High-level expression of anti FLAG tag antibody in plants / 生物工程学报

Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.

Immunogenicity Studies of Various Experimental Vaccines in Chickens

In this paper, the main objective was to raise chickens’ antibodies against three crucial public health microorganisms: the human immunodeficiency virus-1, Salmonella spp, and Staphylococcus aureus. Immunogens were prepared from the said microorganisms. Chickens were vaccinated either orally or intramuscularly. After a booster immunization, mostly eggs were collected and assess for the presence of specific antibodies. The most important results were the production of a large amount of anti-HIV antibodies in chicken’s eggs, and also the synthesis of anti-protein a antibodies with the ability to inhibit the growth of S. aureus in vitro and to serve as anti-anti-idiotypic antibodies with the capacity of neutralizing the original antigen. Enzyme- linked immune absorbent assays detected the presence of these antibodies as anti-Salmonella antibodies that were critical in reducing the bacterial load in the stomach and caeca compared with a control group. The vaccines were effective and safe, but more laboratory work, and economics have to be carried out to start a human trial.

Staphylococcus hominis: a rare cause of endophthalmitis / Staphylococcus hominis: uma causa rara de endoftalmite

ABSTRACT Staphylococcus hominis (S. hominis) is a coagulase-negative Staphylococci and an infrequent cause of endophthalmitis. Due to its ability to produce biofilm, especially in diabetic patients, strains may acquire antibiotic resistance. We present two cases of S. hominis endophthalmitis, one with acute endophthalmitis after intravitreal bevacizumab injection and one with chronic endophthalmitis following undiagnosed penetrating ocular trauma. Although there are only four published S. hominis endophthalmitis cases in the literature, to the best of our knowledge, there has been no previously published case after intravitreal bevacizumab.

RESUMO Staphylococcus hominis (S. hominis) é um estafilococo coagulase-negativo e uma causa pouco frequente de endoftalmite. Devido à sua capacidade de produzir biofilme, especialmente em pacientes diabéticos, cepas dessa bactéria podem adquirir resistência a antibióticos. Este relato apresenta dois casos de endoftalmite por S. hominis: um de endoftalmite aguda após injeção intravítrea de bevacizumabe e outro de endoftalmite crônica após trauma ocular penetrante não diagnosticado. Embora existam apenas quatro casos de endoftalmite por S. hominis publicados na literatura, até onde sabemos não houve nenhum caso publicado anteriormente após bevacizumabe intravítreo.

Use of Schirmer strips and conjunctival swabs for virus detection on the ocular surface of adults: a scoping review / Fitas de Schirmer e swab conjunctival para detecção de vírus na superfície ocular de adultos: scoping review

ABSTRACT Schirmer strips and conjunctival swabs are used in ophthalmology for the collection of tears and fluids. One of the biggest challenges during the COVID-19 pandemic has been accurate diagnosis and, in some cases, ocular manifestations are among the first symptoms. In this context, this study aimed to collect evidence to support the use of Schirmer strips and conjunctival swabs as a method of sample collection for viral analysis. A literature search was conducted following the Scoping Review protocol defined by The Joanna Briggs Institute. Studies were analyzed regarding virus research, collection methods, and sample analysis. The findings support that viruses can be detected on the ocular surface through analysis of Schirmer strips and conjunctival swabs. However, additional studies with larger samples and time data are necessary to confirm these conclusions.

RESUMO A fita de Schirmer e o swab conjunctival são utilizados na oftalmologia como métodos de coleta para lágrimas e fluidos. Durante a pandemia da COVID-19, um dos desafios foi o diagnóstico correto e se sabe que, em alguns casos, as manifestações oculares podem ser um dos primeiros sintomas. Nesse contexto, este estudo tem como objetivo levantar evidência que destaque o uso de fitas de Schirmer e de swabs conjuntivais como método de coleta para análise viral. Conduziu-se uma revisão de literatura seguindo o protocolo para Scoping Review definido pelo Joanna Briggs Institute. Os pesquisadores analisaram os estudos em busca do vírus pesquisado, os métodos de coleta e os métodos de análise. Vírus podem ser detectados na superfície ocular através da análise de fitas de Schirmer e de swabs conjuntivais, entretanto novos estudos com populações maiores e com definições claras de tempo são necessários para conclusões mais assertivas no tema.

Relación estructura-función de la enzima uroporfirinógeno descarboxilasa: implicancias para la comprensión y el tratamiento de la porfiria cutánea tarda / Structure-function relationship of the enzyme uroporphyrinogen decarboxylase: implications for the understanding and treatment of porphyria cutanea tarda / Relação estrutura-função da enzima uroporfirinogênio descarbioxilase: implicações para a compreensão e tratamento da porfiria cutânea tardia / Reconocimiento a la trayectoria del Prof. Dr. Juan Miguel Castagnino†

Resumen La uroporfirinógeno descarboxilasa humana (UROD-h) es la quinta enzima del camino biosintético del hemo y su actividad deficiente, relacionada a mutaciones en su gen, se encuentra asociada a un subgrupo de porfirias. El objetivo de este trabajo fue estudiar la relación entre la dimerización de la enzima y su actividad enzimática y comprobar si la dimerización de UROD-h es imprescindible tanto para la primera etapa de la reacción (urogen→heptagen), como para la segunda etapa (heptagen→coprogen). Con ese objetivo, se expresó y purificó la UROD-h hasta homogeneidad, se analizó el comportamiento dímero-monómero bajo distintas condiciones que pudieran desplazar el equilibrio de dimerización y se evaluó la actividad enzimática en dichas condiciones. Los resultados obtenidos sugieren que la especie activa para la primera etapa de la reacción es el homodímero y que tanto el dímero como el monómero se comportan como especies activas para la segunda etapa de la reacción. Se propone que mutaciones clínicas como la Y311C, existentes en pacientes con porfiria cutánea tarda, podrían afectar la estabilidad del dímero y podrían ser el blanco para futuras terapias génicas.

Abstract Human uroporphyrinogen decarboxylase (UROD-h) is the fifth enzyme in the heme biosynthetic pathway and its deficient activity, related to mutations in its gene, is associated with a subset of porphyrias. The objective of this work was to study the relationship between the dimerisation of the enzyme and its enzymatic activity and to verify if the dimerisation of UROD-h is essential both for the first stage of the reaction (urogen→heptagen), and for the second stage (heptagen→ coprogen). With this objective, the UROD-h was expressed and purified to homogeneity, the dimer- monomer behaviour was analysed under different conditions, which could shift the dimerisation equilibrium, and the enzymatic activity was evaluated under these conditions. The results obtained suggest that the active species for the first stage of the reaction is the homodimer, and both the dimer and the monomer behaved as active species for the second stage of the reaction. It is proposed that clinical mutations such as Y311C, existing in porphyria cutanea tarda patients, could affect dimer stability and could be the target of future gene therapies.

Resumo A enzima uroporfirinogênio descarboxilase humana (UROD-h) é a quinta enzima da via biossintética do heme e sua atividade deficiente, relacionada com mutações em seu gene, está associada a um subgrupo de porfirias. O objetivo deste trabalho foi estudar a relação entre a dimerização da enzima e sua atividade enzimática e comprovar se a dimerização da UROD-h é imprescindível tanto para a primeira etapa da reação (urogênio→heptagênio), quanto para a segunda etapa (heptagênio→coprogênio). Com esse objetivo, a UROD-h foi expressa e purificada até a homogeneidade, o comportamento de dímero-monômero foi analisado sob diversas condições, que puderam deslocar o equilíbrio de dimerização, e a atividade enzimática foi avaliada em tais condições. Os resultados obtidos sugerem que a espécie ativa para a primeira etapa da reação é o homodímero, e tanto o dímero quanto o monômero se comportam como espécies ativas para a segunda etapa da reação. Propõe-se que mutações clínicas como Y311C, existentes em pacientes com porfiria cutânea tardia, poderiam afetar a estabilidade do dímero e poderiam ser o alvo de futuras terapias gênicas em porfiria cutânea tardia.

Prokaryotic expression,purification and activity of CamA protein with methylase activity from Clostridiodies difficile / 中国生物制品学杂志

@#Objective To express CamA(Clostridiodies difficile adenine methltransferase A,CamA)protein with methylase activity in prokaryotic expression system.Methods The gene sequence of CamA protein of the standard strain of C.difficile1870 was amplified by PCR,cloned into plasmid pGEX-4T-1-MBP,transformed into E.coli HB101 and induced by 1 mmol/L IPTG to express the recombinant target protein. After purification with Amylose Resin,CamA protein was digested by tobacco etch virus(TEV)protease,and verified for its methylase activity using MTase-Glo~(TM)Methyltransferase Assay in vitro.Results PCR sequencing showed that the cloned CamA gene sequence was correct,in which no base mutation occurred. The recombinant expression plasmid pGEX-4T-1-MBP-CamA was digested by EcoRⅠand BamHⅠ,which showed that 1 731 bp of CamA gene was connected to the vector plasmid. The relative molecular mass of the recombinant protein was about 108 800(with 1 MBP tag),and the purity was over 90% after purified with Amylose Resin. Under the condition of20 μmol/L DNA and 20 μmol/L SAM at room temperature for 30 min,0. 5 μg CamA produced SAH at a concentration of about 101. 60 nmol/L and the enzyme activity was(0. 339 ± 0. 027)U/mg.Conclusion The CamA protein of C.difficile has been successfully expressed and purified with methylase activity,which lays a foundation of further study on the function of CamA and its role in the occurrence,development and treatment of C.difficile infection.

Progress in the application of blood purification treatment mode in the treatment of wasp sting patients / 中国医师杂志

Wasp sting is a common emergency in mountainous areas of China, with rapid onset and progression, high mortality rate, and serious harm to public health. Wasp sting can cause mild local reactions in mild cases, and Anaphylaxis or even multiple organ dysfunction in severe cases, of which Acute kidney injury (AKI) is the most common and serious. Blood purification treatment is commonly used for wasp sting patients to maintain renal function, eliminate toxins, and maintain Internal environment stability. The commonly used clinical methods are Hemoperfusion (HP), plasma exchange (PE), and continuous renal replacement therapy (CRRT). At present, there is no clear recommendation for the blood purification treatment mode of wasp sting in China, and there is no clear guidance for its combined treatment mode. This article will review the single and combined use of blood purification treatment models for wasp stings, based on the latest clinical research.

Research progress on continuous blood purification in the treatment of severe acute pancreatitis / 中国医师杂志

Severe acute pancreatitis (SAP) is a common gastrointestinal disease, often accompanied by systemic inflammatory reactions and organ dysfunction. SAP has an acute onset, severe condition, rapid progression, and poor prognosis. The development of SAP is closely related to the excessive release of inflammatory factors. In the comprehensive treatment of SAP, continuous blood purification (CBP) can clear inflammatory mediators, improve the stability of Internal environment, improve organ function, reduce blood lipids, regulate immunity, and significantly improve the condition of SAP patients. It is an important means of treating SAP. This article reviews the research progress of CBP in the treatment of SAP.

A comparative study of the effects of citrate and heparin anticoagulation on coagulation function and efficacy in children with septic shock undergoing continuous blood purification / 中华危重病急救医学

Objective:To compare the effects of citrate and heparin anticoagulation on coagulation function and efficacy in children with septic shock undergoing continuous blood purification (CBP), and to provide guidance for CBP anticoagulation in children with septic shock.Methods:A case control study was conducted. Thirty-seven children with septic shock admitted to the pediatric intensive care unit (PICU) of the First Affiliated Hospital of Gannan Medical University from July 2019 to September 2022 were enrolled as the research subjects. The patients were divided into citrate local anticoagulation group and heparin systemic anticoagulation group according to different anticoagulation methods. The baseline data, the level of coagulation indicators [prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (Fib), D-dimer] before treatment and 1 day after weaning from CBP, serum inflammatory mediators [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), hypersensitivity C-reactive protein (hs-CRP), procalcitonin (PCT)], bleeding complications during CBP and 7-day mortality were collected.Results:A total of 37 cases were enrolled finally, including 17 cases with citric acid local anticoagulation and 20 cases with heparin systemic anticoagulation. There was no statistically significant difference in general data such as gender, age, and body weight of children between the two groups. There were no statistically significant differences in baseline levels of coagulation indicators and inflammatory mediators before treatment of children between the two groups. One day after weaning from CBP, both groups showed varying degrees of improvement in coagulation indicators compared with those before treatment. Compared with before treatment, the PT of the heparin systemic anticoagulation group was significantly shortened after 1 day of weaning (s: 11.82±2.05 vs. 13.64±2.54), APTT and TT were significantly prolonged [APTT (s): 51.54±12.69 vs. 35.53±10.79, TT (s): 21.95±4.74 vs. 19.30±3.33], D-dimer level was significantly reduced (mg/L: 1.92±1.58 vs. 4.94±3.94), with statistically significant differences (all P < 0.05). While in the citrate local anticoagulation group, only APTT was significantly prolonged after treatment compared with that before treatment (s: 49.28±10.32 vs. 34.34±10.32, P < 0.05). There were no statistically significant differences in other coagulation indicators compared with before treatment. Compared with the citric acid local anticoagulation group, the PT of the heparin systemic anticoagulation group was significantly shortened after treatment (s: 11.82±2.05 vs. 13.61±3.05, P < 0.05), and the D-dimer level was significantly reduced (mg/L: 1.92±1.58 vs. 3.77±2.38, P < 0.01). The levels of inflammatory mediators in both groups were significantly reduced 1 day after CBP weaning compared with those before treatment [citric acid local anticoagulation group: hs-CRP (mg/L) was 12.53±5.44 vs. 22.65±7.27, PCT (μg/L) was 1.86±1.20 vs. 3.30±2.34, IL-6 (ng/L) was 148.48±34.83 vs. 202.32±48.62, TNF-α (ng/L) was 21.38±7.71 vs. 55.14±15.07; heparin systemic anticoagulation group: hs-CRP (mg/L) was 11.82±4.93 vs. 21.62±8.35, PCT (μg/L) was 1.90±1.08 vs. 3.18±1.97, IL-6 (ng/L) was 143.81±33.41 vs. 194.02±46.89, TNF-α (ng/L) was 22.44±8.17 vs. 56.17±16.92, all P < 0.05]. However, there was no statistically significant difference between the two groups (all P > 0.05). There was no statistically significant difference in bleeding complication during CBP and 7-day mortality in children between the citrate local anticoagulation group and the heparin systemic anticoagulation group (5.9% vs. 30.0%, 17.6% vs. 20.0%, both P > 0.05). Conclusions:Heparin for systemic anticoagulation and regional citrate anticoagulation can significantly reduce the levels of IL-6, TNF-α, hs-CRP and PCT in children with septic shock, and relieve inflammatory storm. Compared with citric acid local anticoagulation, heparin systemic anticoagulation can shorten the PT and reduce the level of D-dimer in children with septic shock, which may benefit in the prevention and treatment of disseminated intravascular coagulation (DIC).

Effect and safety of double plasma molecular absorption in treatment of patients with severe wasp stings injury: a multicenter historical cohort study / 中华危重病急救医学

Objective:To compare the effect and safety of continuous veno-venous hemofiltration (CVVH)+double plasma molecular absorption (DPMA)+hemoperfusion (HP), CVVH+HP, and CVVH+plasma exchange (PE) in treatment of patient with severe wasp stings injury.Methods:Multicenter, historical cohort study and superiority test were used. From July 2020 to October 2022, patients with wasp sting injury and multiple organ damage admitted to the intensive care units (ICU) of five hospitals were consecutively screened and recruited into the CVVH+DPMA+HP group (intervention group). Propensity score matching was used to establish historical cohorts. Patients with severe wasp sting injury who hospitalized from January 2016 to June 2020 in each ICU were collected and matched 1∶1 with the intervention group, and divided into CVVH+HP group and CVVH+PE group according to their actual hemopurification protocols (historical control groups). The primary outcome was the acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score on days 3 and 7 after initiation of treatment. Secondary outcomes included complications, length of ICU and hospital stays, and all-cause mortality. Multivariate Cox proportional risk regression was used to analyze the prognosis of patients.Results:After propensity score matching, 56 patients in intervention group and each of the two historical control groups were matched successfully. There were no significant differences in age, gender, comorbidities, biochemical test indices and critical illness scores among the groups. After treatment, APACHE Ⅱ score markedly declined in all groups, and the decrease was faster in the intervention group; treatment with DPMA [hazard ratio ( HR) = 1.04, 95% confidence interval (95% CI) was 1.02-1.08, P = 0.00], the decreased levels of body temperature ( HR = 1.02, 95% CI was 1.00-1.03, P = 0.02), serum creatine kinase (CK; HR = 0.98, 95% CI was 0.96-1.00, P = 0.05) and myoglobin (MYO; HR = 2.88, 95% CI was 1.24-6.69, P = 0.01) were independent risk factors for APACHE Ⅱ score decline to the target value (15 scores). There were no significant differences in the incidence of bleeding complications, filter or perfusion thrombosis, blood pressure reduction, catheter-related infection and anaphylaxis among the groups. Conclusion:CVVH+DPMA+HP regimen can significantly reduce the APACHE Ⅱ score of patients with severe wasp sting injury, and the efficacy is superior to CVVH+HP and CVVH+PE regimens, with safety.

Value of bedside high-flow continuous blood purificationcombined with Xuebijing in the treatment of severe sepsis / 中国医师进修杂志

Objective:To investigate the effect of bedside high-flow continuous blood purification (CBP) combined with Xuebijing in the treatment of severe sepsis (SS) and the influence on the patient′s coagulation-fibrinolysis index, immunity index and expression of peripheral blood Toll-like receptor 4 (TLR4).Methods:Ninety-three patients with SS who were admitted and treated in the Lianyungang First People′s Hospitalfrom January 2017 to October 2019 were selected. They were divided into the combined group (51 cases, treatment with bedside high-flow CBP and Xuebijing injection based on bundle therapy) and the control group (42 cases, treatment with Xuebijing injection based on bundle therapy). The changes in coagulation and fibrinolysis index, immunity index, biochemical index such as TLR4 before treatment and after 1 week of treatment were compared between the two groups. The incidences of complications in both groups were statistically analyzed, and the discharge time from ICU, mechanical ventilation time and 28-day mortality were recorded.Results:After 1 week of treatment, the levels of prothrombin time (PT) and activated partial thromboplastin time (APTT) in the two groups were shortened, D-dimer (D-D) and fibrinogen (FIB) were decreased ( P<0.05); and the levels of PT and APTT in the combined group were shorter than those in the control group, the levels of DD and FIB were lower than those in the control group, there were statistical differences ( P<0.05). After 1 week of treatment, the levels of CD 4+ and CD 4+/CD 8+ ratio in both groups were increased ( P<0.05), and the levels of CD 4+ and CD 4+/CD 8+ ratio in the combined group were higher than those in the control group ( P<0.05). After 1 week of treatment, the levels of TLR4, C-reactive protein (CRP), procalcitonin (PCT), white blood cell count (WBC), blood lactate (Lac), blood urea nitrogen (BUN) and serum creatinine (Scr) in both groups were decreased ( P<0.05), meanwhile, the above indexes in the combined group were lower than those in the control group ( P<0.05). The incidence of multiple organ failure and the 28-day mortality rate in the combined group were lower than those in the control group: 3.92%(2/51) vs. 19.05%(8/42), 13.73%(7/51) vs. 30.95%(13/42), there were statistical differences ( P<0.05). The discharge time from ICU and mechanical ventilation time in the combined group were shorter than those in the control group: (12.35 ± 2.14) d vs. (14.17 ± 3.36) d, (7.12 ± 2.23) d vs. (8.51 ± 2.39) d, there were statistical differences ( P<0.05). Conclusions:Bedside high-flow CBP combined with Xuebijing injection in the treatment of SS can improve the patient′s condition, regulate the balance of coagulation and fibrinolysis, avoide the activation of coagulation, inhibite inflammatory response, reduce the expression of TLR4 in peripheral blood, improve immune function, protecte kidney function and promotethe patient′s recovery.

Application of blood purification technology in children with acute and critical diseases / 中国小儿急救医学

Objective:To explore the scope, mode, anticoagulation mode and complications of blood purification in children with acute and critical illness.Methods:A total of 377 times of treatment of 102 children treated with blood purification in PICU at the First Affiliated Hospital of Xinxiang Medical College from January 2018 to December 2020 were retrospectively analyzed.Results:Among 102 critically ill children treated with blood purification, acute and chronic renal failure ranked the first in terms of disease distribution, with 23 cases in total, followed by 16 cases of severe viral encephalitis (meningoencephalitis), 11 cases of septic shock, seven cases of acute poisoning, five cases of severe allergic purpura, five cases of necrotic encephalopathy.In terms of clinical prognosis, 51(50.0%) cases were cured, 29(28.4%) cases were improved, 10(9.8%) cases died, and 12 cases abandoned treatment.In 2019, the blood purification application frequency was the highest, with a total of 47 cases, which was higher than those in 2018 and 2020( P<0.05). Continuous veno-venous hemofiltration was used in the largest number of children, with a total of 56 cases.There was a statistically significant difference in the application ratio of this mode during 3 years ( P<0.05), while there was no statistically significant difference in the application ratio of other modes.In terms of the selection of anticoagulation methods, the proportions of systemic anticoagulation and extracorporeal anticoagulation had significantly difference among different years( P<0.05), and the application of extracorporeal anticoagulation had increased year by year.There was no statistically significant difference in the proportion of patients without anticoagulants.The incidence of complications of blood purification was the highest in 2019, with catheter related thrombus in the majority (30 person-times), followed by hypothermia, catheter filter coagulation, hematoma formation, catheter related infection, hypotension, heparin-induced thrombocytopenia, etc.There was statistically significant difference in the total complications among different years( P<0.05). Conclusion:Blood purification is widely used in children with acute and critical illness, with a variety of diseases.The most commonly used mode is continuous veno-venous hemofiltration and in vitro anticoagulation.Catheter-related thrombosis is the most common complication.

Blood purification in critically ill children: a large single-center eight-year retrospective study / 中国小儿急救医学

Objective:To compare the characteristics of patients undergoing blood purification treatment in PICU of a children′s tertiary hospital during 8 years, so as to analyze the changes in the development of blood purification technology in children in East China.Methods:Patients who received blood purification treatment in PICU of Children′s Hospital of Fudan University from 2014 to 2021 were included and divided into two study periods: 2014-2017 and 2018-2021.The clinical characteristics and treatment parameters of patients were collected and analyzed.Results:A total of 1 029 patients were included in the study, of which 103 were combined with extracorporeal membrane oxygenation.The 28-day survival rate of 926 patients treated with pure blood purification was 55.7%.Among them, patients with younger age, lower body weight, using mechanical ventilation, using vasoactive drugs before blood purification, and patients with multiple organ dysfunction syndrome had a higher distribution in the death group than those in survival group( P<0.05). During 8 years, a total of 3 688 cases of blood purification were performed.The main mode was continuous veno-venous hemodiafiltration (CVVHDF) (68.6%), followed by therapeutic plasma exchange (TPE) (23.8%) and hemoperfusion (HP) (4.8%); the main indication was acute kidney injury (AKI) (29.3%), followed by severe inflammatory disease (26.2%) and acute liver failure (16.2%). Compared with 2014-2017, the number of blood purification treatments in 2018-2021 increased by 47.4%, and the survival rate of patients increased significantly (48.7% vs. 58.1%, P<0.05). The distribution of blood purification patterns and indications also changed( P<0.05). The proportions of TPE (20.5% vs. 26.0%) and HP (3.1% vs. 6.0%) increased, while the proportion of CVVHDF (71.9% vs. 66.4%) decreased significantly.The proportions of AKI (29.8% vs. 38.9%) and refractory immune diseases (8.4% vs. 15.2%) were significantly higher, while severe inflammatory diseases (29.2% vs. 24.2%) and acute liver failure (19.6% vs. 13.8%) had declined. Conclusion:From 2014 to 2021, the number of blood purifications performed in our center increased significantly.Although the distribution of indications and patterns have also changed significantly, the overall survival rate is significantly improved.However, standardized practice still needs to be strengthened.

Research progress in culture, purification and isolation of synovial type A cells in vitro and their biological functions / 国际生物医学工程杂志

The temporomandibular joint (TMJ) is surrounded by a joint capsule, the smooth inner layer of which is called the synovial membrane. Synovium is involved in various intraarticular diseases, and it is a key area of joint disease. Synovial type-A cells are located in the lining layer of the synovial membrane, mostly on the side of the membrane close to the joint cavity. They have a strong phagocytic effect, and their main role is to remove the degradation products of the intra-articular and extracellular matrix. Various intra-articular diseases will affect the synovium, which is the key area of joint disease. The method of cell culture in vitro can effectively simulate the growth environment of cells in vivo and can accurately understand the effects of single and multiple factors on synovial cells, which has become a basic research method. In this review paper, the latest research progress in human temporomandibular joint type-A synoviocytes is reviewed from the aspects of cell origin, in vitro culture, cell purification, and cell biological function.

Identification of involatile chemical components from Moutai-flavored distiller’s grains / 中国药科大学学报

@#In order to study the involatile chemical components in Moutai-flavored distiller’s grains, the Moutai-flavored distiller’s grains were extracted with 75% ethanol, followed by extraction with petroleum ether, ethyl acetate, and n-butanol. Silica gel, ODS, sephadex LH-20, and preparative HPLC were used to separate and identify the petroleum ether and ethyl acetate layers.ESI-MS and NMR were used to identify the compounds, which were respectively identified as pentadecanoic acid (1), palmitic acid (2), trans-2-decenoic acid (3), n-nonyl octadecanoate (4), ethyl octadecanoate (5), ethyl linoleate (6), luric acid (7), 1, 3-dicaprylyl-2-linoleylglycerin (8), cyclic (phenylalanine-proline) (9), cyclo-(proline-leucine) (10), 3, 6-bis-(2-methylpropyl)-2,5-dione piperazine (11), 4-hydroxyphenethyl alcohol (12), 2,4-dihydroxybenzoic acid (13), stigmasterol (14), 2-furancarboxylic acid (15), valine (16), L-alanine acyl-L-proline (17), dihydroquercetin (18), 5, 7, 3'', 4''-tetrahydroxyflavonoids (19), quercetin (20), and naringenin (21). Compounds 1-21 were isolated from distiller’s grains for the first time.

Recombinant porcine interferon-gamma expressed in CHO cells and its antiviral activity / 生物工程学报

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×107 U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.

Production and antigenicity analysis of a recombinant insulinoma associated protein-2 in HEK293 cells / 生物工程学报

Insulinoma-associated protein-2 (IA-2) is a transmembrane glycoprotein belonging to the tyrosine phosphatase-like protein family as well as an important autoantigen in the diagnosis of type 1 diabetes. IA-2 products have been marketed in Europe and the United States. At present, commercially available IA-2 antigens are either the recombinant IA-2ic domain or the IA-2 naturally extracted from bovine islets. However, the recombinant IA-2 antigen displays weak positive in clinic practice, which often results in occasional detection failures, thus cannot completely replace the naturally extracted IA-2 antigen. In this study, an HEK293 expression system was used to explore the production of recombinant IA-2. An IA-2 transmembrane fragment (IA-2 TMF) located at amino acid position 449-979, also known as the natural membrane protein form of IA-2, was produced in HEK293 through transfection, and both the expression conditions and dissolution conditions of the membrane protein were also optimized. The purified membrane protein yield was 0.78 mg/L cell culture. Subsequently, the antigen activity of IA-2 TMF was compared with RSR rhIA-2 through enzyme linked immunosorbent assay. The serum of 77 type 1 diabetes patients and 32 healthy volunteers were detected. Receiver operating characteristic curve (ROC) curve was used to characterize the sensitivity and specificity of the test results. The results showed that the sensitivity of IA-2 TMF was 71.4% (55/77), while the sensitivity of RSR rhIA-2 was 63.6% (49/77), and the specificity of both antigens were all 100%. There was no significant difference in specificity between the two antigens, but the sensitivity of IA-2 TMF was appreciably better than that of the imported gold standard RSR rhIA-2 antigen. In conclusion, the recombinant IA-2 TMF produced in HEK293 cells can be used as a raw material to develop in vitro diagnostic reagents for type 1 diabetes.

Characterization and identification of primarily cultured astrocytes and microglia / 生物工程学报

The aim of this study was to investigate the growth characteristics of primarily cultured astrocytes and microglia of different generations and then optimize the method for obtaining primary astrocytes and microglia effectively. Primarily cultured microglia were isolated and purified from the cortices of neonatal mice. The proliferation curve of mixed glia cells was measured by Cell Counting Kit-8 (CCK-8) assay, the proportion of astrocytes and microglia was detected by flow cytometry, and the polarization of the two types of glia cells was identified by immunofluorescence staining. Cell growth results showed that the mixed glia cells of P0 and P1 generation had the best proliferative activity; 97.3% of the high purity microglia could be obtained by mechanical shaking at 170 r/min for 30 min, and there was no significant difference in the morphology of ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia and the proportion of M1 and M2 phenotype among the P0, P1 and P2 generations of microglia isolated by the above methods. Moreover, 95.7 % of the high purity astrocytes could be obtained by astrocyte cell surface antigen-2 (ACSA-2) magnetic beads separation, and there was no significant difference in the morphology of glial fibrillary acidic protein (GFAP) positive astrocyte and the proportion of A1 and A2 phenotype among the P0, P1 and P2 generations of astrocyte isolated by the above methods. Taken together, this study observed the growth characteristics of primarily cultured microglia and astrocyte in vitro, and then proved the best generations for purifying microglia and astrocytes. Finally, we optimized the methods of obtaining microglia and astrocyte, and verified that continuous culture within 2 generations will not affect the functional phenotypes of glia cells. These results provide technical support for studying the molecular mechanism of inflammation-associated diseases in nervous system.

Optimization of large-scale production process for human prothrombin complex concentrates / 中国输血杂志

【Objective】 To determine the optimal process conditions for efficiently extracting human prothrombin complex concentrates from human plasma. 【Methods】 Using human plasma as the materials and the yield of prothrombin complex concentrates as the evaluation standard, the preparation process parameters were studied and optimized through design of exporement(DOE), orthogonal experiments, and single factor experiments. 【Results】 The optimal process conditions were as follows: DEAE Sephadex A50 gel was selected, which balanced to pH 7.6, and then the amount of 1.7-2.5 g/L of plasma weight is added into the cryoprecipitate supernatant for adsorption for 40 minutes; Washing solution (0.15-0.175 mol/L sodium chloride) with 3 times the volume of gel was washed 3 times, and eluent (0.5-2.0 mol/L sodium chloride) was washed 3 to 5 times; Add stabilizer (heparin 35 IU, sodium chloride 0.1 mol/L) for ultrafiltration dialysis. 【Conclusion】 By using the optimized process mentioned above, the yield(measured by human coagulation factor IX)can reach 620 000 to 630 000 IU/ton of plasma, which is suitable for large-scale production.

Engineering the plastic degradation enzyme Ple629 from marine consortium to improve its thermal stability / 生物工程学报

Petrochemical-derived polyester plastics such as polyethylene terephthalate (PET) and polybutylene adipate terephthalate (PBAT) have been widely used. However, the difficulty to be degraded in nature (PET) or the long biodegradation cycle (PBAT) resulted in serious environmental pollution. In this connection, treating these plastic wastes properly becomes one of the challenges of environment protection. From the perspective of circular economy, biologically depolymerizing the waste of polyester plastics and reusing the depolymerized products is one of the most promising directions. Recent years have seen many reports on polyester plastics degrading organisms and enzymes. Highly efficient degrading enzymes, especially those with better thermal stability, will be conducive to their application. The mesophilic plastic-degrading enzyme Ple629 from the marine microbial metagenome is capable of degrading PET and PBAT at room temperature, but it cannot tolerate high temperature, which hampers its potential application. On the basis of the three-dimensional structure of Ple629 obtained from our previous study, we identified some sites which might be important for its thermal stability by structural comparison and mutation energy analysis. We carried out transformation design, and performed expression, purification and thermal stability determination of the mutants. The melting temperature (Tm) values of mutants V80C and D226C/S281C were increased by 5.2 ℃ and 6.9 ℃, respectively, and the activity of mutant D226C/S281C was also increased by 1.5 times compared with that of the wild-type enzyme. These results provide useful information for future engineering and application of Ple629 in polyester plastic degradation.