Gene de virulência oipA de Helicobacter pylori como marcador molecular de gastropatias severas / Helicobacter pylori oipa virulence gene as a molecular marker of severe gastropathies

ABSTRACT Background: Helicobacter pylori is an etiologic agent of gastroduodenal diseases. The microorganism, considered a type I carcinogen, affects about 50% of the global population. H. pylori virulence factors are determinant for the clinical outcome of the infection. The outer inflammatory protein A (oipA) gene encodes an outer membrane adhesin and is related to severe gastropathies, such as gastric cancer. Objective: The aim of this study was to evaluate the association of the oipA gene with the severity of gastroduodenal diseases in dyspeptic patients in region Central Brazil. Methods: The polymerase chain reaction (PCR) was used to determine the presence of H. pylori. Samples positives were used for molecular screening of the oipA gene. Gastropathies were categorized as non-severe and severe diseases. Results: Approximately 68% of patients had H. pylori and 36% were infected with H. pylori oipA+ strains. Infection was significantly associated in patients aged over 44 years (P=0.004). However, there was no association between oipA and patients' age (P=0.89). Approximately 46% of patients infected with oipA+ strains had some severe illness. Gastric adenocarcinoma was the most frequent severe gastropathy. The H. pylori oipA genotype was inversely associated with the severity of gastroduodenal diseases (OR=0.247, 95%CI: 0.0804-0.7149 and P=0.007). Conclusion: The characterization of possible molecular markers will contribute to personalized medicine, impacting the prognosis of patients.

RESUMO Contexto: Helicobacter pylori é um agente etiológico de doenças gastroduodenais. O microrganismo, considerado cancerígeno tipo I, afeta cerca de 50% da população mundial. Os fatores de virulência do H. pylori são determinantes para o desfecho clínico da infecção. O gene da proteína inflamatória externa A (oipA) codifica uma adesina da membrana externa e está relacionado a gastropatias severas, como o câncer gástrico. Objetivo: O objetivo deste estudo foi avaliar a associação do gene oipA com a gravidade das doenças gastroduodenais em pacientes dispépticos na região Brasil Central. Métodos: A reação em cadeia da polimerase (PCR) foi utilizada para determinar a presença de H. pylori. Amostras positivas foram utilizadas para triagem molecular do gene oipA. As gastropatias foram categorizadas como doenças não severas e severas. Resultados: Aproximadamente 68% dos pacientes apresentaram H. pylori e 36% estavam infectados com cepas H. pylori oipA+. A infecção foi significativamente associada em pacientes com idade superior a 44 anos (P=0,004). No entanto, não houve associação entre oipA e a idade dos pacientes (P=0,89). Aproximadamente 46% dos pacientes infectados com cepas oipA+ tiveram alguma doença severa. O adenocarcinoma gástrico foi a gastropatia severa mais frequente. O genótipo oipA de H. pylori foi inversamente associado à gravidade das doenças gastroduodenais (OR=0,247, IC95%: 0,0804-0,7149 P=0,007). Conclusão: A caracterização de possíveis marcadores moleculares contribuirá para a medicina personalizada, impactando no prognóstico dos pacientes.

Effects of Pasturella Multocida B: 2 and its immunogens (LPS and OMP) on reproductive hormones in Nili-Ravi Buffaloes / Efeitos de Pasteurella multocida B: 2 e seus imunógenos (LPS e OMP) em hormônios reprodutivos em búfalos Nili-Ravi

Abstract Livestock is a fundamental part of the agriculture industry in Pakistan and contributes more than 11.53% to GDP. Among livestock species, the buffaloes are regarded as the black gold of Pakistan. Being the highest milk producers globally, Nili-Ravi buffaloes are the most famous ones. Buffaloes are affected by many endemic diseases, and "Hemorrhagic septicemia" (HS) is one of them. This study was designed to ascertain the effects of experimental exposure ofP. multocida B:2 (oral) and its immunogens, i.e., LPS (oral and intravenous) and OMP (oral and subcutaneous) on reproductive hormonal profiles in Nili-Ravi buffaloes. Repeated serum samples were collected from the jugular vein of experimental animals for 21 days (0, 02, 04, 08, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 and 504 hours). Hormonal assays to determine the serum concentrations of Gonadotropin-releasing hormone (GnRH), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Estrogen (E2) and progesterone (P4) were performed using (MyBioSource) commercial Elisa kits. The hormonal profile of all treatment groups of the buffalo heifers exhibited significant (P<0.05) variations as compared to the control group (G-1). These results indicate suppression in Nili-Ravi buffaloes' reproductive hormonal profile on exposure to P. multocida B:2 and its immunogens. This influence warrants that exposure to H.S may be a possible reason for delayed puberty and poor reproduction performance in Nili-Ravi buffaloes.

Resumo A pecuária é uma parte fundamental da indústria agrícola no Paquistão e contribui com 11,53% do PIB nacional. Entre as espécies de gado, os búfalos são considerados o ouro negro do Paquistão. Sendo os maiores produtores de leite em todo o mundo, os búfalos Nili-Ravi são os mais famosos. Os búfalos são afetados por muitas doenças endêmicas, entre as quais a "septicemia hemorrágica" (SH). Este estudo busca verificar os efeitos da exposição experimental de P. multocida B:2 (oral) e seus imunógenos, ou seja, LPS (oral e intravenoso) e OMP (oral e subcutâneo), nos perfis hormonais reprodutivos em búfalos Nili-Ravi. Amostras de soro repetidas foram coletadas da veia jugular de animais experimentais por 21 dias (0, 2, 4, 8, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 e 504 horas). Os ensaios hormonais para determinar as concentrações séricas do hormônio liberador de gonadotrofina (GnRH), hormônio foliculoestimulante (FSH), hormônio luteinizante (LH), estrogênio (E2) e progesterona (P4) foram realizados usando kits comerciais Elisa (MyBioSource). O perfil hormonal de todos os grupos de tratamento das novilhas bubalinas apresentou variações significativas (P < 0,05) em relação ao grupo controle (G-1). Esses resultados indicam supressão no perfil hormonal reprodutivo de búfalos Nili-Ravi na exposição a P. multocida B:2 e seus imunógenos. Essa influência garante que a exposição à SH possa ser uma possível razão para o atraso da puberdade e o baixo desempenho reprodutivo em búfalos Nili-Ravi.

Effects of Pasturella Multocida B:2 and its immunogens (LPS and OMP) on reproductive hormones in Nili-Ravi Buffaloes

Abstract Livestock is a fundamental part of the agriculture industry in Pakistan and contributes more than 11.53% to GDP. Among livestock species, the buffaloes are regarded as the black gold of Pakistan. Being the highest milk producers globally, Nili-Ravi buffaloes are the most famous ones. Buffaloes are affected by many endemic diseases, and "Hemorrhagic septicemia" (HS) is one of them. This study was designed to ascertain the effects of experimental exposure ofP. multocida B:2 (oral) and its immunogens, i.e., LPS (oral and intravenous) and OMP (oral and subcutaneous) on reproductive hormonal profiles in Nili-Ravi buffaloes. Repeated serum samples were collected from the jugular vein of experimental animals for 21 days (0, 02, 04, 08, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 and 504 hours). Hormonal assays to determine the serum concentrations of Gonadotropin-releasing hormone (GnRH), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Estrogen (E2) and progesterone (P4) were performed using (MyBioSource) commercial Elisa kits. The hormonal profile of all treatment groups of the buffalo heifers exhibited significant (P 0.05) variations as compared to the control group (G-1). These results indicate suppression in Nili-Ravi buffaloes' reproductive hormonal profile on exposure to P. multocida B:2 and its immunogens. This influence warrants that exposure to H.S may be a possible reason for delayed puberty and poor reproduction performance in Nili-Ravi buffaloes.

Resumo A pecuária é uma parte fundamental da indústria agrícola no Paquistão e contribui com 11,53% do PIB nacional. Entre as espécies de gado, os búfalos são considerados o ouro negro do Paquistão. Sendo os maiores produtores de leite em todo o mundo, os búfalos Nili-Ravi são os mais famosos. Os búfalos são afetados por muitas doenças endêmicas, entre as quais a septicemia hemorrágica (SH). Este estudo busca verificar os efeitos da exposição experimental de P. multocida B:2 (oral) e seus imunógenos, ou seja, LPS (oral e intravenoso) e OMP (oral e subcutâneo), nos perfis hormonais reprodutivos em búfalos Nili-Ravi. Amostras de soro repetidas foram coletadas da veia jugular de animais experimentais por 21 dias (0, 2, 4, 8, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 e 504 horas). Os ensaios hormonais para determinar as concentrações séricas do hormônio liberador de gonadotrofina (GnRH), hormônio foliculoestimulante (FSH), hormônio luteinizante (LH), estrogênio (E2) e progesterona (P4) foram realizados usando kits comerciais Elisa (MyBioSource). O perfil hormonal de todos os grupos de tratamento das novilhas bubalinas apresentou variações significativas (P 0,05) em relação ao grupo controle (G-1). Esses resultados indicam supressão no perfil hormonal reprodutivo de búfalos Nili-Ravi na exposição a P. multocida B:2 e seus imunógenos. Essa influência garante que a exposição à SH possa ser uma possível razão para o atraso da puberdade e o baixo desempenho reprodutivo em búfalos Nili-Ravi.

Diagnostic value of serum signal peptide-CUB-EGF-like domain-containing protein 1 levels in patients with acute appendicitis

SUMMARY OBJECTIVE: Acute appendicitis is one of the most common surgical causes of an acute abdomen among patients admitted to the emergency room due to abdominal pain. The clinical diagnosis of acute appendicitis is usually difficult and is made by evaluating the clinical, laboratory, and radiological findings together. The aim of this study was to investigate the diagnostic potential of signal peptide-CUB-EGF-like domain-containing protein 1 as a biomarker for acute appendicitis. METHODS: A total of 67 adult patients without any comorbidities who presented to the emergency department with abdominal pain and were clinically diagnosed with acute appendicitis were included in the case group. The patients included in the study were classified into the negative appendectomy group and the acute appendicitis group according to their histopathological final diagnosis. In addition, 48 healthy volunteers without comorbidities were included in the control group. Signal peptide-CUB-EGF-like domain-containing protein 1 levels of patients and the control group were measured. RESULTS: According to postoperative histopathological examinations of the patients, 7 (10.4%) patients were diagnosed with negative appendectomy, and 60 (89.6%) patients were diagnosed with acute appendicitis. Signal peptide-CUB-EGF-like domain-containing protein 1 levels were higher in the patients with acute appendicitis than in negative appendectomy patients (p=0.012). Signal peptide-CUB-EGF-like domain-containing protein 1 levels were also higher in the case group compared to the control group (p=0.001). CONCLUSION: The admission signal peptide-CUB-EGF-like domain-containing protein 1 level was significantly higher in adults with acute appendicitis. The SCUBE1 level is a novel but promising biomarker that aids in the diagnosis of acute appendicitis.

Expression, purification and micelle reconstruction of the transmembrane domain of the human amyloid precursor protein for NMR studies / 生物工程学报

The multiple-step cleavage of amyloid precursor protein (APP) generates amyloid-β peptides (Aβ), highly toxic molecules causing Alzheimer's disease (AD). The nonspecific cleavage between the transmembrane region of APP (APPTM) and γ-secretase is the key step of Aβ generation. Reconstituting APPTM under physiologically-relevant conditions is crucial to investigate how it interacts with γ-secretase and for future AD drug discovery. Although producing recombinant APPTM was reported before, the large scale purification was hindered by the use of biological protease in the presence of membrane protein. Here, we expressed recombinant APPTM in Escherichia coli using the pMM-LR6 vector and recovered the fusion protein from inclusion bodies. By combining Ni-NTA chromatography, cyanogen bromide cleavage, and reverse phase high performance liquid chromatography (RP-HPLC), isotopically-labeled APPTM was obtained in high yield and high purity. The reconstitution of APPTM into dodecylphosphocholine (DPC) micelle generated mono dispersed 2D 15N-1H HSQC spectra in high quality. We successfully established an efficient and reliable method for the expression, purification and reconstruction of APPTM, which may facilitate future investigation of APPTM and its complex in more native like membrane mimetics such as bicelle and nanodiscs.

Relationship between BNIP3L and mitochondrial dysfunction in hippocampus of mice with sepsis-associated encephalopathy / 中华麻醉学杂志

Objective:To evaluate the relationship between B-cell lymphoma/adenovirus E1B19 kDa-interacting protein 3-like protein (BNIP3L)/adenovirus E1B-interacting protein and mitochondrial dysfunction in the hippocampus of mice with sepsis-associated encephalopathy (SAE).Methods:One hundred and eighty C57BL/6J mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=45 each) using a random number table method: control group (C group), sham operation group (Sham group), SAE group, and SAE+ BNIP3L agonist carfilzomib group (SC group). The sepsis model was developed by cecal ligation and puncture (CLP) in anesthetized animals. In SC group, carfilzomib 2 mg/kg was intraperitoneally injected at 2 h after CLP. Twenty mice in each group were selected, and the survival at 7 days after operation was recorded. Eight surviving mice in each group were selected at 1 week after CLP for Morris water maze test. The remaining mice were sacrificed at 24 h after surgery, and the hippocampal tissues were harvested for determination of the expression of BNIP3L (by immunofluorescence) and BNIP3L in mitochondrial protein (by Western blot) and for microscopic examination of the morphological structure of mitochondria. The mitochondrial ATP content was measured by fluorescein-fluorescence enzyme luminescence method, and the mitochondrial membrane potential (MMP) was measured by fluorescence spectrophotometry. Results:Compared with C and Sham groups, the survival rate was significantly decreased, the escape latency was prolonged, the time of staying at the original platform quadrant was shortened, and the number of crossing the original platform region was decreased, the expression of BNIP3L in the hippocampal mitochondria was down-regulated, the MMP and content of mitochondrial ATP were decreased ( P<0.05), the intensity of fluorescence of BNIP3L in the hippocampus was decreased, and the damage to mitochondrial ultrastructure was marked in SAE group. Compared with SAE group, the survival rate was significantly increased, the escape latency was shortened, the time of staying at the original platform quadrant was prolonged, and the number of crossing the original platform region was increased, the expression of BNIP3L in the hippocampal mitochondria was up-regulated, the MMP and content of mitochondrial ATP were increased ( P<0.05), the intensity of fluorescence of BNIP3L in the hippocampus was decreased, and the damage to mitochondrial ultrastructure was attenuated in SC group. Conclusions:BNIP3L-mediated mitochondrial dysfunction may be involved in the mechanism of SAE developed in mice.

Development of fibroblast activation protein-targeted molecular probes in non-neoplastic diseases / 中华核医学与分子影像杂志

Fibroblast activation protein (FAP) is an important molecular marker of cancer-associated fibroblasts (CAFs). FAP is selectively expressed in more than 90% of epithelial carcinomas, but barely expressed in normal tissues. In recent years, a variety of radiolabeled molecular probes based on FAP inhibitor (FAPI) have been developed and used for imaging of malignant tumors. FAP is also highly expressed in some non-neoplastic diseases related to chronic inflammation and tissue remodeling, including arthritis, atherosclerosis, fibrosis of myocardial infarction, cirrhosis, and idiopathic pulmonary fibrosis. FAPI imaging shows a potential in these diseases. This paper reviews the current status of radionuclide labeled FAPI and the application of which in non-neoplastic diseases.

Usefulness of the outer membrane proteins of Shigella sonnei in developing an antibody-based immunoassay for the diagnosis of shigellosis

@#Conventional culture method and biochemical tests remain as the ‘gold standard’ method for the identification of S. sonnei which are time-consuming. We have discovered previously the potential of three OMPs of S. sonnei (33.3 kDa, 43.8 kDa and 100.3 kDa) as biomarkers in the diagnostic test for shigellosis. Here, we evaluated the performance of the outer membrane proteins of S. sonnei for the development of an antibody-based immunoassay for the detection of S. sonnei infections. All threetarget proteins were specifically recognized when probed with S. sonnei sera. In addition, another two potential proteins of molecular weight 29.0 kDa and 88.2 kDa in size were also exclusively recognized by the IgA when probed with S. sonnei sera. The optimized ELISA demonstrated higher sensitivity and specificity which exceeded 86.0%. In conclusion, the identified target proteins showed great potential as diagnostic biomarkers for the detection of S. sonnei infections in patients.

Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor

P-glycoprotein (P-gp) highly expressed in cancer cells can lead to multidrug resistance (MDR) and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment. In this study, we established a label-free and detergent-free system combining surface plasmon resonance (SPR) biosensor with styrene maleic acid (SMA) polymer membrane proteins (MPs) stabilization technology to screen potential P-gp inhibitors. First, P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes (SMALPs). Following that, SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system, and the affinity between P-gp and small molecule ligand was determined. The methodological investigation proved that the screening system had good specificity and stability. Nine P-gp ligands were screened out from 50 natural products, and their affinity constants with P-gp were also determined. The in vitro cell verification experiments demonstrated that tetrandrine, fangchinoline, praeruptorin B, neobaicalein, and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin (Adr). Moreover, tetrandrine, praeruptorin B, and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp. This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system. SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp. The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification.

Role of STING signaling pathway in CORM-3-induced reduction of hepatocyte pyroptosis and apoptosis in a rat model of hepatic ischemia-reperfusion / 中华麻醉学杂志

Objective:To evaluate the role of stimulator of interferon genes (STING) signaling pathway in carbon monoxide (CO)-releasing molecule-3 (CORM-3)-induced reduction of hepatocyte pyroptosis and apoptosis in a rat model of hepatic ischemia-reperfusion.Methods:Forty-eight clean-grade healthy male Sprague-Dawley rats, aged 9-11 weeks, weighing 320-380 g, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (S group), ischemia-reperfusion group (IR group), CORM-3 group (C group) and STING agonist ADU-S100 group (A group).Hepatic ischemia-reperfusion injury models were developed by reversible ligation of left middle hepatic artery, portal vein and bile duct branches for 45 min, followed by reperfusion in anesthetized animals in IR, C and A groups.In group C, CORM-3 4 mg/kg was injected into the femoral vein immediately after reperfusion.The equal volume of normal saline containing dimethyl sulfoxide was injected into the femoral vein in S, IR and A groups.At 1.5 h after injection into the femoral vein, ADU-S100 10 mg/kg was intraperitoneally injected in A group, and the equal volume of normal saline was given instead in S, IR and C groups.The serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were determined at 3 h of reperfusion.The rats were sacrificed at 12 h of reperfusion, and liver tissues were collected for determination of the content of CO (by colorimetry), expression of interleukin-1beta (IL-1β), IL-18, Bcl-2, Bax, interferon regulatory factor 3 (IRF3), phosphorylated IRF3 (p-IRF3), STING, NOD-like receptor protein 3 (NLRP3), aspirin D (GSDMD) and activated caspase-1 (by Western blot), and pyroptosis and apoptosis rates of hepatocytes (by immunofluorescence staining).The liver injury was scored. Results:Compared with group S, the serum ALT and AST concentrations, liver injury score, CO content, and pyroptosis and apoptosis rates of hepatocytes were significantly increased, and the expression of IL-1β, IL-18, p-IRF3, STING, NLRP3, GSDMD and activated caspase-1 was up-regulated, and the Bcl-2/Bax ratio was decreased in group IR ( P<0.05).Compared with group IR, the serum ALT and AST concentrations, liver injury score, and pyroptosis and apoptosis rates of hepatocytes were significantly decreased, the CO content was increased, the expression of IL-1β, IL-18, p-IRF3, STING, NLRP3, GSDMD and activated caspase-1 was down-regulated, and the Bcl-2/Bax ratio was increased in group C ( P<0.05).Compared with group C, the serum ALT and AST concentrations, liver injury score, and pyroptosis and apoptosis rates of hepatocytes were significantly increased, the CO content was decreased, the expression of IL-1β, IL-18, p-IRF3, STING, NLRP3, GSDMD and activated caspase-1 was up-regulated, and the Bcl-2/Bax ratio was decreased in group A ( P<0.05). Conclusions:The mechanism by which CORM-3 attenuates hepatocyte pyroptosis and apoptosis may be related to the inhibition of activation of STING signaling pathway in a rat model of hepatic ischemia-reperfusion.

Relationship between cGAS-STING signaling pathway and ferritinophagy in early stage of cerebral ischemia-reperfusion in mice / 中华麻醉学杂志

Objective:To evaluate the relationship between the second messenger cyclic GMP-AMP (cGAS)-cyclic GMP-AMP receptor stimulator of interferon genes (STING) signaling pathway and ferritinophagy in the early stage of cerebral ischemia-reperfusion (I/R) in mice.Methods:Twenty-four clean-grade healthy male C57BL/6 mice, aged 6-8 weeks, weighing 21-25 g, were divided into 4 groups ( n=6 each) using a random number table method: sham group, cerebral I/R injury group (CIRI group), cerebral I/R injury + cGAS inhibitor group (CIRI + RU group), and cerebral I/R injury + cGAS inhibitor + overexpressed nuclear receptor coactivator 4 (NCOA4) group (MCAO + RU + LV-NCOA4 group). The model of cerebral I/R injury was developed using the middle cerebral artery occlusion (MCAO) in anesthetized animals.In CIRI+ RU group, cGAS inhibitor 5 mg/kg was intraperitoneally injected at 10 min before reperfusion.In CIRI+ RU+ LV-NCOA4 group, NCOA4-overexpressing lentivirus (1×10 9 TU/ml) 2 μl was injected into the ventricle at 7 days before MCAO, and the other operations were the same as those previously described in CIRI+ RU group.After 6 h of reperfusion, the neurological function deficits were assessed and scored, then the mice were sacrificed, and brains were removed for determination of the cerebral infarct size (by TTC method), MDA content (by TBA method), activity of SOD (by WST-1 method), and expression of cGAS, STING, NCOA4, ferritin, and microtubule-associated protein 1 light chain 3B (LC3B) (by Western blot). Results:Compared with Sham group, the neurological function deficit score and cerebral infarct size were significantly increased, SOD activity was decreased, MDA content was increased, the expression of cGAS, STING, NCOA4 and LC3B was up-regulated, and the expression of ferritin was down-regulated in CIRI group ( P<0.05). Compared with CIRI group, the neurological function deficit score and cerebral infarct size were significantly decreased, SOD activity was increased, MDA content was decreased, the expression of cGAS, STING, NCOA4 and LC3B was down-regulated, and the expression of ferritin was up-regulated in CIRI+ RU group ( P<0.05). Compared with CIRI+ RU group, the neurological function deficit score and cerebral infarct size were significantly increased, SOD activity was decreased, MDA content was increased, the expression of cGAS, STING, NCOA4 and LC3B was up-regulated, and the expression of ferritin was down-regulated in CIRI group ( P<0.05), and no significant change was found in the expression of cGAS and STING in CIRI+ RU+ LV-NCOA4 group ( P>0.05). Conclusions:The cGAS-STING signaling pathway can promote the over-activation of ferritinophagy, enhance oxidative stress, and thus induce early CIRI in mice.

Relationship between long-term learning and memory impairment induced by sevoflurane anesthesia and PSD-95/Kalirin-7/Rac1 signaling pathway in neonatal rats / 中华麻醉学杂志

Objective:To evaluate the relationship between long-term learning and memory impairment induced by sevoflurane anesthesia and postsynaptic density protein-95 (PSD-95)/Kalirin-7/Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling pathway in neonatal rats.Methods:Sixty SPF male Wistar rats, aged 7 days, weighing 12-18 g, were divided into 5 groups ( n=12 each) using a random number table method: control group (group C), 1% sevoflurane anesthesia for 2 h group (group S 1), 1% sevoflurane anesthesia for 4 h group (group S 2), 2% sevoflurane anesthesia for 2 h group (group S 3) and 2% sevoflurane anesthesia for 4 h group (group S 4). Morris water maze test was performed at 4, 8 and 12 weeks after anesthesia.The rats were sacrificed after the last Morris water maze test, and the hippocampal tissues were obtained for microscopic examination of the pathological changes (using HE staining), neuron apoptosis (by TUNEL staining), and expression of PSD-95, Kalirin-7 and Rac1 protein and mRNA (by Western blot and quantitative real-time polymerase chain reaction). The apoptosis rate was calculated. Results:Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the time of stay in the target quadrant was shortened, and the apoptosis rate of hippocampal neurons was increased at 4th, 8th and 12th weeks after anesthesia, phosphorylated Rac1/Rac1 ratio was decreased, and the expression of PSD-95 and Kalirin-7 protein and mRNA was down-regulated in S 1, S 2, S 3 and S 4 groups ( P<0.05). Compared with group S 4, the escape latency was significantly shortened, the number of crossing the original platform was increased, the time of stay in the target quadrant was prolonged, and the apoptosis rate of hippocampal neurons was decreased, phosphorylated Rac1/Rac1 ratio was increased, the expression of PSD-95 and Kalirin-7 protein and mRNA was up-regulated, and the histopathological changes of hippocampal tissues were attenuated in S 1, S 2 and S 3 groups ( P<0.05). Conclusions:The mechanism by which sevoflurane anesthesia induces long-term learning and memory impairment may be related to inhibition of activity of PSD-95/Kalirin-7/Rac1 signaling pathway in hippocampi of neonatal rats.

68Ga-DOTA-VAP molecular probe targeted glucose-regulated protein 78 for specific microPET/CT imaging of tumors / 中华核医学与分子影像杂志

Objective:To evaluate the specificity of 68Ga-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA)-Ser-Asn-Thr-Arg-Val-Ala-Pro (SNTRVAP, VAP) molecular probe targeting glucose-regulated protein 78(GRP78) in vivo and in vitro and the feasibility of 68Ga-DOTA-VAP microPET/CT imaging in the diagnosis of GRP78-positive tumors. Methods:68Ga-DOTA-VAP was prepared by the combination of bifunctional chelating agent DOTA and VAP, followed by 68Ga labeling. Western blotting experiment was perfomed to detect the expression of GRP78 in U87MG, BxPC-3, and 293T cell lines, at the same time, cold polypeptide blocked experiments were conducted to verify the specific binding of 68Ga-DOTA-VAP to cells. U87MG and BxPC-3 subcutaneous transplantation tumor mouse models were established and the biodistribution of 68Ga-DOTA-VAP were explored in vivo. The imaging effect of 68Ga-DOTA-VAP in GRP78-positive tumor-bearing mouse models was evaluated by microPET/CT. Independent-sample t test, one-way analysis of variance and Dunnett t test were used for data analysis. Results:68Ga-DOTA-VAP was easily prepared with labeling yield and radiochemical purity >98%. It had good stability in vitro, and its radiochemical purity was still (98.27±0.22)% after 2 h. GRP78 was highly expressed in U87MG and BxPC-3 cells, but lowly expressed in 293T cells ((0.78±0.02), (0.53±0.05) and (0.36±0.03), F=102.22, P<0.001; t values: 0.43, 0.18, both P<0.01). The uptake of 68Ga-DOTA-VAP in U87MG cells was higher than that in BxPC-3 cells (5 154.00±216.70 vs 4 344.00±60.88; t=3.10, P=0.027). Excessive unlabeled VAP polypeptide could significantly reduce the uptake of 68Ga-DOTA-VAP both in U87MG and BxPC-3 cells (3 324.00±54.14, 3 270.00±131.10; t values: 8.19, 7.43, both P<0.01). The uptake of 68Ga-DOTA-VAP in U87MG tumor tissue was higher than that in BxPC-3 tumor tissue ((1.98±0.20) vs (1.30±0.08) percentage activity of injection dose per gram of tissue (%ID/g); t=5.48, P=0.005), while co-injection of excessive unlabeled VAP polypeptide significantly reduced the uptake in U87MG and BxPC-3 tumors ((0.99±0.02) and (0.62±0.05) %ID/g; t values: 8.32, 12.25, both P<0.05). MicroPET/CT imaging showed that 68Ga-DOTA-VAP could clearly display U87MG and BxPC-3 tumors, and U87MG had a better imaging effect. The tumors could not be clearly visualized after co-injection of excessive VAP polypeptide. Conclusion:68Ga-DOTA-VAP molecular probe binds with GRP78 specifically and can reflect the expression level of GRP78 in vivo, which may be a promising probe for the specific imaging diagnosis of GRP78-positive tumors.

Automatic synthesis and in vivo imaging of Al 18F-NOTA-FAPI-04 / 中华核医学与分子影像杂志

Objective:To automatically synthesize Al 18F-1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA)-fibroblast activation protein inhibitor (FAPI)-04, perform PET/CT imaging in vivo, and evaluate its diagnostic efficacy on tumors. Methods:Al 18F-NOTA-FAPI-04 was produced in All-in-one automatic synthesis module and its quality control was conducted by high performance liquid chromatography (HPLC) equipped with a radioactive detector. Al 18F-NOTA-FAPI-04 PET/CT imaging was performed in normal BALB/c mice ( n=3) and 4T1 breast cancer models ( n=3) to determine its biodistribution. Then Al 18F-NOTA-FAPI-04 and 18F-FDG PET/CT imaging were performed in a hepatocellular carcinoma patient (male, 51 years old). Results:The synthesis time of Al 18F-NOTA-FAPI-04 was about 35 min, and the radiochemical yield was (25.2±1.9)% (attenuation correction, n=3). The product was colorless transparent solution with pH value of 7.0-7.5, and the specific activity was (46.11±3.07) GBq/μmol (attenuation correction, n=3). The radiochemical purity was above 99.0% and was still above 98.0% at room temperature after 6 h. PET/CT imaging in mice showed that physiological uptake of Al 18F-NOTA-FAPI-04 was mainly in biliary system and bladder, and Al 18F-NOTA-FAPI-04 highly concentrated in tumor xenografts. PET/CT imaging in the patient showed that Al 18F-NOTA-FAPI-04 obtained high tumor background ratio (TBR) values of 4.1, 8.9, 5.4, 4.8, 2.2 in parasternal lymph nodes, anterior diaphragmatic lymph nodes, hilar lymph nodes, pancreaticoduodenal ligament lymph nodes, abdominal aortic lymph nodes, respectively, while TBR values were 1.0, 2.8, 5.0, 2.1, 1.1 by 18F-FDG. Conclusions:Al 18F-NOTA-FAPI-04 can be synthesized with short time, high radiochemical yield and good stability using All-in-one module. Al 18F-NOTA-FAPI-04 PET/CT imaging has high contrast and excellent diagnostic efficacy on tumors.

The association between vesicle-associated membrane protein - 8 A/G gene polymorphism and the risk of acute myocardial infarction / La relación entre el polimorfismo A/G del gen de la proteína de membrana asociada a vesículas-8 y el riesgo de infarto agudo de miocardio

Abstract Introduction Until now, only few studies have reported the correlation between vesicle-associated membrane protein-8 (VAMP-8) A/G gene polymorphism and acute myocardial infarction. Whereas, theoretically, VAMP-8 plays a pivotal role in the pathogenesis of acute myocardial infarction through platelet activation, secretion, and aggregation. Objective To investigate the association between VAMP-8 A/G gene polymorphism and the risk of acute myocardial infarction. Methods A cross-sectional study was carried out at Saiful Anwar General Hospital during June 2013 - May 2014. A Mae II enzyme with restriction fragment length polymorphism method was used to genotype VAMP-8 A/G gene polymorphisms in acute myocardial infarction and control groups. A multiple logistic regression test was used to analyze the association between VAMP-8 A/G gene polymorphism and the risk of acute myocardial infarction. Results A total of 35 controls and 97 acute myocardial infarction patients from our Hospital during the period were enrolled for our study. Our results found that VAMP-8 A/G gene polymorphism was not associated with the risk of acute myocardial infarction. Moreover, we also failed to confer the association between VAMP-8 A/G gene polymorphism and both smoking and hypertension among patients with acute myocardial infarction. Furthermore, in the setting of premature acute myocardial infarction, the correlation also failed to confirm. Conclusion In our population, there is no association between VAMP-8 A/G gene polymorphism and the risk of acute myocardial infarction.

Resumen Introducción Hasta la fecha, solo unos pocos estudios han reportado la correlación entre el polimorfismo A/G del gen de la proteína de membrana asociada a vesículas-8 (VAMP-8, por sus siglas en inglés) y el infarto agudo de miocardio. Si bien, en teoría, VAMP-8 juega un papel fundamental en la patogénesis del infarto agudo de miocardio a través de la activación, secreción y agregación plaquetaria. Objetivo Investigar la relación entre el polimorfismo A/G del gen VAMP-8 y el riesgo de infarto agudo de miocardio. Métodos: Se llevó a cabo un estudio transversal en Siful Anwar General Hospital entre junio del 2013 y mayo del 2014. Se utilizó la técnica de polimorfismos de longitud de fragmentos de restricción con la enzima Mae II para genotipificar los polimorfismos A/G del gen VAMP-8 en grupos de infarto agudo de miocardio y de control. Se aplicó una prueba de regresión logística múltiple para analizar la relación entre el polimorfismo A/G del gen VAMP-8 y el riesgo de infarto agudo de miocardio. Resultados Se incluyeron un total de 35 controles y 97 pacientes con infarto agudo de miocardio de nuestro Hospital durante el periodo del estudio. Nuestros resultados encontraron que el polimorfismo A/G del gen VAMP-8 no estaba relacionado con el riesgo de infarto agudo de miocardio. Por otra parte, tampoco pudimos establecer una relación entre el polimorfismo A/G del gen VAMP-8 y tanto tabaquismo como hipertensión en pacientes con infarto agudo de miocardio. Asimismo, en el contexto de infarto agudo de miocardio prematuro, tampoco se confirmó la correlación. Conclusión: En nuestra población, no existe una relación entre el polimorfismo A/G del gen VAMP-8 y el riesgo de infarto agudo de miocardio.

El gran papel de Klotho / The Great Role of Klotho / O grande papel de Klotho

Resumen: Klotho es una proteína transmembrana de un solo paso que consta de 1012 aminoácidos y se expresa fuerte y débilmente en células epiteliales renales tubulares distales y proximales, respectivamente. Existen cuatro grupos de proteínas Klotho. El gen αKlotho se expresa abundantemente en riñones, glándulas paratiroides, plexo coroideo, así como en la corteza cerebral, la médula espinal, el cerebelo, el hipotálamo, la hipófisis, las glándulas paratiroides, el ovario, los testículos, las células epiteliales del seno, la placenta, el páncreas, el oído interno, el endotelio vascular del músculo liso y el intestino. Klotho exhibe múltiples funciones, además de la excreción de fosfato, incluida la mejora del estrés oxidativo y la inhibición de vías de señalización del factor de crecimiento de insulina, Wnt/β-catenina, la transformación del factor de crecimiento-β1 y el objetivo mecanicista de la señalización de rapamicina, de modo que se obtiene un importante papel dentro de un sinnúmero de eventos patológicos como, por ejemplo, el que causó la reciente pandemia generada por el COVID-19. Tanto nuevos trabajos como anteriores en humanos y ratones (p. ej., el impacto de Klotho en cuadros de lesión pulmonar aguda) proporcionan una fuerte justificación para examinar más a fondo el papel del Klotho en la salud y el envejecimiento.

Abstract: Klotho is a single-pass transmembrane protein consisting of 1012 amino adds and it is strongly and weakly expressed in distal and proximal renal tubular epithelial cells, respectively. There are four groups of Klotho proteins. The αKlotho gene is abundantly expressed in kidneys, parathyroid glands, choroid plexus, as well as in the cerebral cortex, spinal cord, cerebellum, hypothalamus, pituitary gland, parathyroid glands, ovary, testis, breast epithelial cells, placenta, pancreas, inner ear, smooth muscle vascular endothelium, and intestine. Klotho exhibits multiple functions in addition to phosphate excretion, including enhancement of oxidative stress and inhibition of insulin growth factor signaling pathways, Wnt/β-catenin, transforming growth factor-β1 and mechanistic target of rapamycin, thus eliciting an important role within a myriad of pathological events such as, for example, the one caused by the recent COVID-19 pandemic. Both new and previous work in humans and mice (e.g., the impact of Klotho on acute lung injury) provide a strong rationale to further examine the role of Klotho in health and aging.

Resumo: Klotho é uma proteína transmembrana de urna etapa que consiste em 1012 aminoácidos e é forte e fracamente expressa em células epiteliais renais tubulares distais e proximais, respectivamente. Existem quatro grupos de proteínas Klotho. O gene αKlotho é abundantemente expresso em rins, glándulas paratireóides, plexo coróide, bem como no córtex cerebral, medula espinhal, cerebelo, hipotálamo, glândulas pituitárias, ovário, testículos, células epiteliais do seio, placenta, páncreas, ouvido interno, endotélio vascular do músculo liso e intestinal. Klotho apresenta múltiplas funções, além da excreção de fosfato, incluindo a melhora do estresse oxidativo e inibição das vias de sinalização do fator de crescimento insulínico, WNT/β-catenina, transformação do fator de crescimento-β1 e o objetivo mecanicista da sinalização da rapamicina, de modo que um papel importante dentro de inúmeros eventos patológicos, como o que causou a recente pandemia gerada pela COVID-19. Estudos novos e anteriores em humanos e camundongos (por exemplo, o impacto do Klotho na lesão pulmonar aguda) fornecem urna forte justificativa para examinar melhor o papel do Klotho na saúde e no envelhecimento.

Quinine oxidoreductase 1 inhibits the secretion of IL-8 in Mycoplasma pneumoniae lipid-associated membrane proteins stimulated the THP-1 monocyte line / 中华微生物学和免疫学杂志

Objective:To investigate the effects and regulation mechanism of lipid-associated membrane proteins (LAMPs) derived from Mycoplasma pneumoniae( Mp) on the expression of quinine oxidoreductase 1 (NQO-1) in human monocyte cell line THP-1 cells, and to know the effect of NQO-1 to interleukin 8 secretion in LAMPs stimulated cells, so as to better understand the regulation mechanism upon Mp infection. Methods:Mp were cultivated and the precipitate was collected to extract LAMPs. The cytotoxicity of LAMPs to THP-1 cells was analyzed by using CCK8 test. THP-1 cells were cultured in vitro with different concentrations of LAMPs for different times, and the expression of NQO-1 protein was detected by Western blot. Nrf2 siRNA was used to investigate the role of Nrf2 in NQO-1 expression in LAMPs induced cells, and NQO-1 inhibitor Diminutol was performed to test whether they blocked interleukin 8 (IL-8) secretion when treated with LAMPs in THP-1 cells. Results:LAMPs extracted from Mp had no cytotoxicity to THP-1 cells. The expression of NQO-1 protein in LAMPs-stimulated THP-1 cells showed a dose-dependent and time-dependent manner. The production of NQO-1 protein reached peaks when treated with 5.0 μg/ml or 7.5 μg/ml of LAMPs for 12 h. Silencing of Nrf2 by siRNA significantly decreased NQO-1 production, and blocking NQO-1 by Dim increased the level of IL-8 in LAMPs-stimulated cells. Conclusions:LAMPs derived from Mp induced the expression of NQO-1 protein in THP-1 cells via Nrf2, and NQO-1 can inhibit IL-8 secretion in LAMPs stimulated monocytes.

Prenatal diagnosis of Joubert syndrome caused by de novo mutations of CPLANE1 gene / 中华围产医学杂志

We report a case of fetal cerebellar vermis dysplasia diagnosed prenatally by ultrasonography. Ultrasonography of the 27-year-old woman at 20 +6 gestational weeks revealed partial separation of the cerebellar vermis (Dandy-Walker variants), unclosable upper and lower lips, and polydactyly, based on which a preliminary diagnosis of multiple fetal malformations was made. Karyotype and chromosomal microarray (CMA) analysis of the amniotic fluid showed no abnormality. After genetic counseling, amniocentesis was performed again for a whole-exome sequencing test. The results suggested that there are compound heterozygous variations of c.3435G>A(P.W1145X) and c.2941C>G(p. p981A) in the exon 19 and exon 17 of the CPLANE1 gene, which were both de novo mutations and inherited from the father and mother, respectively. The fetus was diagnosed as Joubert syndrome. Given the facial and limb deformities and a significant risk of neurological abnormalities of the fetus, the patient and her family decided to terminate the pregnancy.

Fetal familial hemophagocytic lymphohistiocytosis: a case report and literature review / 中华围产医学杂志

Objective:To discuss the clinical characteristics and genetic diagnosis of fetal familial hemophagocytic lymphohistiocytosis (FHL).Methods:Clinical data of a case of fetal FHL from Children's Hospital, Capital Institute of Pediatrics was analyzed, and related FHL cases at home and abroad were retrieved from PubMed, CNKI, and Wanfang databases using terms including "fetus", "neonate", and "familial hemophagocytic lymphohistiocytosis", from the establishment of the database to January 3, 2021, to summarize the characteristics of this disease.Results:This index case was found with fetal splenomegaly, free fluid in the abdominal cavity, and enlargement of the ventricle at 39 +3 weeks of gestation, and presented with fever, tachypnea, hepatosplenomegaly, skin ecchymosis and petechia, and lymphadenectasis after birth. Laboratory examination revealed pancytopenia, abnormal liver function, elevated ferritin and triglyceride, and decreased fibrinogen levels. CD107a excitation experiment showed decreased degranulation function of NK cell (ΔCD107a<5%). Hemophagocytosis was observed in the bone marrow smear. Genomic DNA sequence analysis demonstrated compound heterozygous mutations of c.118-308C>T and c.3002T>C in the UNC13D gene. All the above findings led to the diagnosis of FHL3. Despite chemotherapy with dexamethasone and cyclosporin, and symptomatic treatment after admission without hematopoietic stem cell transplantation, the baby died on day 52. A total of 15 papers related to fetal FHL, including 20 infants, were retrieved. Among these 21 cases (including the index case), the main clinical symptoms were fetal edema and hepatosplenomegaly, which may be accompanied by fetal distress and increased amniotic fluid volume, and postnatal fever, dyspnea, rash, and central nervous system involvement. Laboratory and imaging examination results were consistent with the diagnostic criteria for hemophagocytic hyperplasia. As far as we know, the reported fetal FHL gene mutations were PRF1 (FHL2) and UNC13D gene mutation (FHL3), in which reduced expression of perforin and granzyme can be detected, respectively. Dexamethasone, cyclosporin, etoposide, and other chemotherapy and symptomatic treatment are the primary treatments currently, and alternative therapies include intrauterine chemotherapy in the third trimester and postnatal hematopoietic stem cell transplantation. Among the 21 cases, including the index case, intrauterine death occurred in four cases, 13 children died at different times after birth, and only four children survived, among which the eldest one was 12 years old. Conclusions:FHL is a condition with atypical early signs, high mortality rate and treatment difficulties. Fetal FHL should be considered in differential diagnosis in fetuses with edema or hepatosplenomegaly besides hemolysis, infection, autoimmune diseases, and hereditary problems. Therefore, with immunotechnology and gene sequencing, early diagnosis and treatment can be prompted to improve the prognosis of this group of population.

Comparison of clinical utility of 18F-FAPI-42 and 18F-FDG PET/CT imaging in the diagnosis of newly diagnosed lung cancer / 中华核医学与分子影像杂志

Objective:To compare the clinical utility of 18F-fibroblast activating protein inhibitor (FAPI)-42 and 18F-fluorodeoxyglucose (FDG) PET/CT imaging in newly diagnosed lung cancer patients. Methods:From May 2020 to September 2021, the images of 43 lung cancer patients (32 males, 11 females, age: 37-80 years) who pathologically confirmed and received 18F-FDG and 18F-FAPI-42 PET/CT within 2 weeks in the First Affiliated Hospital of Guangzhou Medical University were prospectively analyzed. The maximum standardized uptake value (SUV max) of 18F-FDG and 18F-FAPI-42 and the number of lesions detected by 2 imaging methods were compared by using paired t test and Wilcoxon rank sum test. Results:The 43 newly diagnosed lung cancer patients included 35 adenocarcinoma, 2 squamous cell carcinoma, 4 small cell lung cancer, and 2 high-grade neuroendocrine tumors. 18F-FAPI-42 had a very high tumor uptake (SUV max: 12.24±3.97) and lesion detection rate (positive rate: 100%(37/37)) in primary lung adenocarcinoma and squamous cell carcinoma. The uptake of 18F-FAPI-42 in lymph node (10.13±5.43), pleura (6.75(4.96, 8.58)) and bone lesion (7.18(4.33, 9.66)) were significantly higher than those of 18F-FDG (6.35±3.30, 2.69(1.81, 5.00), 4.38(2.96, 6.36); t=12.19, z values: 5.47, 5.79, all P<0.001). In lung adenocarcinoma and squamous cell carcinoma, although the uptake of 18F-FAPI-42 in brain metastases was significantly lower than that of 18F-FDG (0.72(0.15, 1.82) vs 6.53(4.65, 9.34); z=6.42, P<0.001), the tumor/background (T/B) ratio was significantly higher than that of 18F-FDG (3.54(1.15, 14.88) vs 0.96(0.77, 1.04); z=6.05, P<0.001). In lung adenocarcinoma and squamous cell carcinoma, the number of lesions detected by 18F-FAPI-42 PET/CT was significantly more than that of 18F-FDG (lymph node: 6.0(2.3, 11.5) vs 4.5(2.0, 10.8); brain: 2.0(1.0, 3.0) vs 0.0(0.0, 0.0); pleura: 6.0(2.8, 10.0) vs 4.0(0.8, 5.5); z values: 2.16, 3.10, 2.04, all P<0.05). However, in high-grade neuroendocrine tumors and small cell lung cancer, the SUV max of 18F-FAPI-42 in primary lesions (8.05±2.60), lymph node lesions (5.98±2.21) and brain lesions (0.44(0.13, 0.82)) were lower than those of 18F-FDG (16.28±5.17, 12.30±5.47, 4.94(4.84, 6.25); t values: 3.58, 7.52, z=3.06, all P<0.05). Conclusions:In lung adenocarcinoma and squamous cell carcinoma, 18F-FAPI-42 has a very high tumor uptake and lesion detection rate in primary tumor. In addition, compared with 18F-FDG PET/CT, 18F-FAPI-42 PET/CT shows clearer tumor contours and more lesions. Therefore, 18F-FAPI-42 is more suitable for preliminary staging of lung adenocarcinoma and squamous cell carcinoma than 18F-FDG, while the opposite is true in small cell lung cancer and high-grade neuroendocrine tumors.