Evolution of chemistry and selection technology for DNA-encoded library

DNA-encoded chemical library (DEL) links the power of amplifiable genetics and the non-self-replicating chemical phenotypes, generating a diverse chemical world. In analogy with the biological world, the DEL world can evolve by using a chemical central dogma, wherein DNA replicates using the PCR reactions to amplify the genetic codes, DNA sequencing transcripts the genetic information, and DNA-compatible synthesis translates into chemical phenotypes. Importantly, DNA-compatible synthesis is the key to expanding the DEL chemical space. Besides, the evolution-driven selection system pushes the chemicals to evolve under the selective pressure, i.e., desired selection strategies. In this perspective, we summarized recent advances in expanding DEL synthetic toolbox and panning strategies, which will shed light on the drug discovery harnessing in vitro evolution of chemicals via DEL.

Consideraciones éticas y científicas del inicio de la vida sobre el aborto eugenésico en enfermedades genéticas incapacitantes en Perú

El artículo expone conceptos actuales biológicos, así como algunos planteamientos filosóficos acerca del inicio de la vida, que se examinarán en relación con la necesidad del aborto eugenésico. Se presenta el concepto de "exdurantismo", en el que la combinación del nuevo genoma nuclear y mitocondrial hacen único al individuo; además, se concibe al complejo genómico como "integrador somático" que dirige el desarrollo embrionario, y se muestra la problemática en el caso de las violaciones que provocan embarazos, el aborto eugenésico en enfermedades genéticas incapacitantes o la utilización de embriones posterior a la fertilización in-vitro. Se plantea que la dignidad de la persona comienza desde la concepción. Frente a la detección de una enfermedad genética, grave o letal, se debe ofrecer soporte económico y social, de diagnóstico y tratamiento; además, desde la salud pública, una mayor inversión para plantear estrategias de tamizaje, diagnóstico, manejo e investigación.

The article presents current biological concepts, as well as some philosophical approaches to the beginning of life, which will be examined in relation to the need for eugenic abortion. The concept of "exdurantism" is presented, in which the combination of the new nuclear and mitochondrial genome makes the individual unique; furthermore, the genomic complex is conceived as a "somatic integrator" that directs embryonic development, and the problematic is shown in the case of rape that causes pregnancies, eugenic abortion in incapacitating genetic diseases or the use of embryos after in-vitro fertilization. The dignity of the person begins at conception. When a serious or lethal genetic disease is detected, economic and social support, diagnosis and treatment should be offered; in addition, public health should invest more in screening, diagnosis, management and research strategies.

O artigo apresenta conceitos biológicos atuais, bem como algumas abordagens filosóficas sobre o início da vida, que serão examinados em relação à necessidade do aborto eugênico. É apresentado o conceito de "exdurantismo", no qual a combinação do novo genoma nuclear e mitocondrial torna o indivíduo único; além disso, o complexo genômico é concebido como um "integrador somático" que dirige o desenvolvimento embrionário, e é mostrada a problemática no caso de estupro que resulta em gravidez, aborto eugênico em doenças geneticamente incapacitantes ou o uso de embriões após a fertilização in vitro. Argumenta-se que a dignidade da pessoa começa na concepção. Diante da detecção de uma doença genética, grave ou letal, devem ser oferecidos apoio econômico e social, diagnóstico e tratamento, bem como maior investimento em saúde pública em estratégias de triagem, diagnóstico, gestão e pesquisa.

In-vitro Anti-hyperglycemic Evaluation of Hydroalcoholic Extract of Delonix regia Bark

Anti-hyperglycemic agents is a substance that helps a person with diabetes control their level of glucose (sugar) in the blood. It includes insulin and oral anti-hyperglycemic agents. Diabetes is a metabolic disorder characterized by increased blood glucose levels leading to other major complications. Thus, obtaining these anti hyperglycemic agents through easily available flora is necessary. Delonix regia , a tree cultivated worldwide, has also been used as traditional medicine in various disorders. Aim of the project work was to evaluate the anti-hyperglycemic activity in the hydroalcoholic extract of D. regia bark for the treatment of hyperglycemia. The collected bark was dried, powdered and extracted through cold maceration method. The extract was further concentrated to obtain a gummy mass of the hydroalcoholic extract. The extract was subjected to phytochemical analysis through conventional chemical tests and GC-MS. After the identification of the phytoconstituents, they were studied for their clinically proven properties. In-vitro anti-hyperglycemic studies were carried out through assays like alpha-amylase inhibition assay and alpha-glucosidase inhibition assay. The results of the extract were compared with results of standard acarbose. The IC 50 standard values in alpha-amylase inhibition assay and ?-glucosidase inhibition assay were 98.77 and 84.33 ?g/mL, respectively. The IC 50 values of hydroalcoholic extract of D. regia bark in alpha-amylase and alpha-glucosidase inhibition assay were 167 and 116.31 ?g/mL, respectively. From the study, the hydroalcoholic extract of bark of D. regia exhibit anti-hyperglycemic activity compared to standard acarbose.

Biomechanical Characteristics of Idiopathic Pulmonary Fibrosis / 医用生物力学

Idiopathic pulmonary fibrosis (IPF) is a common chronic interstitial fibrotic disease. During the fibrosis process, myofibroblasts are abnormally activated, collagen is deposited in large quantities and the biomechanical characteristics of lung tissue are significantly altered. In this paper, a systematic review about the changes in lung tissues, cellular biomechanical properties and biomechanical signals during the process of IPF was presented, and the in vitro reproduction of biomechanical features and therapeutic strategies for targeting biomechanics wassummarized, so as to provide references for clinical prevention and treatment of IPF.

Study of resveratrol against bone loss by using in-silico and in-vitro methods / Estudo do resveratrol contra a perda óssea usando métodos in silico e in vitro

By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa-Β ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of −6.9, −7.6, −7.1, −6.9, −6.7, and −7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 μg / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 μg / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.

Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa-Β [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de −6,9, −7,6, −7,1, −6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 μg / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 μg / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.

Study of resveratrol against bone loss by using in-silico and in-vitro methods

Abstract By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa- ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of 6.9, 7.6, 7.1, 6.9, 6.7, and 7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 g / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 g / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.

Resumo Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa- [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de 6,9, 7,6, 7,1, 6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow-derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 g / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 g / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.

Study of resveratrol against bone loss by using in-silico and in-vitro methods / Estudo do resveratrol contra a perda óssea usando métodos in silico e in vitro

Abstract By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa-Β ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of −6.9, −7.6, −7.1, −6.9, −6.7, and −7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 μg / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 μg / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.

Resumo Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis ​​pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa-Β [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de −6,9, −7,6, −7,1, −6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow-derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 μg / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 μg / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.

Emerging trends in organ-on-a-chip systems for drug screening

New drug discovery is under growing pressure to satisfy the demand from a wide range of domains, especially from the pharmaceutical industry and healthcare services. Assessment of drug efficacy and safety prior to human clinical trials is a crucial part of drug development, which deserves greater emphasis to reduce the cost and time in drug discovery. Recent advances in microfabrication and tissue engineering have given rise to organ-on-a-chip, an in vitro model capable of recapitulating human organ functions in vivo and providing insight into disease pathophysiology, which offers a potential alternative to animal models for more efficient pre-clinical screening of drug candidates. In this review, we first give a snapshot of general considerations for organ-on-a-chip device design. Then, we comprehensively review the recent advances in organ-on-a-chip for drug screening. Finally, we summarize some key challenges of the progress in this field and discuss future prospects of organ-on-a-chip development. Overall, this review highlights the new avenue that organ-on-a-chip opens for drug development, therapeutic innovation, and precision medicine.

Phytochemical Analysis and In-vitro Antioxidant Activities of Some Selected Higher Fungi from Oyo State, South West of Nigeria

Higher-fungi (Hf) of the polypore mushrooms are considered to have unique secondary metabolites, making them reservoirs of therapeutically significant bioactive compounds. Phytochemical and antioxidant properties of the Hf were accessed in this study. Four Hf, which were found in several wild locations in Oyo state, Nigeria, were collected. At the University of Ibadan Botany Department Laboratory, the species of the four Hf were determined. In-vitro antioxidant activity were assessed using the 1,1-Diphenyl-2-picrylhydrazyl (DPPH), Ferric Reducing Antioxidant Power (FRAP), and Hydrogen peroxide (H2O2) assays using methanol extracts of air-dried and powdered Hf. Results were presented as Mean SEM, graphs were created in Excel, one-way ANOVA was used for the analysis, and p ?0.05 was regarded as significant. These Hf were identified as Lycoperdon rimlatum (Lr) FFUI1, Trametes versicolor (Tv) FFUI2, Ganoderma lucidum (Gl) FFUI3, and Daedelia quarcina (Dq) FFUI4 and were recorded on the NCBI with accession numbers EU833664.1, JQ621899, JQ520179.1 and KP171209.1, respectively. All the Hf showed positive tests for the presence of saponin, tannin, alkaloid, terpenoid, carbohydrate,tannin and flavonoids. The Hf showed antioxidant activities, the highest DPPH inhibition was by Tv (94.48%), FRAP was by Gl (0.16 mg/g) and H2O2 inhibition was by Lr (70.90%). The antioxidant activities observed were due to the presence of useful phytochemicals making them therapeutically significant.

Parámetros para la evaluación visual e instrumental del color dental en estudios in-vitro. Revisión de la literatura / Parameters for visual and instrumental assessment of tooth colour in in-vitro studies. Literature review

Objetivo: establecer los parámetros para la evaluación visual e instrumental del color dental en estudios in-vitro a partir de la literatura científca publicada entre 2015 y 2021. Métodos: se realizó la búsqueda en las bases de datos: PubMed, Web of Science, Science Direct, Scopus, Scielo y Lilacs; también en el motor de búsqueda Google Académico y las bibliotecas de las editoriales Wiley y Springer. Las palabras clave utilizadas fueron tooth, color, in-vitro, color perception, shade matching, thresholds, appearance, surrounding, "CIELAB" y "CIEDE2000". Teniendo en cuenta los criterios de elegibilidad, se seleccionaron los estudios de acuerdo al título, resumen y texto completo. Resultados: la búsqueda arrojó un total de 37 publicaciones que se agruparon en tres tópicos: 1. toma de color visual: condiciones ambientales, observadores y nivelación; 2. toma de color instrumental: instrumentos; y 3. procesamiento de datos: cálculo de la diferencia de color y umbrales de perceptibilidad (PT) y aceptabilidad (AT). Conclusiones: los aspectos más importantes en la evaluación visual son la iluminación, el ambiente para registro (sitio, entorno y fondo alrededor de la muestra), las condiciones geométricas de visualización, los observadores y el uso de guías. En la evaluación instrumental es relevante elegir el aparato apropiado de acuerdo con su precisión y reproducibilidad, como los espectroradiómetros y los espectrofotómetros de uso clínico. Se presenta el procesamiento de datos para establecer las variaciones de cada coordenada, las diferencias de color (ΔE): CIELAB y CIEDE2000, los umbrales y los lineamientos.

Objective: To establish the parameters for the visual and instrumental evaluation of tooth color in in-vitro studies based on the scientifc literature published between 2015 and 2021. Methods: The search was carried out in the databases of PubMed, Web of Science, Science Direct, Scopus, Scielo, Lilacs; search engine Google Scholar and publishers' library of Wiley and Scielo, using the keywords "tooth", "color", "in vitro", "color perception", "shade matching", "thresholds", "appearance", "surrounding", "CIELAB", and "CIEDE2000". The literature was selected according to the title, abstract and full text taking into the eligibility criteria. Results: It yielded a total of 37 publications, which were grouped into three topics: 1. visual color acquisition: environmental conditions for color acquisition, observers and levelling. 2. instrumental color sampling: instruments. 3. Data processing: Calculation of color diference and perception thresholds (PT) and acceptability thresholds (AT). Conclusions: The most important aspects in the visual assessment are lighting, the environment for color registration (site, environment and background around the sample), the geometric conditions of visualization, the observers and the use of guides. Regarding the instrumental assessment of color, the appropriate devices must be chosen according to its precision and reproducibility, being the spectrophotometers and spectroradiometers the most precise ones. It is presented how the data processing is carried out to establish the variations of each coordinate, the color diferences (ΔE): CIELAB and CIEDE2000, thresholds and guidelines.

Association of raised serum progesterone level with ovulation trigger and histology of endometrium in stimulated cycles

Abstract This was a forthcoming study of those patients, who undergo in-vitro fertilization (IVF) and freeze-all embryo, who acquiesce for the study. The number of participated patients (n=350) in this study, underwent for IVF. The blood sample was collected from patients to evaluate the level of serum progesterone in vacuum vials on the day of ovulation trigger. After 36 hrs of ovulation trigger, ovum picked up was done. Quantitative methods were used to estimate the level of serum progesterone through the electrochemiluminescence immunoassay and correlation of serum progesterone with embryo transfer (ET) outcomes. Main outcome of this current study was to evaluate the value of mean serum progesterone level i.e.0.868± 0.712 ng/ml and 0.88±0.723 ng/ml was found in case of pregnancy positive and negative respectively, at p=0.216 value. In antagonist (n=40) and agonist (n=310) cases, it was 8(20%) and 37(11.94%) PL occurrence was noted at p=0.143 respectively. An overall value of the premature lutenization (PL) occurrences was 13.63% and 15.25% observed in both positive and negative cases of pregnancy at p=0.216 respectively. This study concluded that 12.66% of PL occurrences were recorded in the case of IVF. Study results proved, there were no significant effect of PL on pregnancy outcomes.

Development and optimization of cosolvent-based blended Sertraline orodispersible films - A step to personalized medicine

Abstract Personalized medicine is gaining importance in pharmacotherapeutics as it allows tailoring the drug treatment to achieve the best patient response. Orodispersible film (ODF) is easy to formulate in hospitals, produces dose flexibility to suit an individual needs, particularly for patients suffer from swallowing issues or prohibited to take fluids. Sertraline Hydrochloride (SRT) was solubilized in several cosolvents, then different SRT ODFs based on five hydrophilic polymers namely; polyvinyl alcohol (PVA), hydroxylethyl cellulose (HEC), hydroxypropyl methylcellulose E5 LV (HPMC E5 LV), sodium alginate (NaAlg) and gelatin at two concentrations (2% and 4%) were developed and characterized. The outcomes were exposed to response surface analysis to obtain the desirability results to obtain the optimized formulation. Blended ODFs were developed from 4% PVA and 2% HEC in different blends and then potassium chloride (KCl) as a pore-forming agent was added to the best formulation to investigate its dissolution enhancement effect. F14 containing 4% PVA: 2% HEC 2:1 with 5% KCl showed best physicochemical properties of suitable pH (5.6), disintegration time (6 sec), good folding endurance which released 91 % SRT after 15 min. SRT ODF is an encouraging delivery system in the course of personalized medicine for the management of depression.

Design of experiments assisted the development of inclusion complexes of ramipril using hydrophilic carriers for enhancement of solubility and dissolution rate

Abstract The goal of the present study was to develop inclusion complexes and polymers dispersions of ramipril prepared by physical mixing, kneading, co-evaporation, and solvent evaporation methods to enhance drug solubility and dissolution rate, and thereby to reduce drug dose and side effects using selected hydrophilic carriers such as β-CD, PVP-K25, PEG 4000, and HPMC K100M. The prepared formulations were characterized for solubility and in-vitro drug release studies. The systematic optimization of formulations was performed using I-Optimal experimental design by selecting factors such as type of carriers (X1), drug: carrier ratio (X2), and method of preparation (X3), and response variables including percent yield (Y1), solubility (Y2), Carr's index (Y3) and drug release in 30 min (Y4). Mathematical modeling was carried out using a quadratic polynomial model. The inclusion complex formulation (F27) was selected as an optimized batch by numerical desirability function and graphical optimization with the help of design space. The inclusion complex prepared by the co-evaporation method showed maximum drug solubility and released in pH 6.8 phosphate buffer compared to pure and other formulations. The inclusion complex is a feasible approach to improve the solubility, dissolution rate, bioavailability, and minimization of drugs' gastrointestinal toxicity upon oral administration of ramipril.

A study on gene mutation of coagulation factor Ⅺ protein secretion disorder and its mechanism / 中华检验医学杂志

Objective:To investigate the molecular pathogenesis of a newly discovered gene mutation in a family with hereditary coagulation factor Ⅺ(FⅪ) deficiency.Methods:The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in September 2021 due to "calculus of intrahepatic duct". The patient had no symptoms of spontaneous bleeding.The clinical data and blood samples of the proband and her family members (10 persons in 3 generations) were collected.The activated partial thromboplastin time (APTT) and FⅪ activity (FⅪ:C) were performed by the one-stage clotting assay. FⅪ antigen (FⅪ:Ag) were detected by enzyme linked immunosorbent assay (ELISA). Genomic DNA extracted from peripheral blood cells of subjects was used as template to analyze F11 gene mutation by DNA direct sequencing. Bioinformatics software was used to analyze the effects of mutations on protein structure and function. Wild-type and mutant FⅪ protein expression vectors were constructed and transient transfected into HEK293T cells. The total RNA was extracted from positive transfected cells and then reversely transcribed into cDNA. The mRNA expression level of F11 gene in transfected cells was detected by real-time fluorescence quantitative PCR (qRT-PCR). The content of FⅪ:Ag and the expression of FⅪ protein in transfected cell lysates and culture supernatant were detected by ELISA and western blot.Results:The APTT of the proband was significantly prolonged to 107.9s (reference range 29.0-43.0s), while FⅪ:C and FⅪ:Ag were significantly decreased to 2% (reference range 84%-122%) and 5% (reference range 76%-127%), respectively. Gene sequencing analysis indicated that the proband had c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation in exon 6 and 13 of the F11 gene, respectively. Bioinformatics analysis showed that the amino acids at site 161 of FⅪ protein were threonine (Thr) in the matrix composed of five different species, indicating that Thr161 site was highly conserved among homologous genes in different species. p.Thr161Met heterozygous mutation affected the stability of local intermolecular structure of FⅪ protein. In vitro expression experiments of p.Thr161Met mutation showed that FⅪ protein had a normal synthesis in the cells but secretion dysfunction.Conclusions:c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation were mainly responsible for the decrease of FⅪ in this family. p.Thr161Met mutation was first reported in the world and did not affect the normal synthesis of FⅪ protein, but caused secretion dysfunction.

Bridging the structure gap between pellets in artificial dissolution media and in gastro-intestinal tract in rats

Changes in structure of oral solid dosage forms (OSDF) elementally determine the drug release and its therapeutic effects. In this research, synchrotron radiation X-ray micro-computed tomography was utilized to visualize the 3D structure of enteric coated pellets recovered from the gastrointestinal tract of rats. The structures of pellets in solid state and in vitro compendium media were measured. Pellets in vivo underwent morphological and structural changes which differed significantly from those in vitro compendium media. Thus, optimizations of the dissolution media were performed to mimic the appropriate in vivo conditions by introducing pepsin and glass microspheres in media. The sphericity, pellet volume, pore volume and porosity of the in vivo esomeprazole magnesium pellets in stomach for 2 h were recorded 0.47, 1.55 × 108 μm3, 0.44 × 108 μm3 and 27.6%, respectively. After adding pepsin and glass microspheres, the above parameters in vitro reached to 0.44, 1.64 × 108 μm3, 0.38 × 108 μm3 and 23.0%, respectively. Omeprazole magnesium pellets behaved similarly. The structural features of pellets between in vitro media and in vivo condition were bridged successfully in terms of 3D structures to ensure better design, characterization and quality control of advanced OSDF.

The effect of timing of oocyte denudation from oocyte retrieval in the total fertilization failure among in vitro fertilization - Intracytoplasmic sperm injection cycles

@#<p>Objective: This study aimed to evaluate the effect of oocyte incubation after retrieval in TFF among IVF-ICSI and identify factors affecting total fertilization failure (TFF).</p><p>Methods: This is a retrospective cohort study, involving 995 IVF cycles using the antagonist protocol that were clustered into three timings of oocyte denudation from retrieval : Group 1: <1 hour, Group 2: ?1 hour to <2hours and Group 3: ?2hours. Other variables considered were etiology of infertility, female age, days of stimulation and total number of oocytes retrieved.</p><p>Results: Overall TFF was 4.5%. TFF among groups were 4.8%, 5.8% and 3.2%, respectively. Multiple logistic regression analysis showed that oocyte incubation prior to denudation for ? 2 hours tend to decrease TFF incidence. Among factors studied, male factor infertility and a low number of oocytes adversely affect TFF.</p><p>Conclusion: Timing of incubation of oocyte did not significantly affect the occurrence of TFF. Among factors studied, male factor infertility and a low number of oocytes adversely affect TFF.</p>

Sperm-specific protein ACTL7A as a biomarker for fertilization outcomes of assisted reproductive technology / 亚洲男科学杂志(英文版)

Obtaining high-quality embryos is one of the key factors to improve the clinical pregnancy rate of assisted reproductive technologies (ART). So far, the clinical evaluation of embryo quality depends on embryo morphology. However, the clinical pregnancy rate is still low. Therefore, new indicators are needed to further improve the evaluation of embryo quality. Several studies have shown that the decrease of sperm-specific protein actin-like 7A (ACTL7A) leaded to low fertilization rate, poor embryo development, and even infertility. The aim of this study was to study whether the different expression levels of ACTL7A on sperm can be used as a biomarker for predicting embryo quality. In this study, excluding the factors of severe female infertility, a total of 281 sperm samples were collected to compare the ACTL7A expression levels of sperms with high and low effective embryo rates and analyze the correlation between protein levels and in-vitro fertilization (IVF) laboratory outcomes. Our results indicated that the ACTL7A levels were significantly reduced in sperm samples presenting poor embryo quality. Furthermore, the protein levels showed a significant correlation with fertilization outcomes of ART. ACTL7A has the potential to be a biomarker for predicting success rate of fertilization and effective embryo and the possibility of embryo arrest. In conclusion, sperm-specific protein ACTL7A has a strong correlation with IVF laboratory outcomes and plays important roles in fertilization and embryo development.

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use

ABSTRACT: Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF- (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.

RESUMO: O plasma rico em plaquetas (PRP) é uma alternativa terapêutica promissora, pois as plaquetas são ricas em fatores de crescimento com ação na regeneração de tecidos. No entanto, o PRP fresco não pode ser armazenado por longos períodos. Esse trabalho teve como objetivo desenvolver um protocolo de obtenção de PRP liofilizado canino capaz de manter a viabilidade pós reconstituição. Portanto, foram testados diversos protocolos de extração e liofilização. Para validação do PRP canino liofilizado foi analisada a concentração dos fatores de crescimento VEGF e TGF- antes e após o processo de liofilização, a estrutura tridimensional do PRP liofilizado reconstituído em forma de gel por microscopia eletrônica e seu efeito in vitro na proliferação de células-tronco mesenquimais. Os resultados demonstraram que o PRP liofilizado apresentou concentrações terapêuticas adequadas dos fatores de crescimento VEGF e TGF- (9,1pg/ml e 6161,6pg/ml, respectivamente). O gel de PRP reconstituído após liofilização apresentou uma durabilidade in vitro de 10 dias, sua estrutura tridimensional mostrou-se semelhante ao PRP fresco e proporcionou intensa proliferação de células-tronco mesenquimais durante o cultivo. O teste de imunogenicidade não demonstrou evidências de reação imune. Os achados foram promissores, sugerindo a possibilidade de uso de PRP canino liofilizado para o mercado. Novos estudos in vivo e in vitro deverão ser conduzidos para comprovação terapêutica.

Design and experiment of online monitoring system for long-term culture of embryo / 生物医学工程学杂志

In the study of embryo development process, the morphological features at different stages are essential to evaluate developmental competence of the embryo, which can be used to optimize and improve the system for