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This systematic review and meta-analysis aimed to compare perioperative and oncologic outcomes in patients with pancreatic ductal adenocarcinoma (PDAC) treated with robotic-assisted surgery versus open laparotomy. The study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Randomized controlled trials (RCTs) and cohort studies up to June 15, 2024, were identified using PubMed, EMBASE, and Google Scholar. Additionally, reference lists of included studies, relevant review articles, and clinical guidelines were manually searched. The primary outcomes evaluated were length of stay, 90-day mortality, postoperative pancreatic fistula (POPF), and Post-pancreatectomy haemorrhage (PPH). Secondary outcomes included estimated blood loss, reoperation rate, lymph node yield, and operative time. The final analysis included 10 retrospective cohort studies involving 23,272 patients (2,179 robotic-assisted and 21,093 open surgery). There were no significant differences between the two procedures in terms of postoperative pancreatic fistula, Post-pancreatectomy haemorrhage, lymph node yield, and operative time. However, patients undergoing robotic-assisted surgery had shorter lengths of stay, lower 90-day mortality, and less estimated blood loss compared to those undergoing open surgery. The reoperation rate was higher for the robotic-assisted group. Robotic-assisted surgery for pancreatic ductal adenocarcinoma is safe and feasible. Compared to open surgery, it offers better perioperative and short-term oncologic outcomes, but with a higher risk of reoperation.
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Carcinoma Ductal Pancreático , Tempo de Internação , Pancreatectomia , Neoplasias Pancreáticas , Procedimentos Cirúrgicos Robóticos , Procedimentos Cirúrgicos Robóticos/métodos , Procedimentos Cirúrgicos Robóticos/efeitos adversos , Humanos , Carcinoma Ductal Pancreático/cirurgia , Carcinoma Ductal Pancreático/mortalidade , Neoplasias Pancreáticas/cirurgia , Neoplasias Pancreáticas/mortalidade , Pancreatectomia/métodos , Resultado do Tratamento , Tempo de Internação/estatística & dados numéricos , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Duração da Cirurgia , Fístula Pancreática/etiologia , Fístula Pancreática/epidemiologia , Perda Sanguínea Cirúrgica/estatística & dados numéricos , Reoperação/estatística & dados numéricos , Laparotomia/métodosRESUMO
BACKGROUND: Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1+ cells in the cranial sutures play an important role in bone growth. However, whether Gli1+ cells in facial sutures differentiate into bone under distraction force is unknown. METHODS: 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1+ cell lineage tracing model was used to observe the distribution of Gli1+ cells and explore the role of Gli1+ cells in facial bone remodeling. RESULTS: Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1+ cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2+ cells. Hedgehog signaling was upregulated in Gli1+ cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1+ cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1+ cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1+ cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1+ cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force. CONCLUSIONS: Our study indicates that the primary cilia of Gli1+ cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1+ cells in zygomaticomaxillary sutures.
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Diferenciação Celular , Cílios , Suturas Cranianas , Proteínas Hedgehog , Osteogênese , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco , Animais , Camundongos , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Osteogênese/fisiologia , Cílios/metabolismo , Suturas Cranianas/metabolismo , Camundongos Endogâmicos C57BL , Osteogênese por Distração/métodos , Proliferação de CélulasRESUMO
The developmental toxicity effects of neonicotinoid pesticides such as clothianidin have not been fully explored in agricultural applications. This is particularly noteworthy because such pesticides significantly impact the survival rates of invertebrates, with arthropod larvae being particularly vulnerable. This study aimed to address this research gap by specifically investigating the toxicological effects of clothianidin on the developmental stages of the larvae of the economically important aquaculture species Penaeus vannamei. In these experiments, shrimp eggs were exposed to seawater containing different concentrations of clothianidin beginning at N1, and each phase was observed and analyzed to determine its toxic impact on larval development. These results revealed that clothianidin induces an increase in deformity rates and triggers abnormal cell apoptosis. It also significantly reduced survival rates and markedly decreased body length and heart rate in the later stages of larval development (P3). Transcriptomic analysis revealed disruptions in larval DNA integrity, protein synthesis, and signal transduction caused by clothianidin. To survive prolonged exposure, larvae may attempt to maintain their viability by repairing cell structures and enhancing signal transduction mechanisms. This study offers the first empirical evidence of the toxicity of clothianidin to arthropod larvae, underscoring the impact of environmental pollution on aquatic health.
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Guanidinas , Inseticidas , Larva , Neonicotinoides , Penaeidae , Tiazóis , Animais , Larva/efeitos dos fármacos , Neonicotinoides/toxicidade , Guanidinas/toxicidade , Tiazóis/toxicidade , Inseticidas/toxicidade , Penaeidae/efeitos dos fármacos , Penaeidae/crescimento & desenvolvimento , Poluentes Químicos da Água/toxicidade , Apoptose/efeitos dos fármacosRESUMO
Cycloxaprid, a new neonicotinoid pesticide, poses ecological risks, particularly in aquatic environments, due to its unique action and environmental dispersal. This study investigated the ecotoxicological effects of various concentrations of cycloxaprid on Penaeus vannamei over 28 days. High cycloxaprid levels significantly altered shrimp physiology, as shown by changes in the hepatosomatic index and fattening. Indicators of oxidative stress, such as increased serum hemocyanin, respiratory burst, and nitric oxide, as well as decreased phenol oxidase activity, were observed. Additionally, elevated activities of lactate dehydrogenase, succinate dehydrogenase, and isocitrate dehydrogenase indicated disrupted energy metabolism in the hepatopancreas. Notably, analyses of the nervous system revealed marked disturbances in neural signaling, as evidenced by elevated acetylcholine, octopamine, and acetylcholinesterase levels. Transcriptomic analysis highlighted significant effects on gene expression and metabolic processes in the hepatopancreas and nervous system. This study demonstrated that cycloxaprid disrupts neural signaling and oxidative balance in P. vannamei, potentially affecting its growth, and provides key insights into its biochemical and transcriptomic toxicity in aquatic systems.
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Penaeidae , Poluentes Químicos da Água , Animais , Poluentes Químicos da Água/toxicidade , Penaeidae/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Neonicotinoides/toxicidade , Piridinas/toxicidade , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/metabolismo , Inseticidas/toxicidade , Compostos Heterocíclicos com 3 AnéisRESUMO
Dinotefuran, a neonicotinoid insecticide, may impact nontarget organisms such as Decapoda P. vannamei shrimp with nervous systems similar to insects. Exposing shrimp to low dinotefuran concentrations (6, 60, and 600 µg/L) for 21 days affected growth, hepatosomatic index, and survival. Biomarkers erythromycin-N-demethylase, alanine aminotransferase, and catalase increased in all exposed groups, while glutathione S-transferase is the opposite; aminopyrin-N-demethylase, malondialdehyde, and aspartate aminotransferase increased at 60 and 600 µg/L. Concentration-dependent effects on gut microbiota altered the abundance of bacterial groups, increased potentially pathogenic and oxidative stress-resistant phenotypes, and decreased biofilm formation. Gram-positive/negative microbiota changed significantly. Metabolite differences between the exposed and control groups were identified using mass spectrometry and KEGG pathway enrichment. N-acetylcystathionine showed potential as a reliable dinotefuran metabolic marker. Weighted correlation network analysis (WGCNA) results indicated high connectivity of cruecdysone in the metabolite network and significant enrichment at 600 µg/L dinotefuran. The WGCNA results revealed a highly significant negative correlation between two key metabolites, caldine and indican, and the gut microbiota within co-expression modules. Overall, the risk of dinotefuran exposure to non-target organisms in aquatic environments still requires further attention.
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Microbioma Gastrointestinal , Guanidinas , Nitrocompostos , Penaeidae , Animais , Penaeidae/genética , Penaeidae/metabolismo , Penaeidae/microbiologia , Neonicotinoides/toxicidade , Neonicotinoides/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Oxirredutases N-Desmetilantes/farmacologiaRESUMO
Genome walking, a molecular technique for obtaining unknown flanking genomic sequences from a known genomic sequence, has been broadly applied to determine transgenic sites, mine new genetic resources, and fill in chromosomal gaps. This technique has advanced genomics, genetics, and related disciplines. Here, an efficient and reliable genome walking technique, called primer extension refractory PCR (PER-PCR), is presented. PER-PCR uses a set of primary, secondary, and tertiary walking primers. The middle 15 nt of the primary walking primer overlaps with the 3' parts of the secondary and tertiary primers. The 5' parts of the three primers are heterologous to each other. The short overlap allows the walking primer to anneal to its predecessor only in a relaxed-stringency PCR cycle, resulting in a series of single-stranded DNAs; however, the heterologous 5' part prevents the creation of a perfect binding site for the walking primer. In the next stringent cycle, the target single strand can be extended into a double-stranded DNA molecule by the sequence-specific primer and thus can be exponentially amplified by the remaining stringent cycles. The nontarget single strand fails to be enriched due to the lack of a perfect binding site for any primer. PER-PCR was validated by extension into unknown flanking regions of the hyg gene in rice and the gadR gene in Levilactobacillus brevis CD0817. In summary, in this study, a new practical PER-PCR method was constructed as a potential alternative to existing genome walking methods.
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DNA , Genômica , Reação em Cadeia da Polimerase/métodos , Genômica/métodos , DNA de Cadeia SimplesRESUMO
The electrochemical reduction of CO2 to HCOOH is considered one of the most appealing routes to alleviate the energy crisis and close the anthropogenic CO2 cycle. However, it remains challenging to develop electrocatalysts with high activity and selectivity towards HCOOH in a wide potential window. In this regard, Ag/Bi2O2CO3 was prepared by an in situ electrochemical transformation from Ag/Bi2O3. The Ag/Bi2O2CO3 catalyst achieves a faradaic efficiency (FE) of over 90% for HCOOH in a wide potential window between -0.8 V and -1.3 V versus the reversible hydrogen electrode (RHE). Moreover, a maximum FE of 95.8% and a current density of 15.3 mA cm-2 were achieved at a low applied potential of -1.1 V. Density functional theory (DFT) calculations prove that the high catalytic activity of Ag/Bi2O2CO3 is ascribed to the fact that Ag can regulate the electronic structure of Bi, thus facilitating the adsorption of *OCHO and hindering the adsorption of *COOH. This work expands the in situ electrochemical derivatization strategy for the preparation of electrocatalysts.
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Here, we describe a protocol based on semi-site-specific primer PCR (3SP-PCR) to access unknown flanking DNA sequences. We specify the guidelines for designing primers for 3SP-PCR. We also describe experimental procedures for the 3SP-PCR, along with PCR product purification and subsequent sequencing and analysis. For complete details on the use and execution of this protocol, please refer to Wei et al.1.
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Genoma , Sequência de Bases , Reação em Cadeia da Polimerase/métodos , Primers do DNA/genéticaRESUMO
Previous studies on the efficacy of trans-sutural distraction osteogenesis (TSDO) to treat midface hypoplasia caused by cleft lip and palate (CLP) have mainly focused on objective measurements while ignoring the subjective feelings of patients. This study aimed to analyse the changes in and correlation between computed tomography (CT) measurements and FACE-Q scores in patients who underwent TSDO by performing a comprehensive evaluation from both objective and subjective perspectives. This retrospective study included 25 patients with an average age of 10.7 years who had midface hypoplasia caused by CLP and underwent TSDO between August 2018 and December 2022. The average follow-up time was 18.8 ± 7.7 months. Facial morphology and CT measurements, including A-CR, N-Aâ¥HR, the SNA angle and the L-ZA, indicated significant improvements in midface concavity (all p < 0.0001). All FACE-Q scores (except for facial function) exhibited a significant increase. The ΔA-CR, ΔN-Aâ¥HR, and ΔSNA angle were strongly correlated with specific aspects of the FACE-Q-Appearance items, including the ΔFACE-Q-Appearance of the cheeks (all p < 0.0001), the ΔFACE-Q-Appearance of the face (all p < 0.0001), the ΔFACE-Q-Appearance of the jaws (all p < 0.01), the ΔSatisfaction with decision (all p < 0.0001) and the ΔSatisfaction with outcome (all p < 0.001). However, the ΔA-CR, ΔN-Aâ¥HR, and ΔSNA were weakly correlated with other FACE-Q-Health-related quality of life and function items. These findings suggest that both CT findings and FACE-Q scores have their own emphases and advantages. It is necessary to establish an integrated curative effect evaluation model that combines FACE-Q scores with CT measurements to evaluate both the physical health and psychological status of patients.
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Fenda Labial , Fissura Palatina , Osteogênese por Distração , Humanos , Criança , Osteogênese por Distração/métodos , Fenda Labial/diagnóstico por imagem , Fenda Labial/cirurgia , Fissura Palatina/diagnóstico por imagem , Fissura Palatina/cirurgia , Estudos Retrospectivos , Qualidade de Vida , Tomografia Computadorizada por Raios X/métodos , Maxila/cirurgiaRESUMO
BACKGROUND: The flap closed-incisions healing after 3D-printed prosthesis implantation in Chronic Osteomyelitis with Soft Tissue Defects (COSTD) is critical. This study aimed to explore the safety and effectiveness of Negative Pressure Wound Therapy (NPWT) in promoting flap closed-incisions healing. METHODS: Retrospective analysis of clinical data was performed, including baseline, surgical and hospitalization information. The efficacy of NPWT was assessed by comparing the ASEPSIS scores, Visual Analogue Scale (VAS), Activity of Daily Living Scale (ADLS), and Lower Extremity Functional Scale (LEFS), as well as the major postoperative complications. RESULTS: The study included 20 patients, 13 received conventional dressing (Control group) and 7 received NPWT treatment (NPWT group). These two groups exhibited a notable disparity in the distribution of ASEPSIS scores, and the median scores were 24 in Control group and 9 in NPWT group (p = 0.001). Eight patients in the Control group experienced major incisional complications, including 7 cases of exudation, 3 cases of infection, 2 cases of non-healing, and 1 case of dehiscence, while none were observed in the NPWT group (p = 0.015). The VAS, ADLS, and LEFS scores were significantly improved in the NPWT group compared to the Control group (p = 0.003, 0.017, and 0.043, respectively). CONCLUSIONS: The study findings suggest that NPWT applied to the healing process of flap closed-incisions after 3D prosthesis implantation in patients with COSTD can reduce the occurrence of postoperative major complications and promote the recovery of lower limb function and daily activities, which should be recommended for clinical practice.
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Tratamento de Ferimentos com Pressão Negativa , Osteomielite , Humanos , Infecção da Ferida Cirúrgica/etiologia , Estudos Retrospectivos , Complicações Pós-Operatórias , Implantação de Prótese/efeitos adversos , Osteomielite/cirurgia , Osteomielite/complicações , Impressão TridimensionalRESUMO
BACKGROUND: Amputees suffer from symptomatic neuroma and phantom limb pain. Regenerative peripheral nerve interface (RPNI) has recently been regarded as an effective method to prevent neuroma after amputation. However, the verifications of RPNI efficacy are mostly based on subjective evaluation, lacking objective approaches. This study aims to unveil the effect of RPNI on preventing neuroma formation and provide evidence supporting the efficacy of RPNI based on ultrasound. METHODS: Amputees of lower limb at Peking University People's Hospital from July 2020 to March 2022 were analyzed retrospectively. The clinical data collected consisted of general information, pathology of primary disease, history of limb-salvage treatment, amputation level of nerve, pain scales such as the Numerical Rating Scale (NRS) and the Manchester Foot Pain and Disability Index (MFPDI). Three months after amputation, the transverse diameter, anteroposterior diameter, and cross-sectional area of neuromas in stump nerves at the end of residual limbs were measured using ultrasound and compared to adjacent normal nerves. RESULTS: Fourteen patients were enrolled in the study, including 7 in the traditional amputation group (TA group) and 7 in the RPNI group. There was no significant difference in basic information and amputation sites between the two groups. The NRS and MFPDI scores of patients in RPNI group were significantly lower than those in TA group, and decreased with the follow-up time increasing, indicating that RPNI could reduce symptomatic neuroma pain. The comparison of preoperative ultrasound and postoperative pathology showed ultrasound could reflect the size of neuroma in vivo. Independent-sample t tests indicated that the ratios of anteroposterior diameter, transverse diameter and area of the cross section of both the neuroma and adjacent normal nerve obtained via ultrasound were significantly reduced in the RPNI group. CONCLUSION: This study suggested that RPNI can effectively prevent the formation of symptomatic neuroma after amputation using ultrasound.
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Extremidade Inferior , Neuroma , Humanos , Estudos Retrospectivos , Incidência , Extremidade Inferior/diagnóstico por imagem , Extremidade Inferior/cirurgia , Amputação Cirúrgica , Neuroma/diagnóstico por imagem , Neuroma/etiologia , Neuroma/prevenção & controle , Dor , Nervos PeriféricosRESUMO
To investigate whether human umbilical cord mesenchymal stem cell-derived exosomes combined with gelatin methacryloyl (GelMA) hydrogel are beneficial in promoting healing of laser-injured skin wounds in mice. Supernatants of cultured human umbilical cord mesenchymal stem cells (HUC-MSCs) were collected to obtain human umbilical cord MSC-derived exosomes (HUC-MSCs-Exos), which were combined with GelMA hydrogel complex to treat a mouse fractional laser injury model. The study was divided into PBS group, EX (HUC-MSCs-Exos) group, GEL (GelMA hydrogel) group and EX+GEL (HUC-MSCs-Exos combined with GelMA hydrogel) group. The healing of laser-injured skin in each group was observed by gross view and dermatoscopy, and changes in skin structure, angiogenesis and proliferation-related indexes were observed during the healing process of laser-injured skin in each group. The results of the animal experiments showed that the EX and GEL groups alone and the EL+EX group exhibited less inflammatory response compared to the PBS group. The EX and GEL groups showed marked tissue proliferation and favourable angiogenesis, which promoted the wound healing well. The GEL+EX group had the most significant promotion of wound healing compared to the PBS group. qPCR results showed that the expression levels of proliferation-related factors, including KI67 and VEGF and angiogenesis-related factor CD31, were significantly higher in the GEL+EX group than in the other groups, with a time-dependent effect. The combination of HUC-MSCs-Exos and GelMA hydrogel is beneficial in reducing the early inflammatory response of laser-injured skin in mice and promoting its proliferation and angiogenesis, which in turn promotes wound healing.
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Exossomos , Células-Tronco Mesenquimais , Humanos , Camundongos , Animais , Hidrogéis/uso terapêutico , Exossomos/metabolismo , Cicatrização/fisiologia , Modelos Animais de Doenças , Cordão UmbilicalRESUMO
Gamma-aminobutyric acid (GABA) has positive effects on many physiological processes. Lactic acid bacterial production of GABA is a future trend. This study aimed to produce a sodium-ion-free GABA fermentation process for Levilactobacillus brevis CD0817. In this fermentation, both the seed and fermentation media used L-glutamic acid instead of monosodium L-glutamate as the substrate. We optimized the key factors influencing GABA formation, adopting Erlenmeyer flask fermentation. The optimized values of the key factors of glucose, yeast extract, Tween 80, manganese ion, and fermentation temperature were 10 g/L, 35 g/L, 1.5 g/L, 0.2 mM, and 30 °C, respectively. Based on the optimized data, a sodium-ion-free GABA fermentation process was developed using a 10-L fermenter. During the fermentation, L-glutamic acid powder was continuously dissolved to supply substrate and to provide the acidic environment essential for GABA synthesis. The current bioprocess accumulated GABA at up to 331 ± 8.3 g/L after 48 h. The productivity of GABA was 6.9 g/L/h and the molar conversion rate of the substrate was 98.1%. These findings demonstrate that the proposed method is promising in the fermentative preparation of GABA by lactic acid bacteria.
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To investigate the role of GLI1 on skin proliferation and neovascularization during skin expansion in mice. We constructed GLI1-cre/R26-Tdtomato and GLI1-cre/R26-mtmg gene-tagged skin expansion mouse models. Using a two-photon in vivo imaging instrument to observe the changes in the number and distribution of GLI1(+) cells during the expansion process and to clarify the spatial relationship between GLI1(+) cells and blood vessels during the expansion process. In vitro proliferation assays were performed to further validate the effects of SHH (sonic hedgehog) and its downstream component GLI1 on cell proliferation viability. Finally, qRT-PCR was used to verify the changes in proliferation, angiogenesis-related factors, SHH signalling pathway-related factors, and the role of GLI1 cells in the process of skin expansion in mice. The number of GLI1(+) cells increased during dilation and were attached to the outer membrane of the vessel. The epidermis was thickened and the dermis thinned after the dilated skin was taken, while the epidermal thickening was suppressed and the dermis became thinner after the GLI1 cells were inhibited. The non-inhibited group showed a significant increase in PCNA positivity with prolonged dilation compared to the GANT61(GLI specificity inhibitor) inhibited group; CD31 immunofluorescence showed a significant increase in the number of dilated skin vessels and a significant decrease in the number of vessels after treatment with GANT61 inhibitor. In vitro proliferation results showed that SHH signalling activator significantly increased the proliferation viability of GLI1(+) hair follicle mesenchymal stem cells, while GNAT61 significantly inhibited the proliferation viability of GLI1(+) hair follicle mesenchymal stem cells. GLI1 is necessary for proliferation and neovascularization in expansion skin of mice through activation of the SHH signalling pathway.
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Proteínas Hedgehog , Transdução de Sinais , Camundongos , Animais , Proteína GLI1 em Dedos de Zinco/genética , Proteínas Hedgehog/metabolismo , Proliferação de Células , Epiderme/metabolismoRESUMO
Here we describe a protocol for wristwatch PCR, an approach based on wristwatch-like structure formed between walking primers to obtain unknown flanks. We specify the criteria for designing wristwatch primers and gene-specific primers. We detail how to set wristwatch primer permutations to obtain personalized walking outcomes and improve walking efficiency. We describe experimental procedures for isolating a DNA of interest using three rounds of nested wristwatch PCR as well as the subsequent steps for DNA purification, cloning, and sequencing. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).1.
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DNA , Técnicas de Amplificação de Ácido Nucleico , Sequência de Bases , Reação em Cadeia da Polimerase/métodos , Primers do DNA , DNA/genéticaRESUMO
The efficacy of the available genome-walking methods is restricted by low specificity, high background, or composite operations. We herein conceived bridging PCR, an efficient genome-walking approach. Three primers with random sequences, inner walker primer (IWP), bridging primer (BP), and outer walker primer (OWP), are involved in bridging PCR. The BP is fabricated by splicing OWP to the 5'-end of IWP's 5'-part. A bridging PCR set is constituted by three rounds of amplification reactions, sequentially performed by IWP, BP plus OWP, and OWP, respectively pairing with three nested sequence-specific primers (SSP). A non-target product arising from IWP alone undergoes end-lengthening mediated by BP. This modified non-target product is a preferentially formed hairpin between the lengthened ends, instead of binding with shorter OWP. Meanwhile, a non-target product, triggered by SSP alone or SSP plus IWP, is removed by nested SSP. As a result, only the target DNA is accumulated. The efficacy of bridging PCR was validated by walking the gadA/R genes of Levilactobacillus brevis CD0817 and the hyg gene of rice.
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Genome-walking has been frequently applied to molecular biology and related areas. Herein, a simple but reliable genome-walking technique, termed semi-site-specific primer PCR (3SP-PCR), is presented. The key to 3SP-PCR is the use of a semi-site-specific primer in secondary PCR that partially overlaps its corresponding primary site-specific primer. A 3SP-PCR set comprises two rounds of nested amplification reactions. In each round of reaction, any primer is allowed to partially anneal to the DNA template once only in the single relaxed-stringency cycle, creating a pool of single-stranded DNAs. The target single-stranded DNA can be converted into a double-stranded molecule directed by the site-specific primer, and thus can be exponentially amplified by the subsequent high-stringency cycles. The non-target one cannot be converted into a double-strand due to the lack of a perfect binding site to any primer, and thus fails to be amplified. We validated the 3SP-PCR method by using it to probe the unknown DNA regions of rice hygromycin genes and Levilactobacillus brevis CD0817 glutamic acid decarboxylase genes.
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The eutrophication of coastal water has been a critical environmental problem in China's offshore areas. How to effectively assess the status of coastal waters is key for pollution treatment and environmental protection. In recent years, eutrophication-symptom-based and multi-indicator methods, termed "phase II" methods, have been gradually adopted to assess the eutrophication status in some coastal waters in China and have achieved success. The cumulative quantile is typically selected to determine the characteristic value of an indicator in "phase II" methods. The influence of small-scale damaged water bodies on eutrophication assessment may be exaggerated, which often leads to the overassessment of the eutrophication status. In this study, the area ratio method was integrated into the assessment of the estuarine trophic status (ASSETS) method in order to assess the eutrophication status of Xiamen Bay in 2016. The results indicated that, in 2016, the eutrophication status of Xiamen Bay coastal waters was moderate and exhibited spatiotemporal variation. The area ratio method can effectively reduce the effect of small-scale coastal waters with extremely high eutrophication on the overassessment of eutrophication at the broader scale, allowing the eutrophication status to be better reflected, even with limited observation data. The centralized distribution of pollution sources and poor hydrodynamic conditions are the main reasons for the aforementioned phenomenon. Controlling the pollution discharge from the Jiulong River in flood seasons is key to reducing eutrophication in Xiamen coastal waters.
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Baías , Monitoramento Ambiental , Monitoramento Ambiental/métodos , Eutrofização , Rios , Água , ChinaRESUMO
Various PCR-based genome-walking methods have been developed to acquire unknown flanking DNA sequences. However, the specificity and efficacy levels, and the operational processes, of the available methods are unsatisfactory. This work proposes a novel walking approach, termed differential annealing-mediated racket PCR (DAR-PCR). The key to DAR-PCR is the use of primer-mediated intra-strand annealing (ISA). An ISA primer consists of a 5' root homologous to the known sequence and a heterologous 3' bud. In the single low-stringency cycle, the ISA primer anneals to a site on an unknown region and extends towards the sequence-specific primer (SSP) 1 site, thereby forming a target single-stranded DNA bound by the SSP1 complement and the ISA primer. In the subsequent more stringent cycles, its complementary strand is accumulated, owing to the differential annealing between the moderate-stringency ISA primer and the high-stringency SSP1. The accumulation of this strand provides an opportunity for ISA mediated by the ISA primer root. A loop-back extension subsequent to ISA occurs, creating a racket-like DNA with the known region positioned at both ends of the unknown sequence. This DNA is exponentially amplified during the secondary PCR driven by an SSP pair inner to SSP1. DAR-PCR was validated as an efficient walking method by determining unknown flanking sequences in Lactobacillus brevis and Oryza sativa.
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Midfacial hypoplasia is a common maxillofacial deformity in patients with cleft lip and palate, which requires surgical treatment. However, trans-sutural distraction osteogenesis (TSDO) remains some disadvantages, including difficulty in accurate location of surgical path, prolonged operation time, and excess surgical bleeding. This study aimed to evaluate the application of an optical surgical navigation system (OSNS) developed for TSDO. Six consecutive patients with midfacial hypoplasia who required TSDO were included in the study. Preoperatively, a head computed tomography was performed, and the data were imported into Mimics software (version: 20.0.0.691, Materialise Inc, Belgium) to design a three-dimensional simulation of the surgical approach. TSDO was performed with the use of OSNS. The accuracy and results of the procedure were initially evaluated by comparing the preoperative and postoperative periods. The patients included five boys and one girl, with an average age of 10 years; five with postoperative cleft lip and palate, and one without combined cleft lip and palate. The surgical procedure was successful, with a postoperative follow-up of 4-5 months. All patients demonstrated good treatment results without complications. In conclusioin, OSNS-assisted TSDO can noninvasively correct midfacial dysplasia, improve surgical precision, reduce bleeding and obtain better clinical results. OSNS can guide the TSDO safely and effectively.