls, and most of the genes containing differentially methylated loci in promoter regions were associated with neural disorders. When we further compared the genomic DNA methylation profiles of 7.5-days postconception (dpc) embryos between the biopsy and control group, we found the whole genome low methylation in the biopsied group, suggesting that blastomere biopsy was an obstacle to de novo methylation during early embryo development. Further analysis on mRNA profiles of 4.5-dpc embryos indicated that reduced expression of de novo methylation genes in biopsied embryos may impact de novo methylation. In conclusion, we demonstrate an abnormal neural development and function in mice generated after blastomere biopsy. The impaired epigenetic reprogramming during early embryo development may be the latent mechanism contributing to the impairment of the nervous system in the biopsied mice, which results in a hypomethylation status in their brains.

Characterization of the amantadine-resistant H5N1 highly pathogenic avian influenza variants isolated from quails in Southern China.

Highly pathogenic H5N1 avian influenza viruses have spread in poultry and wild birds in Asia, Europe, and Africa since 2003. To evaluate the role of quails in the evolution of influenza A virus, we characterized three H5N1 viruses isolated from quails (QA viruses) in southern China. Phylogenetic analysis indicated that three QA viruses derived from the A/goose/Guangdong/1/96-like lineage and most closely related to HA clade 4 A/chicken/Hong Kong/31.4/02-like viruses. Molecular analysis suggested that QA viruses and clade 4 H5N1 viruses carried consistent residue signatures, such as the characteristic M2 Ser31Asn amantadine-resistance mutation, implying a common origin of these viruses. As revealed by viral pathogenicity tests, these QA viruses could replicate in intranasally infected mice, but were not lethal to them, showing low pathogenicity in mammals. However, they killed all intravenously inoculated chickens, showing high pathogenicity in poultry. Results from amantadine sensitivity tests of wild-type QA viruses and their reverse genetic viruses demonstrated that all QA viruses were resistant to amantadine, and the M2 Ser31Asn mutation was determined as the most likely cause of the increased amantadine-resistance of H5N1 QA viruses. Our study confirmed experimentally that the amino acid at residue 31 in the M2 protein plays a major role in determining the amantadine-resistance phenotype of H5N1 influenza viruses. Our findings provide further evidence that quails may play important roles in the evolution of influenza A viruses, which raises concerns over possible transmissions of H5N1 viruses among poultry, wild birds, and humans.

Embedded 3D printing of UV-curable thermosetting composites with continuous fiber.

Extrusion-based 3D printing methods with in-nozzle impregnation mechanisms have been extensively employed in the fabrication of continuous fiber composites. This study presents an innovative embedded 3D printing technique that addresses significant challenges associated with existing methods. The technique utilizes a deposition nozzle to precisely write continuous fibers below the resin. A laser beam is directed onto the resin surface, which simultaneously cures the resin around the fiber bundle. The printing method demonstrates its advantages in producing high-quality composite samples with well-aligned fibers, minimized void density, and outstanding mechanical properties. More importantly, it introduces several capabilities that are highly desirable in the fabrication of contemporary composites, but unattainable with existing methods, including the dynamic control of fiber volume fractions and the ability to change matrix materials during printing. Furthermore, it enables the printing of filaments along curved pathways and printing of overhanging filaments for hollow features without support materials. The developed printing method exhibits versatility in working with different commercially available feedstock resins and reinforcement fibers. It is anticipated to be an impactful approach for the future development of thermosetting composites with diverse structural or multifunctional applications.

Characteristics and related factors of nonfatal injuries among adolescents and college students in Shenzhen City of China.

BACKGROUND: Injuries impact adolescents and young adults in unique ways. The purpose of this study was to determine the incidence rate of nonfatal injuries, and identify characteristics and risk factors for the injuries among adolescents and college students in Shenzhen, China. METHODS: A total of 4,138 students from 79 classes were selected using a purposive sampling method in 2010. The questionnaire included personal demographics, behavioral factors, and self-perceived agrypnia. Stepwise multivariate logistic regression models were used to explore the risk factors of injury. RESULTS: The annual incidence rate of nonfatal injuries was 13.5%. Injuries were significantly correlated with gender (boys vs. girls, adjusted odds ratio [OR], 1.58, 95% confidence interval [CI], 1.30-1.93) and self-perceived agrypnia (sometimes vs. no, adjusted OR, 1.64, 95% CI, 1.31-2.05; often vs. no, adjusted OR, 2.34, 95% CI, 1.74-3.14), attending PE class ( >2 classes/week vs. ≤ 2 classes/week, adjusted OR, 1.25, 95% CI, 1.04-1.51), sexual behaviors (yes vs. no, adjusted OR, 1.46, 95% CI, 1.03-2.07), physical fighting (yes vs. no, adjusted OR, 1.84, 95% CI, 1.49-2.28), alcohol consumption (yes vs. no, adjusted OR, 1.29, 95% CI, 1.06-1.59), unsafe cycling (yes vs. no, adjusted OR, 1.47, 95% CI, 1.20-1.80) and skating in unsafe places (yes vs. no, adjusted OR, 1.57, 95% CI, 1.10-2.24). Additionally, falls were the leading cause of injuries, and gymnasiums of schools were the most-reported places where injuries occurred. CONCLUSIONS: Nonfatal injuries have turned into a pressing public health problem among adolescents and college students in Shenzhen, China. Strategies targeting the risk factors may be effective for the prevention of injuries.