Structural basis for substrate recognition by a S-adenosylhomocysteine hydrolase Lpg2021 from Legionella pneumophila.

S-Adenosyl-l-homocysteine hydrolase (SAHH) is a crucial enzyme that governs S-adenosyl methionine (SAM)-dependent methylation reactions within cells and regulates the intracellular concentration of SAH. Legionella pneumophila, the causative pathogen of Legionnaires' disease, encodes Lpg2021, which is the first identified dimeric SAHH in bacteria and is a promising target for drug development. Here, we report the structure of Lpg2021 in its ligand-free state and in complexes with adenine (ADE), adenosine (ADO), and 3-Deazaneplanocin A (DZNep). X-ray crystallography, isothermal titration calorimetry (ITC), and molecular docking were used to elucidate the binding mechanisms of Lpg2021 to its substrates and inhibitors. Virtual screening was performed to identify potential Lpg2021 inhibitors. This study contributes a novel perspective to the understanding of SAHH evolution and establishes a structural framework for designing specific inhibitors targeting pathogenic Legionella pneumophila SAHH.

Crystal structure of an aspartate aminotransferase Lpg0070 from Legionella pneumophila.

Legionella pneumophila aspartate aminotransferase (Lpg0070) is a member of the transaminase and belongs to the pyridoxal 5'-phosphate (PLP)-dependent superfamily. It is responsible for the transfer of α-amino between aspartate and α-ketoglutarate to form glutamate and oxaloacetate. Here, we report the crystal structure of Lpg0070 at the resolution of 2.14 Å and 1.7 Å, in apo-form and PLP-bound, respectively. Our structural analysis revealed the specific residues involved in the PLP binding and free form against PLP-bound supported conformational changes before substrate recognition. In vitro enzyme activity proves that the absence of the N-terminal arm reduces the enzyme activity of Lpg0070. These data provide further evidence to support the N-terminal arm plays a crucial role in catalytic activity.

[Effect of Calcified Lymph Nodes on Thoracoscopic Lobectomy in Chronic Obstructive Pulmonary Disease Patients with Lung Cancer].

Objective To observe the effect of calcified lymph nodes on video-assisted thoracoscopic surgery (VATS) lobectomy in the chronic obstructive pulmonary disease (COPD) patients with lung cancer. Methods A retrospective analysis was conducted on the COPD patients with lung cancer who underwent VATS lobectomy in the Department of Thoracic Surgery in the First Affiliated Hospital of Hebei North University from May 2014 to May 2018.The patients were assigned into a calcified lymph node group and a control group according to the presence or absence of calcified lymph nodes in CT,and the size,morphology,and calcification degree of the lymph nodes were recorded.The operation duration,intraoperative blood loss,chest tube retention time,hospitalization days,and overall complication rate were compared between the two groups. Results The 30 patients in the calcified lymph node group included 17 patients with one calcified lymph node and 13 patients with two or more calcified lymph nodes,and a total of 65 calcified lymph nodes were recorded.The calcified lymph nodes with the size ≤5 mm were the most common (53.8%),and complete calcification was the most common form (55.4%) in lymph node calcification.The mean operation duration had no significant difference between the calcified lymph node group and the control group (t=-1.357,P=0.180).The intraoperative blood loss (t=-2.646,P=0.010),chest tube retention time (t=-2.302,P=0.025),and hospitalization days (t=-2.274,P=0.027) in the calcified lymph node group were higher than those in the control group. Conclusion Calcified lymph nodes increase the difficulty and risk of VATS lobectomy in the COPD patients with lung cancer.The findings of this study are conducive to predicting the perioperative process of VATS lobectomy.

[Risk Factor Analysis of Abdominal Aortic Enlargement after Thoracic Endovascular Aortic Repair Using Two-Stent Graft Implantation for Stanford Type B Aortic Dissection].

Objective: To explore the risk factors of abdominal aortic enlargement (AAE) after thoracic endovascular aortic repair using two-stent graft implantation (TEVAR-TSI) for Stanford type B aortic dissection. Methods: The clinical and imaging data of patients who underwent TEVAR-TSI for Stanford type B aortic dissection in the First Affiliated Hospital of Hebei North University from January 2013 through September 2020 were retrospectively collected and analyzed. CT angiography (CTA) scans were performed before the procedure. Follow-up CTA scans were scheduled and performed in 1, 3, 6, and 12 months after the procedure and annually thereafter. The primary outcome was AAE. The risk factors of AAE after TEVAR-TSI were selected and survival analysis and multivariate logistic regression were conducted accordingly. Results: A total of 146 patients were regularly followed up at our hospital, with the median followup time of the entire cohort being 48 months (ranging from 12 to 84 months). During the followup period after TEVAR-TSI, the incidence of AAE was 19.9% (29/146). A total of 29 patients developed AAE (the AAE group), while 117 patients did not develop AAE (the non-AAE group). There were a total of 27 deaths, including 13 in the non-AAE group versus 14 in the AAE group. Distal aortic reoperation was performed on 10 patients, including 4 in the non-AAE group versus 6 in the AAE group. The cumulative long-term survival and freedom from distal aortic reoperation of the non-AAE group were both significantly better those of the AAE group ( P<0.05). Logistic multivariate regression analysis showed that independent risk factors of AAE after TEVAR-TSI included the following, partial thrombosis of the false lumen (odds ratio [ OR]=4.090, 95% confidence interval [ CI]: 1.539-10.867, P=0.005), the longer cumulative diameter of residual intimal tear above the level of the lowest renal arteries ( OR=1.290, 95% CI: 1.164-1.429, P=0.000), and shorter cumulative diameter of residual intimal tear below the level of the lowest renal arteries ( OR=0.487, 95% CI: 0.270-0.878, P=0.017). Conclusion: The prognosis of patients who developed AAE after TEVAR-TSI was not good. During followup visits, as precautions against the development of AAE, close attention should be paid to partial thrombosis of the false lumen, cumulative diameter of residual intimal tear above the level of the lowest renal arteries, and cumulative diameter of residual intimal tear below the level of the lowest renal arteries.

[The Characteristics of Aortic Remodeling after Thoracic Endovascular Aortic Repair using Two-stent Graft Implantation for Stanford Type B Aortic Dissection].

OBJECTIVE: To investigate the characteristics of aortic remodeling after thoracic endovascular aortic repair using two-stent graft implantation (TEVAR-TSI) for Stanford B aortic dissection. METHODS: The clinical and imaging data of 128 patients who underwent TEVAR-TSI for Stanford B aortic dissection in the First Affiliated Hospital of Hebei North University from January 2013 through May 2019 were retrospectively collected. CT images were obtained before (T 0) TEVAR-TSI and, 1 week (T 1), 3 months (T 2), 6 months (T 3), 1 year (T 4) after TEVAR-TSI. The maximum diameter of the true lumen and false lumen in the short axis view was accessed at five levels: L 1: the level of primary tear entry, L 2: the level of the bronchial bifurcation, L 3: the level of the distal of the first stent-graft, L 4: the level of the celiac trunk, L 5: the level of the lowest renal arteries. The false lumen thrombosis in the thoracic aorta and abdominal aorta were assessed at different times, the false lumen and true lumen changes in diameter were evaluated between the preoperative and postoperative CT scan. RESULTS: The stented segment of the descending thoracic aorta was evaluated (L 1-L 3): The true lumen diameter showed an increasing trend and the false lumen diameter showed an decreasing trend at levels L 1, L 2, and L 3, the change of true lumen diameter was positively correlated with the follow-up time ( r=0.721, 0.827, 0.893, P<0.05), and the change rate of true lumen diameter was positively correlated with the follow-up time ( r=0.763, 0.818, 0.902, P<0.05), and the change of false lumen diameter was negatively correlated with the follow-up time ( r=-0.750, -0.927, -0.934, P<0.05), and the change rate of false lumen diameter was negatively correlated with the follow-up time (-0.774, -0.935, -0.952, P<0.05). When the unstented segment of the abdominal aorta was evaluated (L 4-L 5), the average true lumen diameter at the level of celiac trunk increased significantly at 1 year by 13.7% ( P=0.007), however, the average false lumen diameter did not change over time ( P=0.406). The average true lumen diameter and false lumen diameter at the level of the lowest renal arteries increased over time as well, the average true lumen increased by 10.1%, and the average false lumen increased by 13.6% ( P=0.048, 0.017). Besides, the complete false lumen thrombosis rate of the stented segment of the descending thoracic aorta was higher than that of the unstented segment of the abdominal aorta.e complete false lumen thrombosis rate of the stented segment of the descending thoracic aorta was higher than that of the unstented segment of the abdominal aorta. CONCLUSION: After receiving TEVAR-TSI, Stanford type B aortic dissection patients had high thrombosis absorption rate in the thoracic aortic segment covered by stent, and the aortic remodeling was more ideal. The aortic remodeling effect in the abdominal aortic segment not covered was not ideal, and the inner diameter of the abdominal aorta tended to increase. Therefore, close follow-up monitoring should be conducted.

Suppression of the CD28/B7 pathway reduces the occurrence and development of myasthenia gravis and cytokine levels.

BACKGROUND: Myasthenia gravis (MG) is an antibody-mediated, autoimmune neuromuscular disease. Reports have indicated that the CD28/B7 ligand interactions play a crucial role during primary immune responses. Hence, the aim of the present study was to investigate the possible effects of the CD28/B7 pathway on the occurrence and development of MG and its associated cytokine factors. METHODS: An experimental autoimmune myasthenia gravis (EAMG) was initially established by immunization of Lewis rats with acetylcholine receptor (AChR) α97-116 peptide. Then the rats were treated with dexamethasone and CTLA4-Ig (used for inhibiting the CD28/B7 pathway). Serum levels of AChR IgG and AChR IgG2b were then detected using ELISA. The clinical features, muscle contraction function, AChR content, expression of CD28, CTLA4, B7.1 and B7.2 in mononuclear cells of peripheral blood and the secretion of cytokines (INF-γ, IL-2, IL-10 and IL-12) in serum of rats were measured. Finally, lymphocyte proliferation upon CTLA4 IgG treatment was examined in vitro. RESULTS: Inhibition of the CD28/B7 pathway and dexamethasone were found to significantly improve clinical symptoms of EAMG rats, reduce serum levels of AChR IgG, AChR IgG2b, INF-γ, IL-2, IL-10 and IL-12, the expression of CD28, CTLA4, B7.1 and B7.2 in mononuclear cells of peripheral blood, and enhance muscle contraction function and AChR content in the muscle in vivo. Meanwhile, CTLA4 IgG could abolish the increased lymphocyte proliferation following AChR stimulation in vitro. CONCLUSION: Overall, the suppression of the CD28/B7 pathway by CTLA4-Ig can have the potential to retard the occurrence and development of MG.

Anti-Relapse effects of donor natural killer cells and IL-2 gene modification on allogeneic hematopoietic stem cell transplantation in acute leukemia.

BACKGROUND: Patients with acute leukemia (AL) refractory to induction or reinduction chemotherapy show poor prognoses if they do not undergo allogeneic hematopoietic stem-cell transplantation (AHSCT). The present study aims to investigate whether donor natural killer (NK) cells and interleukin-2 (IL-2) gene modification exert anti-relapse effects on AHSCT after establishing a mouse model of AL. METHODS: C57BL/6 (H-2b) mice were selected as donor mice to obtain NK cells and hematopoietic stem cells, while BALB/c (H-2d) mice were selected as the recipient mice for AHSCT. The AHSCT-treated mice were then injected with the donor NK cells, recombinant adenovirus expressing IL-2 (AdIL-2), or the NK cells infected by AdIL-2. Flow cytometry was performed to detect the cell transplantation rate, immune cell number, and cell immunogenicity. Enzyme-linked immunosorbent assay (ELISA) was employed to quantify the secretion of IL-2 in spleen cells, and the level of peripheral blood factors, including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-35, transforming growth factor ß (TGF-ß), and IL-10. RESULTS: In our experiments, promotional effects of NK cells and AdIL-2 were found on cell transplantation rate, immune reconstitution ability, cell immunogenicity, IL-2 secretion, as well as increased peripheral blood factor levels in the recipient mice treated with AHSCT, with improved pathological changes observed. Moreover, the aforementioned changes were further promoted in the AHSCT-treated recipient mice injected with the AdIL-2-infected NK cells. CONCLUSIONS: These results uncover that the donor NK cells and IL-2 gene modification could inhibit the relapse of AL mice underwent AHSCT, hereby providing a new target for leukemia treatment.

Hexameric structure of the ATPase motor subunit of magnesium chelatase in chlorophyll biosynthesis.

Magnesium chelatase (MgCh) is a heterotrimeric enzyme complex, composed of two AAA+ family subunits that can assembly into a double ring structure and a large catalytic subunit. The small AAA+ subunit has ATPase activity and can self-oligomerize into a ring structure, while the other AAA+ subunit lacks independent ATPase activity. Previous structural studies of the ATPase motor subunit of MgCh from a bacteriochlorophyll-synthesizing bacterium have identified a unique ATPase clade, but the model of oligomeric assembly is unclear. Here we present the hexameric structure of the MgCh ATPase motor subunit from the chlorophyll-synthesizing cyanobacterium Synechocystis sp. PCC 6803. This structure reveals details of how the hexameric ring is assembled, and thus provides a basis for further studying the heterotrimeric complex.

Ligustrazine eases lung cancer by regulating PTEN and Wnt/ß-catenin pathway.

BACKGROUND: Lung cancer (LC) is the most common malignant tumor in the world. Ligustrazine (LSZ), an alkaloid, is an active ingredient extracted from chuanxiong Hort. Earlier studies suggested that LSZ plays a crucial role in lung diseases. The study aimed to investigate the detailed mechanism of LSZ on LC. METHODS: Protein expression was determined by qPCR, western blotting and immunohistochemistry. Cell viability was determined by CCK8 assay. Invasiveness was detected by Transwell assay. Cell growth was determined by clone formation assay. Male BALB/c nude mice were randomly divided into 2 groups (n=10): LSZ group and Control group. RESULTS: Results showed that PTEN is low-expressed in lung tumor tissues and LC cells (H1650, A549, H1299, and PC-9). Appropriate doses of LSZ could significantly promote the expression of PTEN in LC cells. Besides, LSZ inhibits invasion and proliferation of H1299 cells. However, knockdown PTEN counteracts the inhibitory effect. Animal experiments suggest that LSZ inhibits tumor formation in vivo. Mechanistically, western blotting shows that LSZ treatment significantly decreased the levels of Wnt and ß-catenin, while increasing the levels of PTEN and GSK-3ß. CONCLUSIONS: Collectively, these results demonstrate that LSZ ameliorates LC by down-regulating PTEN level and blocking Wnt/ß-catenin signaling pathway.

[Thoracoscopic and laparoscopic esophagectomy in the treatment of esophageal carcinoma: clinical analysis of 350 cases].

OBJECTIVE: To investigate the clinical efficacy and learning curve of thoracoscopic and laparoscopic esophagectomy(TLE) in the treatment of esophageal carcinoma. METHODS: Clinical data of 350 consecutive patients with esophageal carcinoma undergoing TLE in the West China Hospital between February 2008 and October 2013 were retrospectively analyzed. Patients in the early stage(n=150) were chronologically and evenly divided into three groups, and perioperative outcomes were compared between the three groups in order to evaluate the learning curve. RESULTS: There were no intraoperative deaths. Intraoperative complications occurred in 29(8.3%) patients. Thirteen(3.7%) cases were converted to open operation including 9 thoracotomy and 4 laparotomy. The mean operative time was 332.5(range 230-780) min and the mean blood loss was 160.8(range 15-4000) ml. The tumor free resection margins (R0) were completely in 333(95.1%) cases. The mean lymph nodes harvested was 21.6(range 6-42). The average length of postoperative hospital stay was 11.6(range 7-93) d. Postoperative complications occurred in 75(21.4%) patients, and 3(0.8%) patients died within 30 days after surgery. Compared with TLE 1 group, TLE 2 group presented shorter operative time, less blood loss, shorter postoperative hospital stay, lower postoperative complication rate, and more lymph nodes harvested(all P<0.05). Only blood loss was less in TLE 3 group as compared to TLE 2 group, and other perioperative results were found to be of no statistical differences between the two groups. There was no significant difference in 30-day mortality between the three groups. CONCLUSIONS: TLE is a technically feasible and safe procedure and provides comparable oncological outcomes with open esophagectomy, thus suggesting that TLE is an alternative approach in the treatment of esophageal carcinoma. A plateau of TLE skill can be reached after 50 surgical procedures.

[Clinical value of thoracoscopic extensive mediastinal lymphadenectomy in the surgical treatment of esophageal carcinoma].

OBJECTIVE: To explore the safety and feasibility of extensive mediastinal lymphadenectomy during thoracoscopic esophagectomy in the treatment of esophageal carcinoma. METHODS: Clinical data of 125 patients with esophageal carcinoma undergoing thoracoscopic and laparoscopic esophagectomy(TLE) in West China Hospital of Sichuan University between May 2009 and December 2011 were retrospectively analyzed. Patients were divided into 2 groups: non-extensive mediastinal lymphadenectomy group(non-extensive group, n=53) and extensive mediastinal lymphadenectomy group(extensive group, n=72). Perioperative outcomes of these two groups were compared. RESULTS: No significant differences were found in the time of thoracic operation, length of intensive care unit stay and postoperative hospital stay, postoperative complication and the overall mortality between the two groups. Compared with non-extensive group, extensive group showed less blood loss during thoracic operation [(140.6±62.1) ml vs. (167.7±69.2) ml, P=0.023], more thoracic lymph nodes harvested (12.2±4.2 vs. 9.0±4.1, P<0.01). Seventeen patients (23.6%) in extensive group were found to have positive recurrent laryngeal nerve lymph nodes, which resulted in upstaging of TNM in 7 patients(9.7%). The incidence of recurrent laryngeal nerve palsy was higher in extensive group as compared to non-extensive group, but the difference was not statistically significant (6.9% vs. 1.9%, P=0.240). CONCLUSION: Extensive mediastinal lymphadenectomy during thoracoscopic esophagectomy is a feasible and safe procedure for esophageal carcinoma patients, which can increase the number of harvested lymph node and the accuracy of tumor staging.

[Minimally invasive esophagectomy for esophageal carcinoma: clinical analysis of 160 cases].

OBJECTIVE: To explore the feasibility, safety and clinical application value of minimally invasive esophagectomy (MIE). METHODS: Clinical data of 160 patients undergoing minimally invasive approach in the West China Hospital of Sichuan University between February 2008 and December 2011 were analyzed retrospectively. RESULTS: There were 140 males and 20 females with a mean age of 59.6 years. Approaches to esophagectomy were thoracoscopic and laparoscopic esophagectomy (n=139), thoracoscopic and mediastinoscopic esophagectomy (n=3), laparoscopic-assisted Ivor Lewis resection (n=15), thoraco-laparoscopic Ivor Lewis resection (n=3). The mean operative time was 364 (range 230-780) min and the mean blood loss was 286.2 (range 20 to 4000) ml. The tumor free resection margins (R0) were completely in 152 cases (95.0%). The mean lymph node harvested was 19.4 (range 6-39). There were 11 (6.9%) cases converted to open operation including 9 thoracotomy and 2 laparotomy. The intraoperative complication rate was 11.3% (18/160). The average length of intensive care unit (ICU) stay was 22.1(range 0 to 430) h and the average length of postoperative hospital stay was 13.1 (range 7-93 d). Postoperative complication occurred in 34.4% of patients. The 30-day mortality was 1.2% (2/160) and the overall mortality was 2.5% (4/160). CONCLUSION: MIE is technically feasible and safe for the treatment of esophageal carcinoma, which provides good or even better outcomes than open approach.

Comparison of open three-field and minimally-invasive esophagectomy for esophageal cancer.

The aim was to compare the early outcomes between thoracoscopic and laparoscopic esophagectomy (TLE) and open three-field esophagectomy for esophageal cancer. We retrospectively analyzed clinical data from 96 patients with esophageal cancer who underwent TLE, and 78 patients who underwent open three-field esophagectomy from March 2008 to September 2010. All the operations were successful. There was no significant difference between TLE and open three-field esophagectomy with regard to the number of lymph nodes procured (17.75±5.56 vs. 18.03±6.20, P>0.05), complications (32.3% vs. 46.2%, P>0.05), and operative mortality (2.1% vs. 3.8%, P>0.05). However, hospital stay was significantly shorter in the TLE group than the open esophagectomy group (12.64±8.82 vs. 17.53±6.40 days, P<0.01), and the TLE group had significantly less blood loss (346.68±41.13 vs. 519.26±47.74 ml, P<0.01). This showed that TLE for esophageal cancer offers results as good as or better than those with open three-field esophagectomy.