Training nurses in an international emergency medical team using a serious role-playing game: a retrospective comparative analysis.

BACKGROUND: Although game-based applications have been used in disaster medicine education, no serious computer games have been designed specifically for training these nurses in an IEMT setting. To address this need, we developed a serious computer game called the IEMTtraining game. In this game, players assume the roles of IEMT nurses, assess patient injuries in a virtual environment, and provide suitable treatment options. METHODS: The design of this study is a retrospective comparative analysis. The research was conducted with 209 nurses in a hospital. The data collection process of this study was conducted at the 2019-2020 academic year. A retrospective comparative analysis was conducted on the pre-, post-, and final test scores of nurses in the IEMT. Additionally, a survey questionnaire was distributed to trainees to gather insights into teaching methods that were subsequently analyzed. RESULTS: There was a significant difference in the overall test scores between the two groups, with the game group demonstrating superior performance compared to the control group (odds ratio = 1.363, p value = 0.010). The survey results indicated that the game group exhibited higher learning motivation scores and lower cognitive load compared with the lecture group. CONCLUSIONS: The IEMT training game developed by the instructor team is a promising and effective method for training nurses in disaster rescue within IEMTs. The game equips the trainees with the necessary skills and knowledge to respond effectively to emergencies. It is easily comprehended, enhances knowledge retention and motivation to learn, and reduces cognitive load.

The effects of biochar derived from feedstock with different Si and Al concentration on soil N2O and CO2 emissions.

Desilicification and allitization is important characteristic of acidic soil. While decrease in soil silicon (Si) may generate Si limitation, the increase of aluminum (Al) will aggravate soil acidification. Biochar has been used in acid soil improvement, which could mitigate nitrous oxide (N2O) emissions and alter soil Si and Al concentration. However, the effect of biochar with different Si and Al concentration on greenhouse gas emissions remains unclear. We evaluated the effects of biochar derived from feedstock with different Si (moso bamboo leaves, BL; rice straw, RS) and Al (Camellia oleifera fruit shell, CFS; C. oleifera leaves, CL) concentration on greenhouse gas emissions and soil acidification. Microbial functional gene abundance associated with N2O emissions were measured to further explore the response of microbiological community. The results showed that BL, RS, CFS and CL significantly increased soil pH (by 19.2%, 16.7%, 18.7% and 24.9%, respectively), decreased soil exchangeable acid and exchangeable Al content, and reduced N2O emission rate of soil with nitrogen (N) (by 14.2%, 27.3%, 25.6% and 38.7%, respectively), which correlated with increase in soil nosZ abundance. BL, RS, CFS and CL increased soil nirK (by 325.6%, 66.7%, 155.8%, and 253.2%, respectively) and nosZ (by 198.6%, 174.1%, 72.2%, and 152.0%, respectively) abundance with N. Structural equation model showed that Si input via biochar application directly reduced N2O emissions, and soil acid-extractable Si is inversely proportional to N2O emission rate. In addition, Si input reduced carbon dioxide (CO2) emissions via indirect effects. Al input via biochar addition indirectly affected N2O and CO2 emissions through mainly indirect effects on other soil factors. In intensive management and production activities, Si-rich biochar can be considered instead of sole addition as fertilizer, which will be beneficial to the sustainable development of agricultural and forestry production in acid soil areas, and mitigation of global change.

Effects of Biofuel Crop Switchgrass (Panicum virgatum) Cultivation on Soil Carbon Sequestration and Greenhouse Gas Emissions: A Review.

Under the macroenvironmental background of global warming, all countries are working to limit climate change. Internationally, biofuel plants are considered to have great potential in carbon neutralization. Several countries have begun using biofuel crops as energy sources to neutralize carbon emissions. Switchgrass (Panicum virgatum) is considered a resource-efficient low-input crop that produces bioenergy. In this paper, we reviewed the effects of switchgrass cultivation on carbon sequestration and greenhouse gas (GHG) emissions. Moreover, the future application and research of switchgrass are discussed and prospected. Switchgrass has huge aboveground and underground biomass, manifesting its huge carbon sequestration potential. The net change of soil surface 30 cm soil organic carbon in 15 years is predicted to be 6.49 Mg ha-1, significantly higher than that of other crops. In addition, its net ecosystem CO2 exchange is about -485 to -118 g C m-2 yr-1, which greatly affects the annual CO2 flux of the cultivation environment. Nitrogen (N) fertilizer is the main source of N2O emission in the switchgrass field. Nitrogen addition increases the yield of switchgrass and also increases the N2O flux of switchgrass soil. It is necessary to formulate the most appropriate N fertilizer application strategy. CH4 emissions are also an important indicator of carbon debt. The effects of switchgrass cultivation on CH4 emissions may be significant but are often ignored. Future studies on GHG emissions by switchgrass should also focus on CH4. In conclusion, as a biofuel crop, switchgrass can well balance the effects of climate change. It is necessary to conduct studies of switchgrass globally with the long-term dimension of climate change effects.

Quantitative proteomics identified circulating biomarkers in lung adenocarcinoma diagnosis.

BACKGROUND: Lung cancer (LC) is a common malignant tumor with a high incidence and poor prognosis. Early LC could be cured, but the 5-year-survival rate for patients advanced is extremely low. Early screening of tumor biomarkers through plasma could allow more LC to be detected at an early stage, leading to a earlier treatment and a better prognosis. METHODS: This study was based on total proteomic analysis and parallel reaction monitoring validation of peripheral blood from 20 lung adenocarcinoma patients and 20 healthy individuals. Furthermore, differentially expressed proteins closely related to prognosis were analysed using Kaplan-Meier Plotter and receiver operating characteristic curve (ROC) curve analysis. RESULTS: The candidate proteins GAPDH and RAC1 showed the highest connectivity with other differentially expressed proteins between the lung adenocarcinoma group and the healthy group using STRING. Kaplan-Meier Plotter analysis showed that lung adenocarcinoma patients with positive ATCR2, FHL1, RAB27B, and RAP1B expression had observably longer overall survival than patients with negative expression (P < 0.05). The high expression of ARPC2, PFKP, PNP, RAC1 was observably negatively correlated with prognosis (P < 0.05). 17 out of 27 proteins showed a high area under the curve (> 0.80) between the lung adenocarcinoma and healthy plasma groups. Among those proteins, UQCRC1 had an area under the curve of 0.960, and 5 proteins had an area under the curve from 0.90 to 0.95, suggesting that these hub proteins might have discriminatory potential in lung adenocarcinoma, P < 0.05. CONCLUSIONS: These findings provide UQCRC1, GAPDH, RAC1, PFKP have potential as novel biomarkers for the early screening of lung adenocarcinoma.

Immunogenic Cell Death Activates the Tumor Immune Microenvironment to Boost the Immunotherapy Efficiency.

Tumor immunotherapy is only effective in a fraction of patients due to a low response rate and severe side effects, and these challenges of immunotherapy in clinics can be addressed through induction of immunogenic cell death (ICD). ICD is elicited from many antitumor therapies to release danger associated molecular patterns (DAMPs) and tumor-associated antigens to facilitate maturation of dendritic cells (DCs) and infiltration of cytotoxic T lymphocytes (CTLs). The process can reverse the tumor immunosuppressive microenvironment to improve the sensitivity of immunotherapy. Nanostructure-based drug delivery systems (NDDSs) are explored to induce ICD by incorporating therapeutic molecules for chemotherapy, photosensitizers (PSs) for photodynamic therapy (PDT), photothermal conversion agents for photothermal therapy (PTT), and radiosensitizers for radiotherapy (RT). These NDDSs can release loaded agents at a right dose in the right place at the right time, resulting in greater effectiveness and lower toxicity. Immunotherapeutic agents can also be combined with these NDDSs to achieve the synergic antitumor effect in a multi-modality therapeutic approach. In this review, NDDSs are harnessed to load multiple agents to induce ICD by chemotherapy, PDT, PTT, and RT in combination of immunotherapy to promote the therapeutic effect and reduce side effects associated with cancer treatment.

Transumbilical single-site two incision laparoscopic pyloromyotomy for pediatric hypertrophic pyloric stenosis.

PURPOSE: A new novel technique for the treatment of pediatric hypertrophic pyloric stenosis (HPS), transumbilical single-site laparoscopic pyloromyotomy with a single instrument (TUSSLP), was introduced. TUSSLP was compared with the transabdominal three-site laparoscopic pyloromyotomy (TATSLP) procedure. METHODS: Patients with HPS who underwent TUSSLP and TATSLP between January 2016 and September 2020 were assigned to group A and group B, respectively. The descriptive variables, perioperative clinical characteristics and postoperative follow-up results were retrospectively analyzed and compared between the 2 groups. The primary outcome of this study was the rate of switching to conventional pyloromyotomy. RESULTS: Sixty-four patients were enrolled in this study. Of these patients, 29 (22 males, 7 females, 54.4 ± 22.6 days) who received TUSSLP were assigned to group A. The remaining 35 (28 males, 7 females, 54.5 ± 27.6 days) who received TATSLP were assigned to group B. The data of preoperative patient variables were comparable between the 2 groups (P > 0.05). The mean operative time (ORT) was 28.1 ± 5.6 min in group A, which was not significantly different from 25.8 ± 3.1 min in group B (P = 0.25). The other perioperative features were not significantly different between the 2 groups (P > 0.05). During follow-up (39.1 ± 14.7 m in group A and 35.4 ± 16.1 m in group B, P = 0.51), no significant difference was observed in the overall incidence of vomiting between the 2 groups (P = 0.26). CONCLUSIONS: TUSSLP is a feasible and reliable minimally invasive method for HPS. It has the advantages of an improved cosmetic appearance. The postoperative follow-up results of TUSSLP are comparable with those of TATSLP.

Effect of mutations across reverse transcriptase region on HBV replication and progression of liver diseases in Chinese patients.

It was known that mutations in the RT region were mainly related to nucleot(s)ide analogs resistance. Increasing studies indicated that RT mutations were related to advanced liver diseases (ALD) and had effects on HBV replication, but the distribution characteristics of mutations across RT region in the development of liver diseases and the effect of RT mutations on HBV replication were not fully clarified. HBV RT region was direct-sequenced in 1473 chronic HBV-infected patients. Mutation frequencies were analyzed to identify the specific mutations differing between groups classified by genotypes, loads of HBV DNA, or progression of liver diseases. In the range of rt145-rt290, rt145, rt221, rt222, rt267, and rt271 were the genotype-polymorphic sites, while rt238 was the genotype-specific sites. Mutations at rt163, rt173, rt180, rt181, rt184, rt191, rt199, and rt214 were more frequent among patients with C-genotype HBV, while those at rt220, rt225, rt226, rt269, and rt274 were more frequent among patients with B-genotype HBV. RtM204V/I could reduce the HBV DNA loads while rtQ/L267H/R could increase the HBV DNA loads. RtV214A/E/I (OR 3.94, 95% CI 1.09 to 14.26) was an independent risk factor for advanced liver diseases. In summary, the hotspots of mutations were different between B and C genotypes. Besides the effect on the S region, RT mutations had effects on HBV replication by other unknown ways. RtV214A/E/I was found to be an independent risk factor for ALD, suggesting that mutations at rt214 site could be used as a potential virological marker for the liver disease progression.

Factors Associated with In-Patient Mortality in the Rapid Assessment of Adult Earthquake Trauma Patients.

OBJECTIVE: To date, there is limited evidence for health care providers regarding the determinants of early assessment of poor outcomes of adult in-patients due to earthquakes. This study aimed to explore factors related to early assessment of adult earthquake trauma patients (AETPs). METHODS: The data on 29,933 AETPs in the West China Earthquake Patients Database (WCEPD) were analyzed retrospectively. Then, 37 simple variables that could be obtained rapidly upon arrival at the hospital were collected. The least absolute shrinkage and selection operator (LASSO) regression analyses were performed. A nomogram was then constructed. RESULTS: Nine independent mortality-related factors that contributed to AETP in-patient mortality were identified. The variables included age (OR:1.035; 95%CI, 1.027-1.044), respiratory rate ([RR]; OR:1.091; 95%CI, 1.050-1.133), pulse rate ([PR]; OR:1.028; 95%CI, 1.020-1.036), diastolic blood pressure ([DBP]; OR:0.96; 95%CI, 0.950-0.970), Glasgow Coma Scale ([GCS]; OR:0.666; 95%CI, 0.643-0.691), crush injury (OR:3.707; 95%CI, 2.166-6.115), coronary heart disease ([CHD]; OR:4.025; 95%CI, 1.869-7.859), malignant tumor (OR:4.915; 95%CI, 2.850-8.098), and chronic kidney disease ([CKD]; OR:5.735; 95%CI, 3.209-10.019). CONCLUSIONS: The nine mortality-related factors for ATEPs, including age, RR, PR, DBP, GCS, crush injury, CHD, malignant tumor, and CKD, could be quickly obtained on hospital arrival and should be the focal point of future earthquake response strategies for AETPs. Based on these factors, a nomogram was constructed to screen for AETPs with a higher risk of in-patient mortality.

Teaching Disaster Evacuation Management Education to Nursing Students Using Virtual Reality Mobile Game-Based Learning.

The purpose of this study was to explore the effectiveness of a virtual reality mobile game-based application for teaching disaster evacuation management education to nursing students. A pre-test, post-test, and final-test study design was used to compare traditional lecture group and game group instructional knowledge delivery effectiveness and instructional mode satisfaction. The statistical comparison of pre-test and post-test knowledge and decision-making scores did not reveal significant group differences for short-term improvement ( P ≥ .05); however, final test scores revealed that the virtual reality mobile game-based application group had significantly higher knowledge and decision-making retention scores compared with the traditional lecture group ( P = .000). The game group also had significantly higher instructional mode satisfaction scores for course interest and cooperation with others ( P < .05). The virtual reality mobile game-based application was more effective for teaching nursing students about disaster evacuation management educational training than lecture instruction. The greater satisfaction expressed by nursing students when using this instructional mode suggests that it may better facilitate self-initiated lifelong disaster evacuation learning behaviors.

The Effect of Ferulic Acid-Grafted Chitosan (FA-g-CS) on the Transmembrane Transport of Anthocyanins by sGLT1 and GLUT2.

This work aims to evaluate the effect of ferulic acid-grafted chitosan (FA-g-CS) on the interaction between anthocyanin (ANC) and sGLT1/GLUT2 and their functions in ANC transmembrane transport using Caco-2 cells. The transmembrane transport experiments of ANC showed its low transport efficiency (Papp < 10-6 cm/s), whereas the phenomenon of a significantly rise in anthocyanins transport efficiency was observed with the incubation of FA-g-CS (p < 0.05). In order to investigate the mechanism of FA-g-CS improving ANC transmembrane transport, Caco-2 cells were transfected with small interfering RNA (siRNA) specific for transporters sGLT1 and GLUT2, and incubated with ANC, FA-g-CS, or their combination. Subsequently, Western blot analyses and immunofluorescence staining were carried out to monitor the intracellular sGLT1 and GLUT2 levels. These siRNA-transfected cells, incubated with compounds, indicate that sGLT1 and GLUT2 participated in the ANC transmembrane transport and that FA-g-CS, ANC, or their combination enhance sGLT1/GLUT2 expression. In particular, Caco-2 cells incubated with both FA-g-CS and ANC show significantly increased sGLT1 or GLUT2 expression (>80%) compared with exclusively using FA-g-CS or ANC (<60%). Molecular docking results demonstrate that there is a good binding between FA-g-CS/ANC and sGLT1 or GLUT2. These results highlight that FA-g-CS promotes the transmembrane transport of ANC by influencing the interaction between ANC and sGLT1/GLUT2; the interaction between FA-g-CS and ANC could be another key factor that improves the bioavailability of ANC.

Factors related to early and rapid assessment of in-hospital mortality among older adult trauma patients in an earthquake.

BACKGROUND: There is limited evidence for emergency physicians and emergency trauma surgeons regarding the determinants of early and rapid assessment of older adult in-hospital mortality due to earthquakes. This study explored factors related to the early and rapid assessment of the mortality among older adult earthquake trauma patients (OAETPs) and created a screening model. METHODS: Data on 7,308 OAETPs from the West China Earthquake Patients Database were analyzed retrospectively. The 35 variables that can be obtained rapidly on arrival at the hospital were collected. Least absolute shrinkage and selection operator (LASSO) regression analysis was performed. Then, the nomogram for assessing the mortality of OAETPs was constructed. RESULTS: We identified 10 independent mortality-related factors that contributed to the in-hospital mortality of OAETPs. The 10 factors included age (odds ratio [OR]=1.061, 95% confidence interval [CI]:1.031-1.090), dementia (OR=5.146, 95%CI: 1.169-17.856), coronary heart disease (CHD; OR=23.441, 95%CI: 4.799-83.927), malignant tumor (OR=8.497, 95%CI: 3.583-17.967), deep vein thrombosis (DVT; OR=7.110, 95%CI: 1.369-27.168), chronic kidney disease(CKD; OR=11.783, 95%CI: 5.419-24.407), pulse rate (PR; OR=1.036, 95%CI: 1.022-1.048), mean artery pressure (MAP; OR=0.960, 95%CI: 0.945-0.975), Glasgow Coma Scale (GCS; OR=0.864, 95%CI: 0.760-0.972), and Triage Revised Trauma Score (T-RTS, OR=0.485, 95%CI: 0.351-0.696). CONCLUSION: The 10 mortality-related factors could be quickly obtained on hospital arrival and should be the focal point of future earthquake response strategies regarding hospitalized older adults with trauma. A nomogram was constructed based on the factors for screening OAETPs with a higher risk of in-hospital mortality.

Improving Nursing Students' COVID-19 Knowledge Using a Serious Game.

Improving nursing students' understanding of the novel COVID-19 is an essential component of their education. We designed and developed a serious game-based computer learning application that requires nursing students to solve problems through simulated cases. This study aimed to compare knowledge mastery among nursing students who used this teaching method versus those who received online lectures. A retrospective observational study was conducted using preclass, postclass, and final test scores of 130 students retrieved from the university's database of test scores. Both teaching methods produced significant increases in short-term knowledge of COVID-19. There was no statistical difference between the two methods in pre- and postclass scores; however, the serious game group scored higher than the online lecture group in knowledge retention. In summary, the serious game application is a potentially effective method for COVID-19 education among nursing students, particularly in terms of its capacity for improved knowledge retention.

A rare case of synovial sarcoma with lung, heart and adrenal gland metastasis: a caution for patients and clinicians.

A 68-year-old man presented to the Urology Clinic, West China Hospital, Chengdu, with a suspected right adrenal gland mass that had persisted for two months. He had no associated lumbodynia, dizziness or palpitation. Abdominal computed tomography (CT) revealed an uneven density and contrast-enhanced oval-like mass with smooth edges in the right adrenal gland. Laparoscopic right adrenal gland resection followed by histopathology confirmed a diagnosis of metastatic synovial sarcoma. The patient had a history of synovial sarcoma on the right upper leg 3 years previously that was surgically treated, but he had not undergone further treatment. Approximately 1.5 years later, he had undergone surgery for heart and lung metastasis from the synovial sarcoma of the thigh. At 5 months following laparoscopic right adrenal gland resection, abdominal CT showed a significant sign of right adrenal recurrence, and targeted therapy of 12 mg oral anlotinib, daily, was initiated. This relatively rare but alarming case highlights the importance of patient understanding and compliance to treatment.

Mutational analysis of the T4 gp59 helicase loader reveals its sites for interaction with helicase, single-stranded binding protein, and DNA.

Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, whereas the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB.

The integrity of the periplasmic domain of the VirA sensor kinase is critical for optimal coordination of the virulence signal response in Agrobacterium tumefaciens.

The plant pathogen Agrobacterium tumefaciens responds to three main signals at the plant-bacterium interface: phenolics, such as acetosyringone (AS), monosaccharides, and acidic pH (∼5.5). These signals are transduced via the chromosomally encoded sugar binding protein ChvE and the Ti plasmid-encoded VirA/VirG two-component regulatory system, resulting in the transcriptional activation of the Ti plasmid virulence genes. Here, we present genetic and physical evidence that the periplasmic domain of VirA dimerizes independently of other parts of the protein, and we examine the effects of several engineered mutations in the periplasmic and transmembrane regions of VirA on vir-inducing capacity as indicated by AS sensitivity and maximal level of vir-inducing activity at saturating AS levels. The data indicate that helix-breaking mutations throughout the periplasmic domain of VirA or mutations that reposition the second transmembrane domain (TM2) of VirA relieve the periplasmic domain's repressive effects on the maximal activity of this kinase in response to phenolics, effects normally relieved only when ChvE, sugars, and low pH are also present. Such relief, however, does not sensitize VirA to low concentrations of phenolics, the other major effect of the ChvE-sugar and low pH signals. We further demonstrate that amino acid residues in a small Trg-like motif in the periplasmic domain of VirA are crucial for transmission of the ChvE-sugar signal to the cytoplasmic domain. These experiments provide evidence that small perturbations in the periplasmic domain of VirA can uncouple sugar-mediated changes in AS sensitivity from the sugar-mediated effects on maximal activity.

Arabidopsis UEV1D promotes Lysine-63-linked polyubiquitination and is involved in DNA damage response.

DNA damage tolerance (DDT) in budding yeast requires Lys-63-linked polyubiquitination of the proliferating cell nuclear antigen. The ubiquitin-conjugating enzyme Ubc13 and the Ubc enzyme variant (Uev) methyl methanesulfonate2 (Mms2) are required for this process. Mms2 homologs have been found in all eukaryotic genomes examined; however, their roles in multicellular eukaryotes have not been elucidated. We report the isolation and characterization of four UEV1 genes from Arabidopsis thaliana. All four Uev1 proteins can form a stable complex with At Ubc13 or with Ubc13 from yeast or human and can promote Ubc13-mediated Lys-63 polyubiquitination. All four Uev1 proteins can replace yeast MMS2 DDT functions in vivo. Although these genes are ubiquitously expressed in most tissues, UEV1D appears to express at a much higher level in germinating seeds and in pollen. We obtained and characterized two uev1d null mutant T-DNA insertion lines. Compared with wild-type plants, seeds from uev1d null plants germinated poorly when treated with a DNA-damaging agent. Those that germinated grew slower, and the majority ceased growth within 2 weeks. Pollen from uev1d plants also displayed a moderate but significant decrease in germination in the presence of DNA damage. This report links Ubc13-Uev with functions in DNA damage response in Arabidopsis.

Interplay between primase and replication factor C in the hyperthermophilic archaeon Sulfolobus solfataricus.

The heterodimeric primase from the hyperthermophilic archaeon Sulfolobus solfataricus synthesizes long RNA and DNA products in vitro. How primer synthesis by primase is coupled to primer extension by DNA polymerase in this organism is unclear. Here we show that the small subunit of the clamp loader replication factor C (RFC) of S. solfataricus interacted with both the catalytic and non-catalytic subunits of the primase by yeast two-hybrid and co-immunoprecipitation assays. Further, the primase-RFC interaction was also identified in the cell extract of S. solfataricus. Deletion analysis indicated that the small subunit of RFC interacted strongly with the N-terminal domain of the catalytic subunit of the primase. RFC stimulated dinucleotide formation but decreased the amount of primers synthesized by the primase. The inhibition of primer synthesis is consistent with the observation that RFC reduced the affinity of the primase for DNA templates. On the other hand, primase stimulated the ATPase activity of RFC. These findings suggest that the primase-RFC interaction modulates the activities of both enzymes and therefore may be involved in the regulation of primer synthesis and the transfer of primers to DNA polymerase in Archaea.

Biochemical characterization of an ATP-dependent DNA ligase from the hyperthermophilic crenarchaeon Sulfolobus shibatae.

A gene encoding a putative ATP-dependent DNA ligase was identified in the genome of the hyperthermophilic archaeon Sulfolobus shibatae and expressed in Escherichia coli. The 601 amino acid recombinant polypeptide was a monomeric protein capable of strand joining on a singly nicked DNA substrate in the presence of ATP ( K(m)=34 micro mu) and a divalent cation (Mn(2+), Mg(2+), or Ca(2+)). dATP was partially active in supporting ligation catalyzed by the protein, but GTP, CTP, UTP, dGTP, dCTP, dTTP, and NAD(+) were inactive. The cloned Ssh ligase showed an unusual metal cofactor requirement; it was significantly more active in the presence of Mn(2+) than in the presence of Mg(2+) or Ca(2+). Unexpectedly, the native Ssh ligase preferred Mg(2+) and Ca(2+) rather than Mn(2+). Both native and recombinant enzymes displayed optimal nick-joining activity at 60-80 degrees C. Ssh ligase discriminated against substrates containing mismatches on the 3'-side of nick junction and was more tolerant of mismatches at the 5'-end than of those at the penultimate 5'-end. The enzyme showed little activity on a 1-nucleotide gapped substrate. This is the first biochemical study of a DNA ligase from the crenarchaeotal branch of the archaea domain.

A complete sequence of the T. tengcongensis genome.

Thermoanaerobacter tengcongensis is a rod-shaped, gram-negative, anaerobic eubacterium that was isolated from a freshwater hot spring in Tengchong, China. Using a whole-genome-shotgun method, we sequenced its 2,689,445-bp genome from an isolate, MB4(T) (Genbank accession no. AE008691). The genome encodes 2588 predicted coding sequences (CDS). Among them, 1764 (68.2%) are classified according to homology to other documented proteins, and the rest, 824 CDS (31.8%), are functionally unknown. One of the interesting features of the T. tengcongensis genome is that 86.7% of its genes are encoded on the leading strand of DNA replication. Based on protein sequence similarity, the T. tengcongensis genome is most similar to that of Bacillus halodurans, a mesophilic eubacterium, among all fully sequenced prokaryotic genomes up to date. Computational analysis on genes involved in basic metabolic pathways supports the experimental discovery that T. tengcongensis metabolizes sugars as principal energy and carbon source and utilizes thiosulfate and element sulfur, but not sulfate, as electron acceptors. T. tengcongensis, as a gram-negative rod by empirical definitions (such as staining), shares many genes that are characteristics of gram-positive bacteria whereas it is missing molecular components unique to gram-negative bacteria. A strong correlation between the G + C content of tDNA and rDNA genes and the optimal growth temperature is found among the sequenced thermophiles. It is concluded that thermophiles are a biologically and phylogenetically divergent group of prokaryotes that have converged to sustain extreme environmental conditions over evolutionary timescale.