TCS01 Two-Component System Influenced the Virulence of Streptococcus pneumoniae by Regulating PcpA.

Streptococcus pneumoniae relies on two-component systems (TCSs) to regulate the processes of pathogenicity, osmotic pressure, chemotaxis, and energy metabolism. The TCS01 system of S. pneumoniae is composed of HK01 (histidine kinase) and RR01 (response regulator). Previous studies have reported that an rr01 mutant reduced the pneumococcal virulence in rat pneumonia, bacteremia, a nasopharyngeal model, and infective endocarditis. However, the mechanism of TCS01 (HK/RR01) regulating pneumococcal virulence remains unclear. Here, pneumococcal mutant strains Δrr01, Δhk01, and Δrr01&hk01 were constructed, and bacterial adhesion and invasion to A549 cells were compared. RNA sequencing was performed in D39 wild-type and Δrr01 strains, and transcript profile changes were analyzed. Differentially expressed virulence genes in the Δrr01 strain were screened out and identified by quantitative real-time PCR (qRT-PCR). Our results showed that pneumococcal mutant strains exhibited attenuated adhesion and invasion to A549 cells and differential transcript profiles. Results of qRT-PCR identification showed that the differential virulence genes screened out were downregulated. Among those changed virulence genes in the Δrr01 strain, the downregulated expression level of choline binding protein pcpA was the most obvious. Complementation of rr01 and overexpression of pcpA in the Δrr01 strain partially restored both pneumococcal adhesion and invasion, and rr01 complementation made the expression of pcpA upregulated. These findings revealed that rr01 influenced pneumococcal virulence by regulating pcpA.

Advances in colonic manometry in adults with colonic motility disorders / 中华胃肠外科杂志

In recent years, colonic manometry has been gradually introduced into clinical practice. It helps clinicians to gain a better understanding of the physiology and pathophysiology of colonic contractile activity in healthy adults and patients with colonic dysfunction. More and more patterns of colonic motility are being discovered with the help of colonic manometry. However, the clinical significance of these findings still needs to be further investigated. This review enhances our understanding of colonic motility and the current state of development and application of colonic manometry, as well as the limitations, future directions and potential of the technique in assessing the impact of treatment on colonic motility patterns, by analyzing and summarizing the literature related to colonic manometry.

Conservative treatment of supination and external rotation for type Ⅲ and Ⅳankle fracture by bone setting / 中国骨伤

OBJECTIVE@#To explore curative effect of conservative treatment of supination-lateral rotation (SER) with type Ⅲ and Ⅳ ankle fracture by bone setting technique.@*METHODS@#From January 2017 to December 2019, 64 patients diagnosed with SER with type Ⅲ and Ⅳ ankle fracture were treated with manipulative reduction and conservative treatment (manipulation group) and surgical treatment with open reduction and internal fixation (operation group), 32 patients in each group. In manipulation group, there were 17 males and 15 females, aged from 15 to 79 years old with an average of (51.42±13.68) years old;according to Lauge-Hansen classification, there were 8 patients with supination external rotation type Ⅲ and 24 patients with type Ⅳ. In operation group, there were 13 males and 19 females, aged from 18 to 76 years old with an average of (47.36±15.02) years old;7 patients with type Ⅲ and 25 patients with type Ⅳ. Displacement of ankle fracture was measured by Digimizer software, and compared before treatment, 3 and 12 months after treatment between two groups. Lateral medial malleolus displacement, lateral medial malleolus displacement, lateral malleolus displacement, lateral malleolus displacement, lateral malleolus contraction displacement and posterior malleolus displacement were measured and compared between two groups. Mazur score was used to evaluate ankle joint function.@*RESULTS@#All patients were followed up from 12 to 36 months with an average of (17.16±9.36) months. There were statistical differences in lateral medial malleolus displacement, lateral medial malleolus displacement, lateral malleolus displacement, lateral malleolus displacement, lateral malleolus contraction displacement and posterior malleolus displacement in manipulation group before and after reduction(P<0.05). Compared with operation group, there were no statistically significant differences in lateral malleolus shift, lateral malleolus shift, lateral malleolus contraction shift(P>0.05), while there were statistically significant differences in lateral malleolus shift, posterior malleolus shift up and down (P<0.05). Mazur scores of ankle joint at 3 months after treatment in manipulation group and operation group were 68.84±13.08 and 82.53±7.31, respectively, and had statistical differences(P<0.05), while there was no difference in evaluation of clnical effect(P>0.05). There were no differences in Mazur score and evaluation of clnical effect between two groups at 12 months after treatment (P>0.05).@*CONCLUSION@#Bone setting technique could effectively correct lateral displacement of medial malleolus, lateral displacement of medial malleolus, lateral displacement of lateral malleolus and lateral contraction displacement of lateral malleolus in supination lateral rotation type Ⅲ and Ⅳ ankle fracture, and has good long-term clinical effect, which could avoid operation for some patients and restore ankle function after fracture.

Multiple immune function impairments in diabetic patients and their effects on COVID-19.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2, poses a significant threat to public health worldwide, and diabetes is considered a risk factor for the rapid progression and poor prognosis of COVID-19. Limited immune function is a clinical feature of COVID-19 patients, and diabetes patients have defects in innate and adaptive immune functions, which may be an important reason for the rapid progression and poor prognosis of COVID-19 in patients with diabetes. We review the possible multiple effects of immune impairment in diabetic patients on the immune responses to COVID-19 to provide guidance for the diagnosis and treatment of diabetic patients with COVID-19.

Activation of AMPK restored impaired autophagy and inhibited inflammation reaction by up-regulating SIRT1 in acute pancreatitis.

AIMS: Acute pancreatitis (AP) is a common inflammatory disorder with high incidence and mortality. AMPK-SIRT1 pathway is involved in a variety of diseases, but its role in AP remains elusive. This study was aimed to explore the role of AMPK-SIRT1 pathway in AP. MAIN METHODS: AP models in vivo and vitro were constructed by intraperitoneal administration of L-arginine and caerulein-stimulated respectively. Rat serum amylase, IL-6 and TNF-α were determined by ELISA. The expression levels of AMPK, SIRT1, Beclin-1, LC3 and p62 were determined by qRT-PCR and western blot. The number of autophagosome was checked by transmission electron microscope. KEY FINDINGS: Compared with NC rats, serum amylase, IL-6 and TNF-α were increased in AP rats. The expressions of AMPK and SIRT1 were decreased, while Beclin-1, LC3II/Iratio and p62 were markedly increased in AP rats. After activation of AMPK by metformin, expressions of p-AMPKα, SIRT1 were significantly raised, while expressions of Beclin-1, LC3 II/I, p62, TNF-α, IL-6 were reduced, and the number of autophagosome was decreased significantly in caerulein-stimulated AR42J cells. The inhibition of AMPK by compound C obtained opposite results. SIGNIFICANCE: During AP occurrence, p-AMPK and SIRT1 were down-regulated, leading to the accumulation of p62, increase of autophagic vacuoles, damage of autophagy, and the occurrence of inflammation. It hinted that activation of AMPK restored impaired autophagy and inhibited inflammation reaction by up-regulating SIRT1. Our findings might provide important theoretical basis for explaining the pathogenesis of AP and investigating therapeutic target to treat and prevent AP.

Negative Regulation of SIRT1 by IRF9 Involved in Hyperlipidemia Acute Pancreatitis Associated with Kidney Injury.

BACKGROUND: Interferon regulatory factor 9 (IRF9) acts as a negative regulator of sirtuin-1 (SIRT1) to participate in many diseases. However, the role of SIRT1 and IRF9 in hyperlipidemia acute pancreatitis associated with kidney injury is unclear. AIMS: To explore the function of SIRT1 and IRF9 in hyperlipidemia acute pancreatitis associated with kidney injury and provide theoretical guidance for disease diagnosis and treatment. METHODS: Model rats were established by intraperitoneal injection of 20% L-arginine. Apoptosis of kidney tissue was determined by TUNEL staining. Expressions of IRF9, SIRT1, p53, and acetylated p53 were detected by qRT-PCR and Western blot. Dual-Luciferase Reporter Assay was carried out to validate the regulation of IRF9 on SIRT1. RESULTS: Pancreatic and renal injury was more serious, and apoptosis of kidney epithelial cells increased in acute pancreatitis (AP) and hyperlipidemia acute pancreatitis (HLAP) group. IRF9, p53, and acetylated p53 were up-regulated, and SIRT1 was down-regulated in AP and HLAP group (p < 0.05). Down-regulation of SIRT1 was negatively correlated with up-regulation of IRF9 in AP and HLAP group (p < 0.05). Pancreatic and renal injury and kidney epithelial cells apoptosis in HLAP group were more obvious than AP group (p < 0.05). The up-regulation of IRF9 and down-regulation of SIRT1 in HLAP group were more than AP group (p < 0.05). The promoter activity of SIRT1 was repressed by IRF9. CONCLUSION: In pancreatitis associated with kidney injury, IRF9 was a negative regulator of SIRT1, down-regulated the expression of SIRT1, increased acetylated p53, and promoted renal cell apoptosis. Hyperlipidemia further aggravated pancreatic and renal injury and renal cell apoptosis.

A novel function of IRF9 in acute pancreatitis by modulating cell apoptosis, proliferation, migration, and suppressing SIRT1-p53.

Acute pancreatitis (AP) is an inflammatory disease caused by the abnormal activation of pancreatic enzymes in the pancreas, with a considerably high morbidity and mortality. However, the etiological factor and pathogenesis of AP are still unclear. This study was aimed to explore the role and mechanism of interferon regulatory factor 9 (IRF9) in the occurrence of AP and to provide experimental and theoretical foundation for AP diagnosis and treatment. AP model in vitro was established by caerulein-induced group. Small interfering RNA (siRNA) was designed and constructed to silence IRF9 gene. After siRNA transfected and caerulein treated successfully, the expression levels of IRF9, SIRT1, and acetylated p53 (Ac-p53) were determined by qRT-PCR and Western blot. The apoptosis, proliferation, and migration of AR42J cells were checked by flow cytometry, MTT, and transwell assay. Dual-luciferase reporter assay was implemented to validate the regulatory effect of IRF9 on SIRT1. Here, our study showed that the expression of IRF9 and Ac-p53 was increased, SIRT1 was decreased, and cell apoptosis, proliferation, and migration of AR42J cells were increased after caerulein induced. IRF9 gene silencing upregulated SIRT1, downregulated Ac-p53, and inhibited cell apoptosis, proliferation, and migration. Dual-Luciferase reporter assay showed that IRF9 could negatively regulate SIRT1. The potential mechanism was that IRF9 could modulate cell apoptosis, proliferation, migration, and bind the promoter of SIRT1 to repress SIRT1-p53. It hinted that IRF9 showed a novel function in AP by modulating cell apoptosis, proliferation, migration, and suppressing SIRT1-p53. IRF9 might be a good potential treatment target for AP.

An effective immobilized haloalkane dehalogenase DhaA from Rhodococcus rhodochrous by adsorption, crosslink and PEGylation on meso-cellular foam.

Haloalkane dehalogenase DhaA catalyzes the hydrolysis of halogenated compounds by cleavage of the carbon-halogen bond. However, DhaA suffers from poor environmental stability and difficult recovery, which significantly increase the cost of DhaA. Here, an effective enzyme immobilization strategy was developed to overcome the disadvantages of DhaA. DhaA was physically absorbed with amine-functionalized meso-cellular foam (MCF). The MCF-absorbed DhaA (MD) was intermolecularly crosslinked with 8-arm PEG N­hydroxysuccinimide ester and then PEGylated by maleimide-thiol chemistry. DhaA from Rhodococcus rhodochrous was absorbed at a loading capacity of 100 mg/g in MD. The bulk crystallinity and morphology of MCF were largely maintained. The immobilized DhaA (MD-P1-P2) showed a lower Michaelis constant (Km, 0.588 mM) than DhaA (0.905 mM), along with an extremely low leaching ratio of DhaA (1.1%) from MCF. MD-P1-P2 exhibited a high stability in the extreme environmental conditions, as reflected by the remaining activity of 99.8% in 40% (v/v) DMSO for 5 h, 87.3% in 3 M urea solution for 1 h, 25.9% at pH 3.0, and 51.8% at room temperature for 30 days. Thus, our study was expected to develop an effective immobilized DhaA for practical application.

Conjugation with 20 kDa dextran decreases the autoxidation rate of bovine hemoglobin.

Haemoglobin-based oxygen carrier (HBOC) is highly susceptible to autoxidation that renders a series of tissue and cellular toxicities. HBOC is prepared by chemical modification of haemoglobin (Hb), which typically increases the autoxidation rate of Hb. Thus, it is necessary to decrease the autoxidation of HBOC. In the present study, two dextran-bHb conjugates (dex20-bHb and dex40-bHb) were prepared by conjugation with 20 kDa or 40 kDa dextrans, where the thiol group of Cys-93(ß) was reversibly protected. The autoxidation rate of bHb was decreased by conjugation with 20 kDa dextran and maintained by conjugation with 40 kDa dextran. In order to understand the low autoxidation rate of dex20-bHb, the effects of aldehyde modification and dextran on the autoxidation rate of dex20-bHb were investigated. The high oxygen affinity, high tetramer stability and dextran itself were found to decrease the autoxidation rate of dex20-bHb. The conjugated dextran had a predominant effect on decreasing the autoxidation rate of bHb, which was particularly promising for the potential development of safe HBOCs. Conjugation with dextran is of general significance to decrease the oxidation process of the heme-containing proteins, such as Hb and myoglobin.

In vitro and in vivo investigation of the novel Dex-bHb as oxygen carriers.

The development of hemoglobin-based oxygen carriers (HBOCs) is a persistent and urgent need in biomedicine. As a potential HBOCs, Dextran-hemoglobin (Dex-bHb) has been developed over the past years. The novel Dex-bHb, whose thiol group of Cys-93(ß) was reversibly protected, was produced and the characteristics were evaluated in our previous study. Herein, blood compatibility was characterized in terms of the red blood cell aggregation and hemolysis rate in vitro, and Dex-bHb showed no obvious effects. After intravenous administration of Dex-bHb to golden Syrian hamsters with hemorrhages shock, it showed mean arterial pressure recovery, blood flow increase and the organ protection from serious hemorrhage injury. Consequently, Dex-bHb is hopeful to be a safe and available blood substitute.

Clinicopathological features and risk factors of neuroendocrine tumors in 256 patients / 天津医药

Objective To investigate the epidemiology, clinical manifestations, diagnosis, treatment and prognosis of neuroendocrine tumors (NETs). Methods Medical records of 265 patients with neuroendocrine tumors diagnosed and treated in our hospital from January 2006 to August 2015 were collected and retrospectively reviewed in this study. The clinicopathological data including gender, age of onset, initial symptoms, primary site, pathological conditions, diagnosis, treatment, prognosis and follow up were analyzed. Results The gender ratio M/F of the 265 cases was 160:105 (1.5:1), with mean age of (55.8±2.7) years, and the high incidence was in age of 55-65 years. The tumors were located in the colon and rectum (127 cases, 47.9％), lung (59 cases, 22.3％), stomach (21 cases, 7.8％), appendix (15 cases, 5.7％), small intestine (especially in the duodenum and pancreas, 10 cases, 3.8％), mammary gland (11 cases, 4.2％), neck (10 cases, 3.8％) and unknown primary site (12 cases, 4.5％). Patients with different tumor sites showed different symptoms. Patients with colorectal tumors mainly manifested as changes in bowel habits, such as diarrhea, constipation and blood in stool. The main manifestation of patients with primary pulmonary symptoms was cough or bloody sputum. The patients with tumors at stomach, appendix or small intestine showed many discomfort, such as abdominal pain and abdominal distention. Among the 265 cases, 186 patients were diagnosed as phase G1 (70.2％), 54 patients were diagnosed as phase G2 (20.4％) and 25 patients were diagnosed as phase G3 (9.4％). Immunohistochemistry showed that synaptophysin (Syn) was positive in 228 cases (86.4％), chromaffin A (CgA) was positive in 102 cases (38.5％), and C56 was positive in 74 cases (27.9％). A total of 232 patients were treated with surgery (87.5％), 28 patients received radiotherapy or chemotherapy treatment (10.6％) and 5 patients were not treated. One hundred and ninety-eight patients were followed up at least 1 time, and the follow-up rate was 74.7％. The median follow-up time was 38 months. No tumor related death was found in patients with phase G1 during the follow-up, 6 cases of tumor associated death were found in patients with phase G2 and 19 cases of cancer related death were found in patients with phase G3. Metastasis was found in all 23 patients with tumor related death. The survival rate of patients with neuroendocrine tumor (G1+G2) was significantly higher than that of patients with neuroendocrine carcinoma (G3, Log rankχ2=13.774,P<0.01). Conclusion The males have a higher incidence rate of NETs than females. Patients with different tumor sites showed different symptoms. The most common primary sites of NETs are the digestive tract, especially in patients with colorectal cancer. The more late the pathological stage, the worse the prognosis.

Treatment of thoracolumbar fractures with three-dimensional reduction method and external fixation devices under analgesia / 中国骨伤

<p><b>OBJECTIVE</b>To compare the clinical effects between open reduction internal fixation and three-dimensional reduction with external fixation under analgesia in treating fresh thoracolumbar fractures, and explore the simple and effective method for thoracolumbar fractures.</p><p><b>METHODS</b>The clinical data of 40 patients with thoracolumbar fractures who met the inclusion and exclusion criteria in the department of orthopaedics affiliated to Suzhou Hospital of Nanjing University of Chinese Medicine from February 2013 to August 2017 were retrospectively analyzed. According to therapeutic methods, the patients were devided into treatment group and control group, 20 cases in each group. Treatment group was treated by three-dimensional reduction method and external fixation devices under analgesia, and control group was treated by open reduction and common spinal fixation system. In treatment group, there were 9 males and 11 females, aged from 26 to 68 years old with an average of (52.8±11.3) years; and in control group, there were 10 males and 10 females, aged from 26 to 64 years old with an average of(50.6±8.8) years. Anterior vertebral body compression(AVBC), Cobb angle and visual analogue scale(VAS) were measured and compared in two group.</p><p><b>RESULTS</b>All 40 patients finished follow-up. The follow-up time in treatment group was 5 to 37 months with average of (16.1±8.8) months, in control group was 5 to 29 months with an average of(17.3±6.0) months. There was no significant difference between two groups(>0.05). AVBC, Cobb angle, VAS score were obviously improved in all patients after treatment(<0.05), but there were no significant difference between two groups(>0.05).</p><p><b>CONCLUSIONS</b>Clinical effect of two methods was similar in treating thoracolumbar fractures, but three-dimensional reduction and external fixation devices under analgesia has advantage of easy operation, smaller trauma and no need secondary surgery for removed internal fixation.</p>

PEGylation with the thiosuccinimido butylamine linker significantly increases the stability of haloalkane dehalogenase DhaA.

Haloalkane dehalogenase (HLD) can catalyze the hydrolytic dehalogenation of halogenated compounds. However, HLD suffers from the poor stability to resist the environmental stress. PEGylation is an effective approach to enhance the stability of enzymes. The linker is an important stabilization factor of PEGylation. Thus, the linkers of the PEGylated HLD were optimized to improve the stability of HLD in the present study. The PEGylated haloalkane dehalogenase DhaAs with methylamine (Ml), carbamate (Cm) and thiosuccinimido butylamine (Tb) linkers were prepared, respectively. The effects of the Ml, Cm and Tb linkers on the stability of the PEGylated DhaAs were investigated under different environmental stresses. Among the three linkers, the Tb linker showed the highest efficacy to improve the stability of the PEGylated DhaA. The Tb linker significantly increased the thermal stability of the PEGylated DhaA by slowing its structural unfolding, and the pH stability of the PEGylated DhaA by slowing the protonation process. In addition, the PEGylated DhaA with the Tb linker showed the maximum resistance to high ionic strength (1M NaCl) and organic solvent (40% DMSO). PEGylation with the Tb linker is of general interest to effectively improve the stability of proteins, particularly the protein with poor stability.

Percutaneous poking reduction with bone grafting and limited internal fixation for the treatment of calcaneal fractures / 中国骨伤

<p><b>OBJECTIVE</b>To investigate clinical effects of percutaneous poking reduction with bone grafting and limited internal fixation for the treatment of calcaneal fractures.</p><p><b>METHODS</b>From May 2013 to October 2016, 53 patients with closed calcaneal fractures were analyzed, and were divided into treatment group and control group. There were 33 patients in treatment group including 25 males and 8 females, aged from 15 to 82 years old with an average of(44.7±14.2) years old; 17 cases were type II and 16 cases were type III according to Sanders classification; treated by percutaneous poking reduction with bone grafting and limited internal fixation. There were 33 patients in control group, including 20 males aged from 25 to 62 years old with an average of (42.2±11.3) years old; 8 cases were type II and 12 cases were type III according to Sanders classification; treated by open reduction and internal fixation. Imaging indicators, hospital stays and preoperative waiting time were observed and compared, Maryland scoring were applied to evaluate foot function.</p><p><b>RESULTS</b>Fifty-three patients were followed up, and treatment group was followed up from 8 to 40 months with an average of (19.9±7.2) months; while control group was followed up from 12 to 40 months with an average of (21.7±7.7) months, and there were not significant differences between two groups in follow-up time (>0.05). There were no obvious meaning in Böhler angles, Gissane angles between two groups (>0.05). There were significant differences in hospital stays and preoperative waiting time(<0.01). There were no significant differences in Maryland score between treatment group(90.45±5.76) and control group(89.10±6.16).</p><p><b>CONCLUSIONS</b>Percutaneous poking reduction with bone grafting and limited internal fixation for the treatment of calcaneal fractures could obtain satisfied effects, and has advantages of less trauma and complications, rapid recovery and good clinical effects.</p>

Chemical constituents from flower of Gentiana tibetica / 中草药

Objective: To investigate the chemical constituents in flowers of Gentiana tibetica. Methods: The chemical constituents were isolated from the 95% alcohol extract of G. tibetica flowers by silica column chromatography, Sephadex LH-20, C18 and RP-HPLC. Their chemical structures were elucidated on the basis of IR, ESI-MS, 1H-MNR, and 13C-MNR spectroscopic data. Results: Twelve compounds, including chromene, flavones C-glycosides, secoiridoid glycosides, iridoid glycosides, aliphatic alcohol, and disaccharide, were obtained from the 95% alcohol extract of G. tibetica flowers, Their structures were identified as macrophylloside D (1), orientin 7-caffeate (2), 7-O-feruloylorientin (3), isovitexin (4), saponarin (5), isoorientin (6), 6'-O-β-D-glucopyranosyl gentiopicroside (7), sweroside (8), swertiamarine (9), loganic acid (10), 1-heneicosanol (11), and sucrose (12). Conclusion: Compounds 1-12 are isolated from G. tibetica for the first time.