Alda-1 alleviates brain injury after cardiopulmonary resuscitation by regulating acyl-CoA synthetase long-chain family member 4/glutathione peroxidase 4 pathway-mediated ferroptosis in swine / 中华危重病急救医学

OBJECTIVE@#To investigate whether the acetaldehyde dehydrogenase 2 specific activator, Alda-1, can alleviate brain injury after cardiopulmonary resuscitation (CPR) by inhibiting cell ferroptosis mediated by acyl-CoA synthetase long-chain family member 4/glutathione peroxidase 4 (ACSL4/GPx4) pathway in swine.@*METHODS@#Twenty-two conventional healthy male white swine were divided into Sham group (n = 6), CPR model group (n = 8), and Alda-1 intervention group (CPR+Alda-1 group, n = 8) using a random number table. The swine model of CPR was reproduced by 8 minutes of cardiac arrest induced by ventricular fibrillation through electrical stimulation in the right ventricle followed by 8 minutes of CPR. The Sham group only experienced general preparation. A dose of 0.88 mg/kg of Alda-1 was intravenously injected at 5 minutes after resuscitation in the CPR+Alda-1 group. The same volume of saline was infused in the Sham and CPR model groups. Blood samples were collected from the femoral vein before modeling and 1, 2, 4, 24 hours after resuscitation, and the serum levels of neuron specific enolase (NSE) and S100 β protein were determined by enzyme-linked immunosorbent assay (ELISA). At 24 hours after resuscitation, the status of neurologic function was evaluated by neurological deficit score (NDS). Thereafter, the animals were sacrificed, and brain cortex was harvested to measure iron deposition by Prussian blue staining, malondialdehyde (MDA) and glutathione (GSH) contents by colorimetry, and ACSL4 and GPx4 protein expressions by Western blotting.@*RESULTS@#Compared with the Sham group, the serum levels of NSE and S100β after resuscitation were gradually increased over time, and the NDS score was significantly increased, brain cortical iron deposition and MDA content were significantly increased, GSH content and GPx4 protein expression in brain cortical were significantly decreased, and ACSL4 protein expression was significantly increased at 24 hours after resuscitation in the CPR model and CPR+Alda-1 groups, which indicated that cell ferroptosis occurred in the brain cortex, and the ACSL4/GPx4 pathway participated in this process of cell ferroptosis. Compared with the CPR model group, the serum levels of NSE and S100 β starting 2 hours after resuscitation were significantly decreased in the CPR+Alda-1 group [NSE (μg/L): 24.1±2.4 vs. 28.2±2.1, S100 β (ng/L): 2 279±169 vs. 2 620±241, both P < 0.05]; at 24 hours after resuscitation, the NDS score and brain cortical iron deposition and MDA content were significantly decreased [NDS score: 120±44 vs. 207±68, iron deposition: (2.61±0.36)% vs. (6.31±1.66)%, MDA (μmol/g): 2.93±0.30 vs. 3.68±0.29, all P < 0.05], brain cortical GSH content and GPx4 expression in brain cortical was significantly increased [GSH (mg/g): 4.59±0.63 vs. 3.51±0.56, GPx4 protein (GPx4/GAPDH): 0.54±0.14 vs. 0.21±0.08, both P < 0.05], and ACSL4 protein expression was significantly decreased (ACSL4/GAPDH: 0.46±0.08 vs. 0.85±0.13, P < 0.05), which indicated that Alda-1 might alleviate brain cortical cell ferroptosis through regulating ACSL4/GPx4 pathway.@*CONCLUSIONS@#Alda-1 can reduce brain injury after CPR in swine, which may be related to the inhibition of ACSL4/GPx4 pathway mediated ferroptosis.

Síndrome antisintetasa: reporte de un caso / Antisynthetase syndrome: a case repor

El síndrome antisintetasa es una miopatía inflamatoria idiopática (MII) de origen autoinmune, poco frecuente, que se caracteriza por la presencia de autoanticuerpos antisintetasa ARNt (generalmente anti-Jo1), asociado frecuentemente a miositis, enfermedad pulmonar intersticial, poliartritis, manos de mecánico y fenómeno de Raynaud. Se reporta el caso de una mujer de 45 años de edad que presenta este síndrome con características fenotípicas de dermatomiositis y responde de forma favorable luego de la administración del tratamiento con glucocorticoides asociado a metotrexato.

Anti-synthetase syndrome is a rare autoimmune inflammatory myopathy characterized by autoantibodies against tRNA synthetases (most commonly anti-Jo1) with clinical features that include myositis, interstitial lung disease, polyarthritis, mechanic's hands and Raynaud's phenomenon. We report a 45-year-old woman who presented with dermatomyositis phenotypical features and a significant improvement with corticosteroids and metotrexate treatment.

Speckle type POZ adaptor protein (SPOP) and its role in cancer / La proteína adaptadora Speckle-type POZ (SPOP) y su papel en cáncer

Abstract Proteasomal degradation is an essential regulatory mechanism for cellular homeostasis maintenance. The speckle-type POZ adaptor protein (SPOP) is part of the ubiquitin ligase E3 cullin-3 RING-box1 complex, responsible for the ubiquitination and proteasomal degradation of biomolecules involved in cell cycle control, proliferation, response to DNA damage, epigenetic control, and hormone signaling, among others. Changes in SPOP have been associated with the development of different types of cancer, since it can act as a tumor suppressor mainly in prostate, breast, colorectal, lung cancer and liver cancer, due to point mutations and/or reduced expression, or as an oncogene in kidney cancer by protein overexpression. In endometrial cancer it has a dual role, since it can act as a tumor suppressor or as an oncogene. SPOP is a potential prognostic biomarker and a promising therapeutic target.

Resumen La degradación proteosómica es un mecanismo de regulación esencial para el mantenimiento de la homeostasis celular. La proteína adaptadora Speckle-type POZ (SPOP) hace parte del complejo ubiquitin ligasa E3 cullin-3 RING-box1, encargado de la ubiquitinación y degradación proteosomal de biomoléculas involucradas en el control del ciclo celular, proliferación, respuesta al daño de ADN, control epigenético, señalización hormonal, entre otros. Las alteraciones en SPOP han sido asociadas al desarrollo de diferentes tipos de cáncer, ya que puede actuar como supresor tumoral principalmente en cáncer de próstata, mama, colorrectal y pulmón, debido a mutaciones puntuales y/o expresión reducida o como oncogén en cáncer riñón por sobreexpresión de la proteína. En cáncer endometrial tiene un rol dual, ya que puede actuar como supresor tumoral o como oncogén. SPOP es considerado como un potencial biomarcador pronóstico y un objetivo terapéutico prometedor.

Cloning and functional verification of carboxyl CoA ligases(AeCCLs) in Arnebia euchroma / 中国中药杂志

Carboxyl CoA ligases(CCLs) is an important branch of adenylate synthetase gene family, which mainly has two-step catalytic reactions. Firstly, in the presence of adenosine triphosphate, it can catalyze the pyrophosphorylation of carboxylateswith diffe-rent structures to form corresponding acyl adenosine monophosphate intermediates. Secondly, adenosine monophosphate was replaced by free electrons in the mercaptan group of enzyme A or other acyl receptors by nucleophilic attack to form thioesters. In this study, on the basis of the transcriptome database of Arnebia euchroma, two genes were selected, named AeCCL5(XP_019237476.1) and AeCCL7(XP_019237476.1). Bioinformatics analysis showed that their relative molecular weights were 60.569 kDa and 60.928 kDa, theoretical PI were 8.59 and 8.92, respectively. They both have transmembrane domains but without signal peptide. By multiple sequence alignment and phylogenetic tree analysis, we found that the similarity between AeCCLs and other plant homologous proteins was not high, and the substrate binding sites of AeCCLs were not highly conserved. The reasons might be that the sequence and structure need to adapt to the changes of new substrates in the process of evolution. In this study, the full-length of AeCCL5 and AecCCL7 were cloned into the expression vector pCDFDuet-1. The proteins of AeCCL5 and AeCCL7 with His-tag were expressed in Escherichia coli. The proteins of AeCCL5 and AeCCL7 were purified by nickel column. In vitro enzymatic reactions proved that both AeCCL5 and AeCCL7 can participate in the upstream phenylpropane pathway of shikonin biosynthesisby catalyzing 4-coumaric acid to produce 4-coumarin-CoA, and then to synthesis p-hydroxybenzoic acid, which is an important precursor of shikonin biosynthesis in A. euchroma.

Overexpression of a fusion protein of 4-coumaroyl-CoA ligase and polyketide synthase for raspberry ketone production in Chlamydomonas reinhardtii / 生物工程学报

Raspberry ketones have important therapeutic properties such as anti-influenza and prevention of diabetes. In order to obtain raspberry ketone from Chlamydomonas reinhardtii, two enzymes catalyzing the last two steps of raspberry ketone synthesis, i.e. 4-coumaryl-CoA ligase (4CL) and polyketide synthase (PKS1), were fused using a glycine-serine-glycine (GSG) tripeptide linker to construct an expression vector pChla-4CL-PKS1. The fusion gene 4CL-PKS1 driven by a PSAD promoter was transformed into a wild-type (CC125) and a cell wall-deficient C. reinhardtii (CC425) by electroporation. The results showed the recombinant C. reinhardtii strain CC125 and CC425 with 4CL-PKS1 produced raspberry ketone at a level of 6.7 μg/g (fresh weight) and 5.9 μg/g (fresh weight), respectively, both were higher than that of the native raspberry ketone producing plants (2-4 μg/g).

Relationships between CASP8, Fas Gene Polymorphisms and the Prognosis of Patients with Non-Hodgkin's Lymphoma in Han Nationality / 中国实验血液学杂志

OBJECTIVE@#To investigate the relationships between caspase-8 (CASP8), fatty acid synthetase (Fas) gene polymorphisms and prognosis of non-Hodgkin's lymphoma patients in Han nationality.@*METHODS@#The clinical data of 85 patients with non-Hodgkin's lymphoma were analyzed retrospectively. The polymorphisms of CASP8 and Fas gene were detected, and prognosis of the patients were analyzed. The polymorphisms of CASP8 and Fas gene in patients with different prognosis were compared, and the relationships between gene polymorphisms and the poor prognosis of the patients were investigated.@*RESULTS@#The incidence rate of poor prognosis of the patients enrolled in the study was 65.88%. The polymorphisms of CASP8 and Fas genes in the patients with poor or good prognosis were in accordance with Hardy Weinberg's law of genetic balance. The frequencies of GG genotype and G allele at rs 1035142 of CASP8 gene, GA genotype and A allele at rs 1377 of Fas gene in patients with poor prognosis were lower than those of the patients with good prognosis (P<0.05). The frequencies of GT, TT and T alleles at rs 1035142 of CASP8 gene, GG and G alleles at rs 1377 of Fas gene in patients with poor prognosis were higher than those of the patients with good prognosis (P<0.05). The proportions of Ann Arbor stage III-IV and high malignancy in patients with poor prognosis were higher than those of the patients with good prognosis (P<0.05). Logistic multiple regression analysis showed that Ann Arbor stage III-IV, moderate malignant, high malignancy, CASP8 rs 1035142 GT genotype, CASP8 rs 1035142 TT genotype and Fas rs 1377 GG genotype were all the risk factors for the poor prognosis of the patients (P<0.05).@*CONCLUSION@#The poor prognosis rate of non-Hodgkin's lymphoma patients in Han nationality is relatively high, and the risk factors for the prognosis of the patients include Ann Arbor stage III-IV, moderate and high malignancy, CASP8 rs 1035142 GT genotype, CASP8 rs 1035142 TT genotype and Fas rs 1377 GG genotype.

Síndrome antisintetasa en paciente con COP como afección pulmonar / Antisynthetase Syndrome in Patient with COP as Pulmonary Disease

El síndrome antisintetasa es una miopatía inflamatoria autoinmune que puede presentar afectación pulmonar intersticial. La presencia de anticuerpos antisintetasa se relaciona con una mayor incidencia de enfermedad pulmonar intersticial. El patrón imagenológico y anatomopatológico de la EPID es variable, fundamentalmente inflamatorio. En el caso presentado se describe una paciente con miopatía inflamatoria y compromiso pulmonar presentando un patrón tomográfico de neumonía organizativa. Se destaca la importancia de elevar el índice de sospecha de síndrome antisintetasa ante un paciente con compromiso pulmonar y miopatía, siendo fundamental para arribar a un diagnóstico la evaluación multidisciplinaria. Se realiza una revisión de la evidencia al respecto en la discusión del caso

The antisynthetase syndrome is an autoimmune inflammatory miopathy that may show interstitial pulmonary involvement. The presence of antisynthetase antibodies is related to a higher incidence of interstitial pulmonary involvement. The imaging and anatomopathological pattern of diffuse interstitial pulmonary disease (DIPD) is variable, mainly inflammatory. This case describes a female patient with inflammatory miopathy and pulmonary involvement who shows a tomographic pattern of organizing pneumonia. It is important to increase suspicion for antisynthetase syndrome in a patient with pulmonary involvement and miopathy, where multidisciplinary evaluation is fundamental to reach a diagnosis. A review of the evidence is made in the discussion of the case.

Improvement of polyhydroxybutyrate production by deletion of csrA in Escherichia coli

BACKGROUND: Poly-3-hydroxybutyrate (PHB) can be efficiently produced in recombinant Escherichia coli by the overexpression of an operon (NphaCAB) encoding PHB synthetase. Strain improvement is considered to be one of critical factors to lower the production cost of PHB in recombinant system. In this study, one of key regulators that affect the cell growth and PHB content was confirmed and analyzed. RESULT: S17-3, a mutant E. coli strain derived from S17-1, was found to be able to achieve high cell density when expressing NphaCAB with the plasmid pBhya-CAB. Whole genome sequencing of S17-3 revealed genetic alternations on the upstream regions of csrA, encoding a global regulator cross-talking between stress response, catabolite repression and other metabolic activities. Deletion of csrA or expression of mutant csrA resulted in improved cell density and PHB content. CONCLUSION: The impact of gene deletion of csrA was determined, dysfunction of the regulators improved the cell density of recombinant E. coli and PHB production, however, the detail mechanism needs to be further clarified.

Establishment and gene expression analysis of drug-resistant cell lines in hepatocellular carcinoma induced by sorafenib / 北京大学学报(医学版)

OBJECTIVE@#To establish the drug-resistant cell lines of hepatocellular carcinoma (HCC) induced by sorafenib, and to screen out the high expression genes in drug-resistant cell lines of HCC induced by sorafenib, then to explore the genes related to sorafenib resistance in hepatocellular carcinoma.@*METHODS@#The human PLC and Huh7 cell lines were obtained, then the PLC and Huh7 drug-resistant cell lines were induced with sorafenib by using intermittent induction in vitro. CCK8 assay was used to detect the IC50 value of sorafenib for evaluation of drug sensitivity of hepatocellular carcinoma cell lines in PLC and Huh7. All the up regulated genes in PLC and Huh7 drug-resistant cell lines induced by sorafenib were screened out using high-throughput cDNA sequencing (RNA-Seq), Ualcan database was used to analyze the correlations between the up regulated genes in PLC and Huh7 drug-resistant cell lines induced and four clinical biological characteristics of hepatocellular carcinoma, including the gene expressions between normal samples and tumor samples, tumor stage, tumor grade, and patient overall survival, to find the genes that might be involved in the mechanism of sorafenib resistance of hepatocellular carcinoma.@*RESULTS@#All the up regulated genes detected by the using high-throughput cDNA sequencing (RNA-Seq) in PLC and Huh7 drug-resistant cell lines were further screened out by following conditions:(1) genes co-expressed in PLC and Huh7 drug-resistant cells induced by sorafenib, (2) the fold change was more than 4 times and the difference was statistically significant (P <0.05), the top 12 up regulated genes in PLC and Huh7 drug-resistant cell lines were found, which were TPSG1, CBX4, CLC, CLEC18C, LGI4, F2RL1, S100A6, HABP2, C15ORF48, ZG16, FOLH1, and EPCAM. Compared with the correlations between the twelve genes and the clinical biological characteristics by Ualcan database, the potentially significant gene CBX4 was screened out.@*CONCLUSION@#The human PLC and Huh7 drug-resistant cell lines of hepatocellular carcinoma induced by sorafenib were successfully established. CBX4, the gene related to sorafenib resistance in hepatocellular carcinoma, was screened out by the high-throughput cDNA sequencing (RNA-Seq) and further analysis using Ualcan database, which is providing a powerful basis for further research on the mechanism of sorafenib resistance of hepatocellular carcinoma.

Molecular cloning and expression analysis of iridoid synthase genes from Rehmannia glutinosa / 中国中药杂志

Iridoid synthase( IS),the key enzyme in the natural biosynthesis of vegetal iridoids,catalyzes the irreversible cyclization of 10-oxogeranial to epi-iridodial. In this study,we screened the Rehmannia glutinosa transcriptome data by BLASTn with Catharanthus roseus CrIS cDNA,and found four c DNA fragments with length of 1 527,1 743,1 425,1 718 bp,named RgIS1,RgIS2,RgIS3 and RgIS4,respectively. Bioinformatics analysis revealed that the four iridoid synthase genes encoding proteins with 389-392 amino acid residues,protein molecular weights were between 44. 30-44. 74 k Da,and theoretical isoelectric points were between 5. 30 and 5. 87. Subcellular localization predictions showed that the four iridoid synthase were distributed in the cytoplasm. Structure analysis revealed that R. glutinosa iridoid synthases contain six conserved short-chain dehydrogenase/reductase( SDR) motifs,and their 3 D models were composed typical dinucleotide-binding " Rossmann" folds covered by helical C-terminal extensions. Using the amino acid sequences of four R. glutinosa iridoid synthases,phylogenetic analysis was performed,the result indicated that RgIS3,CrIS and Olea europaea OeIS were grouped together,the other R. glutinosa iridoid synthases and fifteen proteins in other plants had close relationship. Real-time fluorescent quantitative PCR revealed that RgIS1 and RgIS3 highly expressed in unfold leaves,however,RgIS2 and RgIS4 highly expressed in stems and tuberous roots,respectively. RgIS3 showed higher expression levels in non-radial striations( nRS) of the two cultivars,and RgIS1 and RgIS2 had higher expression levels in nRS of QH,while RgIS4 had less expression levels in nRS of QH1. RgIS1,RgIS2 and RgIS3 were up-regulated by Me JA treatment,although the time and degree of response differed. Our findings are helpful to reveal molecular function of R. glutinosa iridoid synthases and provide a clue for studing the molecular mechanism of iridoid biosynthesis.

Methionyl-tRNA Synthetase is a Useful Diagnostic Marker for Lymph Node Metastasis in Non-Small Cell Lung Cancer

PURPOSE: Identification of lymph node (LN) metastasis in non-small cell lung cancer (NSCLC) is critical for disease staging and selection of therapeutic modalities. Sometimes it is not possible to obtain LN core tissue by endobronchial ultrasound-guided transbronchial needle aspirate (EBUS-TBNA), resulting in low diagnostic yield. MATERIALS AND METHODS: In this study, 138 specimens were collected from 108 patients who underwent EBUS-TBNA under the suspicion of LN metastasis of NSCLC. Diagnostic yields of anti-CD45 and anti-methionyl-tRNA synthetase (MRS), immunofluorescent (IF) staining on cytology specimens were compared with those of conventional cytology and positron emission tomography-computed tomography (PET-CT). RESULTS: MRS was strongly expressed in NSCLC cells metastasized to LNs, but weakly expressed in cells at the periphery of the LN germinal center. The majority of cells were CD20 positive, although a few cells were either CD3 or CD14 positive, indicating that CD45 staining is required for discrimination of non-malignant LN constituent cells from NSCLC cells. When the diagnostic efficacy of MRS/CD45 IF staining was evaluated using 138 LN cellular aspirates from 108 patients through EBUS-TBNA, the sensitivity was 76.7% and specificity was 90.8%, whereas those of conventional cytology test were 71.8% and 100.0%, respectively. Combining the results of conventional cytology testing and those of PET-CT showed a sensitivity and specificity of 71.6% and 100%, and the addition of MRS/CD45 dual IF data to this combination increased sensitivity and specificity to 85.1% and 97.8%, respectively. CONCLUSION: MRS/CD45 dual IF staining showed good diagnostic performance and may be a good tool complementing conventional cytology test for determining LN metastasis of NSCLC.

Pja2 Inhibits Wnt/β-catenin Signaling by Reducing the Level of TCF/LEF1

Ubiquitination of proteins plays an essential role in various cellular processes, including protein degradation, DNA repair, and cell signaling pathways. Previous studies have shown that protein ubiquitination is implicated in regulating pluripotency as well as fate determination of stem cells. To identify how protein ubiquitination affects differentiation of embryonic stem cells, we analyzed microarray data, which are available in the public domain, of E3 ligases and deubiquitinases whose levels changed during stem cell differentiation. Expression of pja2, a member of the RING-type E3 ligase family, was up-regulated during differentiation of stem cells. Wnt/β-catenin signaling is one of the most important signaling pathways for regulation of the self-renewal and differentiation of embryonic stem cells. Pja2 was shown to bind to TCF/LEF1, which are transcriptional factors for Wnt/β-catenin signaling, and regulate protein levels by ubiquitination, leading to down-regulation of Wnt signaling activity. Based on these results, we suggest that E3 ligase Pja2 regulates stem cell differentiation by controlling the level of TCF/LEF1 by ubiquitination.

The Efficacy of Fibroblast Growth Factor for the Treatment of Chronic Vocal Fold Scarring: From Animal Model to Clinical Application

OBJECTIVES: This study assessed the regenerative efficacy of basic fibroblast growth factor (FGF) in a rabbit model of chronic vocal fold scarring and then confirmed its utility and safety in a prospective trial of patients with this condition. METHODS: FGF was injected three times, at 1-week intervals, into a chronic vocal fold scar created in a rabbit model. After 1 month, mRNA level of procollagen I, hyaluronic acid synthetase 2 (HAS 2), and matrix metalloproteinase 2 (MMP 2) were analyzed by real-time polymerase chain reaction. The relative densities of hyaluronic acid (HA) and collagen were examined 3 months post-injection. From April 2012 to September 2014, a prospective clinical trial was conducted at a tertiary hospital in Korea. FGF was injected into the mild vocal fold scar of 17 consecutive patients with a small glottic gap. The patients underwent perceptual, stroboscopic, acoustic aerodynamic test, and Voice Handicap Index (VHI) survey prior to and 3, 6, and 12 months after FGF injection. RESULTS: FGF injection of the vocal fold scar decreased the density of collagen and increased mRNA level of HAS 2 and MMP 2 expression significantly compared to the control group injected with phosphate buffered solution in a rabbit model (P < 0.05). In the clinical trial, significant improvements in the majority of the subjective and objective voice parameters were registered 3 months after FGF injection and were maintained at 12 months. Complications associated with the FGF injections, such as granuloma, were not observed during the follow-up period. CONCLUSION: Based on the animal model and the prospective clinical trial, vocal fold injections of FGF in patients with mild chronic vocal fold scarring can significantly improve voice quality for as long as 1 year and without side effects. Our results recommend the use of FGF vocal fold injection as an alternative treatment modality for mild chronic vocal fold scarring.

Microproteínas urinarias marcadoras de daño renal en pacientes con glomerulopatías / Urinary microprotein markers of renal disease in patients with glomerulopathies / Microproteínas urinárias marcadoras de lesão renal em pacientes com glomerulopatias

En las glomerulopatías, las Microproteínas Urinarias (MU) se eliminan por distintos mecanismos fisiopatológicos. El objetivo del trabajo fue correlacionar las MU con el daño histológico evaluado en la punción biopsia renal (PBR) de pacientes con diversas glomerulopatías. Se estudiaron 44 orinas espontáneas (33 mujeres y 11 varones) entre 18 y 71 años de edad, por el método de electroforesis bidimensional de uso clínico (2D UC). Las proteínas identificadas se dividieron en 5 grupos y se compararon con lesiones vasculares, glomerulares y túbulointersticiales; estas dos últimas se dividieron en crónicas y agudas. Las del grupo identificado como las "Tres Marías" (fragmento de 35 kDa de la proteína inhibidor de tripsina cadena pesada H4, Prostaglandina H2 sintasa y fragmento de 23 kDa del Perlecan) resultaron no ser marcadoras de daño tubular, sino de alteraciones glomerulares crónicas. La presencia de las mismas, con proteinuria, se observa antes de la caída de la filtración glomerular (<60 mL/min) y correlaciona con el 30% de glomérulos totalmente esclerosados (p<0,001). El grupo Triángulo, en la glomerulopatía, contiene a la Alfa-1 microglobulina (A1m) y a las cadenas livianas libres de Inmunoglobulinas (CLL), e indica lesión glomerular activa. Por lo tanto, las MU en glomerulopatías, responden a lesiones glomerulares activas y crónicas. Los perfiles proteicos urinarios hallados por la 2D UC permitieron conocer el grado de lesión en los distintos compartimentos renales.

In Glomerulopathies, urinary microproteins (UM) are eliminated by different pathophysiological mechanisms. The objective of this work was to correlate the UM with the histological damage evaluated in the renal biopsy through puncture of patients with various glomerular diseases. Forty-four urine samples (33 females, 11 males) aged 18 to 71 years old were studied by the method of two-dimensional electrophoresis of clinical use (2D UC). The identified proteins were divided into 5 groups and were compared with vascular injury, glomerular and tubulointerstitial injury, the latter in chronic and acute cases. The group identified as the "Three Marias" (fragment 35 kDa protein trypsin inhibitor heavy chain H4, prostaglandin H2 synthase and 23 kDa fragment of Perlecan), was not found as marker of tubular damage, but it was found in chronic glomerular disorders. The presence of this same group -with proteinuria- is seen before the collapse of the glomerular filtration rate (<60 mL/min) and it is correlated with 30% of fully sclerotic glomeruli (p<0.001). In glomerulopathy, the Triangle group: Alpha-1 microglobulin (A1m) and free Immunoglobulin light chains (FLC), indicates active glomerular injury. Therefore, the UM in glomerular diseases, respond to active and chronic glomerular lesions. Urinary protein profiles found by the 2D UC made it possible to know the degree of renal injury in different renal compartments.

Nas glomerulopatias, as Micro proteínas Urinárias (MU) são eliminadas através de diferentes mecanismos fisiológicos. O objetivo do trabalho foi relacionar as MU com o dano histológico avaliado na punção biópsia renal (PBR) de pacientes com diversas glomerulopatias. Foram estudadas 44 urinas espontâneas (33 mulheres e 11 homens entre 18 e 71 anos de idade), pelo método de eletroforese bidimensional de uso clínico (2D UC). As proteínas identificadas foram divididas em 5 grupos e comparadas com lesões vasculares, glomerulares e túbulo-intersticiais, estas duas últimas classificadas em crônicas e agudas. O grupo identificado como as "Três Marias" (fragmento de 35 kDa da proteína inibidora de tripsina cadeia pesada H4, Prostaglandina H2 sintase e fragmento de 23 kDa do Perlecam), resultaram não ser marcadoras de dano tubular, mas de alterações glomerulares crônicas. A presença de tais proteínas, com proteinúria, observa-se antes da queda da filtragem glomerular (<60 mL/min) e correlaciona com 30% de glomérulos totalmente esclerosados (p<0,001). O grupo Triângulo, na glomerulopatia, que contém a Alfa-1 microglobulina (A1m) e as cadeias leves livres de Imunoglobulinas (CLL) indicam lesão glomerular ativa. Portanto, as MU em glomerulopatias respondem a lesões glomerulares ativas e crônicas. Os perfis proteicos urinários encontrados pela 2D UC permitiram conhecer o grau de lesão nos diferentes compartimentos renais.

Construction of recombinant strains co-expressing PPK and GMAS for the synthesis of L-theanine / 生物工程学报

Recombinant strains expressing enzymes for ATP regeneration and L-theanine production were constructed and used for the synthesis of L-theanine. The ppk gene encoding polyphosphate kinase (PPK) from Rhodobacter sphaeroides and gmas gene encoding γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays were synthesized, and two recombinant plasmids, pETDuet-ppk+gmas and pET21a-ppk+gmas were constructed for co-expression of PPK and GMAS in Escherichia coli BL21(DE3). SDS-PAGE analysis showed that PPK and GMAS were overexpressed in soluble form in both recombinant strains. GMAS-PPK obtained from the recombinant strain containing pET21a-ppk+gmas was more efficient to synthesize L-theanine. After 24 h at 37 ℃ and pH at 7.0, 86.0% yield of L-theanine was achieved with catalytic amount of ATP. This study extends the application of enzymatic ATP regeneration system. In addition, it provides an efficient method for the biosynthesis of L-theanine.

Acute Respiratory Distress Syndrome as the Initial Clinical Manifestation of an Antisynthetase Syndrome / 결핵및호흡기질환

Antisynthetase syndrome has been recognized as an important cause of autoimmune inflammatory myopathy in a subset of patients with polymyositis and dermatomyositis. It is associated with serum antibody to aminoacyl-transfer RNA synthetases and is characterized by a constellation of manifestations, including fever, myositis, interstitial lung disease, mechanic's hand-like cutaneous involvement, Raynaud phenomenon, and polyarthritis. Lung disease is the presenting feature in 50% of the cases. We report a case of a 60-year-old female with acute respiratory distress syndrome (ARDS), which later proved to be an unexpected and initial manifestation of anti-Jo-1 antibody-positive antisynthetase syndrome. The present case showed resolution of ARDS after treatment with high-dose corticosteroids. Given that steroids are not greatly beneficial in the treatment of ARDS, it is likely that the improvement of the respiratory symptoms in this patient also resulted from the prompt suppression of the inflammatory systemic response by corticosteroids.

Effects of Nephrolithiasis on Serum DNase (Deoxyribonuclease I and II) Activity and E3 SUMO-Protein Ligase NSE2 (NSMCE2) in Malaysian Individuals / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Nephrolithiasis is one of the most common disorders of the urinary tract. The aim of this study was to examine a possible relationship between DNase I/II activity and E3 SUMO-protein ligase NSE2 in the sera of nephrolithiasis patients to evaluate the possibility of a new biomarker for evaluating kidney damage.</p><p><b>METHODS</b>Sixty nephrolithiasis patients and 50 control patients were enrolled in a case-control study. Their blood urea, creatinine, protein levels and DNase I/II activity levels were measured by spectrometry. Serum NSMCE2 levels were measured by ELISA. Blood was collected from patients of the government health clinics in Kuantan-Pahang and fulfilled the inclusion criteria.</p><p><b>RESULTS</b>The result indicated that mean levels of sera NSMCE2 have a significantly increase (P<0.01) in patients compared to control group. Compared with control subjects, activities and specific activities of serum DNase I and II were significantly elevated in nephrolithiasis patients (P$lt;0.01).</p><p><b>CONCLUSION</b>This study suggests that an increase in serum concentrations of DNase I/II and E3 SUMO-protein ligase NSE2 level can be used as indicators for the diagnosis of kidney injury in patients with nephrolithiasis.</p>

Pathology of dogs in Campo Grande, MS, Brazil naturally co-infected with Leishmania infantum and Ehrlichia canis / Patologia de cães naturalmente coinfectados por Leishmania infantum e Ehrlichia canis em Campo Grande, MS, Brasil

Different parasites that commonly occur concomitantly can influence one another, sometimes with unpredictable effects. We evaluated pathological aspects of dogs naturally co-infected with Leishmania infantum and Ehrlichia canis. The health status of the dogs was investigated based on histopathological, hematological and biochemical analyses of 21 animals infected solely with L. infantum and 22 dogs co- infected with L. infantum and E. canis. The skin of both groups showed chronic, predominantly lymphohistioplasmacytic inflammatory reaction. The plasmacytosis in the lymphoid tissues was likely related with the hypergammaglobulinemia detected in all the dogs. The disorganization of extracellular matrix found in the reticular dermis of the inguinal region and ear, characterized by the substitution of thick collagen fibers for thin fibers, was attributed to the degree of inflammatory reaction, irrespective of the presence of parasites. In addition, the histopathological analysis revealed that twice as many dogs in the co-infected group presented Leishmania amastigotes in the ear skin than those infected solely with Leishmania, increasing the possibility of becoming infected through sand fly vectors. Our findings highlight the fact that the health of dogs infected concomitantly with L. infantum and E. canis is severely compromised due to their high levels of total plasma protein, globulins, alkaline phosphatase and creatine kinase, and severe anemia.

A infecção simultânea por parasitas de diferentes espécies pode resultar em alterações imprevisíveis. O presente estudo avaliou a patologia de cães naturalmente coinfectados por Leishmania infantum e Ehrlichia canis. A saúde dos cães foi investigada pelas análises histopatológicas, hematológicas e bioquímicas de 21 cães infectados somente por L. infantum e 22 cães coinfectados por L. infantum e E. canis. Observou-se uma reação inflamatória crônica, predominantemente linfohistioplasmocítica, na pele dos dois grupos. A plasmocitose, encontrada nos tecidos linfóides, provavelmente estava relacionada com a hipergamaglobulinemia observada em todos os cães amostrados. A desorganização da matriz extracelular da derme da região inguinal e da orelha, demonstrada pela substituição das fibras de colágeno espessas por fibras finas, foi relacionada com o grau de reação inflamatória, independente da presença de parasitas. Ainda, observamos duas vezes mais animais do grupo coinfectado apresentando formas amastigotas na pele de orelha pela histopatologia comparado ao número de cães infectados apenas por Leishmania, tornando-os desta forma mais infectivos aos vetores. Nossos resultados ressaltam que a saúde de cães coinfectados estava severamente comprometida devido aos altos níveis de proteína plasmática total, globulinas, fosfatase alcalina, creatina quinase e anemia acentuada.

The first successful live birth following preimplantation genetic diagnosis using PCR for type 1 citrullinemia

Type 1 citrullinemia (CTLN1) is an autosomal recessive inherited metabolic disorder caused by anargininosuccinicnate synthetase deficiency. The patient was a 38-year-old Korean woman who is a carrier for CTLN1 and her first baby was diagnosed with CTLN1. Preimplantation genetic diagnosis (PGD) for CTLN1 in day 3 embryos using polymerase chain reaction was performed for live birth of healthy baby who is no affected with CTLN1. One unaffected blastocyst was transferred. This resulted in a clinical pregnancy and the live birth of healthy male twin. They were confirmed to be unaffected with CTNL1 by post natal diagnosis. This is the first case report of the use of PGD for CTNL1.

Ubiquitin Ligases in Cholesterol Metabolism

To maintain cholesterol homeostasis, the processes of cholesterol metabolism are regulated at multiple levels including transcription, translation, and enzymatic activity. Recently, the regulation of protein stability of some key players in cholesterol metabolism has been characterized. More and more ubiquitin ligases have been identified including gp78, Hrd1, TRC8, TEB4, Fbw7, and inducible degrader of low density lipoprotein receptor. Their working mechanisms and physiological functions are becoming revealed. Here, we summarize the structure, substrates and function of these ubiquitin ligases. Their potential application in drug discovery is also discussed.