Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing

Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.

Avaliação da infecção por Parvovírus Humano B19 em diferentes grupos populacionais: diagnóstico diferencial e aspectos imunopatogênicos da infecção aguda e persistente / Evaluation of Human Parvovirus B19 infection in different population groups: differential diagnosis and immunopathogenic aspects of acute and persistent infection

A infecção pelo Parvovírus B19 (B19V) pode ocorrer em indivíduos imunocompetentes e imunocomprometidos, de todas as faixas etárias, e se caracteriza por ser aguda e autolimitada, podendo levar a quadros de doença exantemática (DE), doença febril aguda (DFA), doença renal crônica (DRC) e falência hepática aguda (FHA). O diagnóstico diferencial de B19V nessas populações, muitas vezes, não ocorre e estudos sobre a prevalência do B19V são antigos e escassos, não refletindo a atualidade. Marcadores da infecção podem ser detectados na circulação e em diferentes tipos de tecidos, inclusive em tecidos não eritroides, por meses ou anos. A infecção pode levar a manifestações clínicas graves, que requer tratamento hospitalar, e a doenças inflamatórias atípicas, como: cardiomiopatia, artrite reumatoide, hepatite e vasculite. No entanto, a detecção de B19V DNA não implica necessariamente na presença de vírions infecciosos e na associação do B19V com essas manifestações atípicas. Dessa forma, o objetivo do trabalho foi otimizar técnicas de PCR em tempo real para quantificação do B19V DNA e de detecção de partículas virais infecciosas, a fim de realizar o diagnóstico diferencial da infecção pelo B19V em pacientes com DE, DFA, DRC e FHA. Para o diagnóstico da infecção, amostras de diferentes populações foram testadas: DE (n=54), DFA (n=60), DRC (n=221), e FHA (n=30). Amostras de soro (e de tecido hepático para FHA) foram submetidas a avaliação de marcadores sorológicos (IgM e IgG anti-B19V) e moleculares do B19V, a fim de determinar a fase da infecção em que o paciente se encontrava. Para a avaliação de marcadores moleculares, a metodologia de PCR quantitativo e em tempo real foi otimizada e permitiu um diagnóstico sensível e específico do B19V DNA. Além disso, a presença de vírions em amostras de pacientes com B19V (n=10) e de macacos cynomolgus (n=4) infectados experimentalmente foram avaliadas por meio da técnica de pré-tratamento das amostras com uma enzima endonuclease. O teste molecular (qPCR) otimizado durante o estudo, apresentou sensibilidade e especificidade de 100%. O ensaio com a endonuclease revelou que a maioria das amostras de soro humano tornou-se B19V DNA negativa após o pré-tratamento, indicando que não eram infecciosas. Foi observado prevalências do B19V DNA em 5,5% dos pacientes com DE; 6,6% em DFA; 65,6% em DRC, e 23,3% em FHA. Como conclusão a técnica de qPCR otimizada no presente estudo foi efetiva para o esclarecimento de casos da infecção por B19V e é adequada para diagnóstico diferencial. Além disso, o teste laboratorial baseado em endonuclease possibilitou a discriminação do B19V DNA (se encapsidado em vírions ou não). Portanto, estes testes podem ser utilizados para esclarecer o papel do B19V como agente etiológico associado a diversas manifestações clínicas. As prevalências encontradas nesse estudo indicam que o B19V está circulando entre os diversos grupos populacionais estudados e deve ser feita uma melhor vigilância da infecção, pois está presente tanto em indivíduos imunocompetentes como em imunocomprometidos. Além disso, os resultados sugerem a importância da inclusão de B19V no diagnóstico laboratorial diferencial, não apenas para fins epidemiológicos, mas também para o manejo adequado do paciente.

Parvovirus B19 (B19V) infection can occur in immunocompetent and immunocompromised individuals of all group ages and is characterized as acute and self limiting, which can lead to rash disease (RD), acute febrile illness (AFI), chronic kidney disease (CKD), and acute liver failure (ALF). Differential diagnosis of B19V in these populations often does not occur and studies on the prevalence of B19V are scarce, outdated, and do not reflect the current situation. B19V markers of acute infection can be detected in the circulation and in different tissue types, including non-erythroid tissues, for months to years and may lead to severe clinical manifestations, requiring hospital treatment, and to atypical inflammatory diseases, such as cardiomyopathy, rheumatoid arthritis, hepatitis, and vasculitis. However, the detection of B19V DNA does not necessarily imply the presence of infectious virions and the causal relation between B19V and atypical manifestations could not be proved yet. Thus, the aim of this study was to standardize the real-time PCR for quantification of B19V DNA and detection of infectious viral particles in order to perform the differential diagnosis of the B19V infection in RD, AFI, CKD, and ALF patients. For the diagnosis of the infection, samples from different populations were tested: RD (n=54), AFI (n=60), CKD (n=221), and ALF (n=30). Serum samples (and hepatic tissue for ALF) were submitted to the evaluation of B19V serological status (anti-B19V IgM and IgG antibodies) and molecular markers, in order to determine the stage of infection in which the patient is. For the evaluation of molecular markers, a quantitative real-time PCR methodology was optimized and allowed a sensitive and specific diagnosis of B19V DNA. In addition, the presence of virions in samples from patients with B19V (n=10) and from cynomolgus monkeys (n=4) experimentally infected were evaluated by endonuclease enzyme pretreatment. The molecular test optimized during the study showed 100% sensitivity and specificity. The endonuclease treatment assay revealed that most human serum samples became negative after pretreatment, as indicative of non-infective particles. Concerning the prevalence of B19V DNA: 5.5% were obtained in patients with RD; 6.6% in AFI; 65.6% in CKD, and 23.3% in ALF. In conclusion, the qPCR technique optimized in the present study was effective for clarifying cases of B19V infection and is suitable for differential diagnosis. In addition, the endonuclease-based laboratory test made it possible to discriminate B19V DNA (whether encapsidated in virions or not). Therefore, these tests can be used to clarify the role of B19V as an etiologic agent associated with several clinical manifestations. The prevalence found in this study indicate that B19V is circulating among the different populational groups that have been studied and better surveillance of the infection should be carried out, as it is present in both immunocompetent and immunocompromised individuals. In addition, the results suggest the importance of including B19V in the differential laboratory diagnosis, not only for epidemiological purposes but also for the proper management of the patient.

Progress in gene editing technologies for Saccharomyces cerevisiae / 生物工程学报

Saccharomyces cerevisiae is one of the most important hosts in metabolic engineering. Advanced gene editing technology has been widely used in the design and construction of S. cerevisiae cell factories. With the rapid development of gene editing technology, early gene editing technologies based on recombinase and homologous recombination have been gradually replaced by new editing systems. In this review, the principle and application of gene editing technology in S. cerevisiae are summarized. Here, we first briefly describe the classical gene editing techniques of S. cerevisiae. Then elaborate the genome editing system of MegNs, ZFNs and TALENs based on endonuclease. The latest research progress is especially introduced and discussed, including the CRISPR/Cas system, multi-copy integration of heterologous metabolic pathways, and genome-scale gene editing. Finally, we envisage the application prospects and development directions of Saccharomyces cerevisiae gene editing technology.

miRNA-296-5p functions as a potential tumor suppressor in human osteosarcoma by targeting SND1 / 中华医学杂志(英文版)

BACKGROUND@#The pathogenesis of osteosarcoma (OS) is still unclear, and it is still necessary to find new targets and drugs for anti-OS. This study aimed to investigate the role and mechanism of the anti-OS effects of miR-296-5p.@*METHODS@#We measured the expression of miR-296-5p in human OS cell lines and tissues. The effect of miR-296-5p and its target gene staphylococcal nuclease and tudor domain containing 1 on proliferation, migration, and invasion of human OS lines was examined. The Student's t test was used for statistical analysis.@*RESULTS@#We found that microRNA (miR)-296-5p was significantly downregulated in OS cell lines and tissues (control vs. OS, 1.802 ± 0.313 vs. 0.618 ± 0.235, t = 6.402, P < 0.01). Overexpression of miR-296-5p suppressed proliferation, migration, and invasion of OA cells. SND1 was identified as a target of miR-296-5p by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of SND1 abrogated the effects induced by miR-296-5p upregulation (miRNA-296-5p vs. miRNA-296-5p + SND1, 0.294 ± 0.159 vs. 2.300 ± 0.277, t = 12.68, P = 0.003).@*CONCLUSION@#Our study indicates that miR-296-5p may function as a tumor suppressor by targeting SND1 in OS.

Development of a CRISPR/Cpf1 gene editing system in silkworm Bombyx mori / 生物工程学报

The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type (V type) CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5'T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1 (AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3A-346 knockout vectors. The efficiency for editing the target genes BmATAD3A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.

Advances of CRISPR/dCas9 system in live cell imaging / 生物工程学报

The study of distinct genes, chromosomes and the spatio-temporal relationships between them is of great significance in genetics, developmental biology and biomedicine. CRISPR/Cas9 has become the most widely used gene editing tool due to its excellent targeting ability. Recently, researchers have developed a series of advanced live cell imaging techniques based on the nuclease-inactivated mutant of Cas9 (dCas9), providing rapid and convenient tools for high-resolution imaging of specific sites in the chromatin and genome. This review summarizes the advances of CRISPR/dCas9 system in live cell imaging from three aspects, including the strategies of cell delivery, optimization of the fluorescence signals, as well as orthogonal and multicolor imaging. Furthermore, we shed light on the development trends and prospects of this field.

Evaluation of nuclear NF-κB, transglutaminase2, and ERCC1 as predictors of platinum resistance in testicular tumors

ABSTRACT Purpose: Testicular germ cells tumor (TGCT) are associated with a high cure rate and are treated with platinum-based chemotherapy. However, a group of testicular cancer patients may have a very unfavorable evolution and insensitivity to the main therapeutic agent chemotherapy (CT) cisplatin. The aim of this study was to evaluate the risk of recurrence and overall survival related to the expression of nuclear factor kappa-B (NF-κB), transglutaminase 2 (TG2) and excision repair cross-complementation group 1 (ERCC1) in patients with TGCT treated with platinum combinations. Patients and Methods: A retrospective study was performed with TGCT patients treated with platinum-based chemotherapy. Immunohistochemical analysis was performed and the expression was correlated with clinical and laboratory data. Results: Fifty patients were included, the mean age was 28.4 years (18 to 45), and 76% were non-seminoma. All patients were treated with standard cisplatin, etoposide and bleomycin or cisplatin, and etoposide. Patient's analyzed immunodetection for NF-κB, TG2, and ERCC1 were positive in 76%, 54% and 42%, respectively. Multivariate analysis identified that positive expressions to ERCC1 and NF-κB are independent risk factors for higher recurrence TGCT after chemotherapy (RR 2.96 and 3.16, respectively). Patients with positive expression of ERCC1 presented a poor overall survival rate for 10-year follow (p=0.001). Conclusions: The expression of ERCC1 and NF-κB give a worse prognosis for relapse, and only ERCC1 had an influence on the overall survival of TGCT patients treated with platinum-based chemotherapy. These may represent markers that predict poor clinical outcome and response to cisplatin.

Regulatory framework of genome-edited products - a review / 生物工程学报

Genome editing is a genetic engineering technique that uses site-directed cleavage activity of specific artificial nucleases and endogenous DNA damage repair activity to generate insertions, deletions or substitutions in the targeted genomic loci. As the accuracy and efficiency of genome editing is improving and the operation is simple, the application of genome editing is expanding. This article provides an overview of the three major genome editing technologies and genome editing types, and the regulatory frameworks for genome-edited products were summarized in the United States, the European Union, and other countries. At the same time, based on the Chinese safety management principles and systems for genetically modified organisms (GMOs), the authors proposed a regulatory framework for genome-edited products. Genome-edited products should first be classified according to whether containing exogenous genetic components such as Cas9 editing enzymes or not. They should be regulated as traditional genetically modified organisms if they do. Otherwise, the regulation of genome-edited products depends on targeted modifications.

Development and challenges of gene editing technology / 生物工程学报

Gene editing is a technique for modifying gene fragments. The novel gene editing technology focuses on the field of artificial nuclease cleavage technology, mainly ZFN technology, TALEN technology, CRISPR technology and base editing technology. The continuous improvement of gene editing technology has promoted the rapid development of agriculture, animal husbandry and biomedicine, but at the same time, technical defects and ethical controversy have brought enormous challenges to its own development. This article will briefly discuss the development and challenges of gene editing technology, as well as the views at home and abroad, and hope to inspire readers to recognize gene editing technology.

Application and optimization of CRISPR/Cas system in bacteria / 生物工程学报

Clustered regular interspaced short palindromic repeats (CRISPR) system has been widely used in recent years. Compared with traditional genome editing technology, CRISPR/Cas system has notable advantages, including high editing efficiency, high specificity, low cost and the convenience for manipulation. Type Ⅱ and Ⅴ CRISPR/Cas system only requires a single Cas9 protein or a single Cpf1 protein as effector nucleases for cutting double-stranded DNA, developed as genome editing tools. At present, CRISPR/Cas9 technology has been successfully applied to the genome editing of eukaryotes such as zebrafish, mice and human cells, whereas limited progress has been made in the genome editing of bacteria. In our review, we describe CRISPR/Cas system, its mechanism and summarize the optimization and progress of genome editing in bacteria.

Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer

Cervical cancer is a public health problem and the molecular mechanisms underlying radioresistance are still poorly understood. Here, we evaluated the modulation of key molecules involved in cell proliferation, cell cycle and DNA repair in cervical cancer cell lines (CASKI and C33A) and in malignant tissues biopsied from 10 patients before and after radiotherapy. The expression patterns of epidermal growth factor receptor (EGFR), excision repair cross-complementation group 1 (ERCC1) and p53 were evaluated in cancer cell lines by quantitative PCR and western blotting, and in human malignant tissues by immunohistochemistry. The mutation status of TP53 gene was evaluated by direct sequencing. Among cell lines, absent or weak modulations of EGFR, ERCC1 and p53 were observed after exposure to 1.8 Gy. Conversely, increased expressions of p53 (5/10 patients; P=0.0239), ERCC1 (5/10 patients; P=0.0294) and EGFR (4/10 patients; P=0.1773) were observed in malignant tissues after radiotherapy with the same radiation dose. TP53 mutations were found only in one patient. Here we show that a single dose of radiotherapy induced EGFR, ERCC1 and p53 expression in malignant tissues from cervical cancer patients but not in cancer cell lines, highlighting the gap between in vitro and in vivo experimental models. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of p53, EGFR and ERCC1 may be part of a radioresistance mechanism.

Relationship between the Asp1104His polymorphism of the nucleotide excision repair gene ERCC5 and treatment sensitivity to oxaliplatin in patients with advanced colorectal cancer in China

OBJECTIVES: To study the relationship between the Asp1104His polymorphism of the nucleotide excision repair gene ERCC5 and treatment sensitivity to oxaliplatin in patients with advanced colorectal cancer (CRC) in China. METHODS: A group of 226 patients in the Department of Gastrointestinal Oncology at Zhejiang Xiaoshan Hospital from July 2011∼December 2016 and a control group of 226 normal healthy individuals were involved in this study. All patients were first diagnosed with advanced CRC and were treated with oxaliplatin-based chemotherapy. The genotype of ERCC5 at the site of amino acid 1104 was determined by a TaqMan probe-based real-time PCR approach. RESULTS: There were no differences in age or gender between the groups, but the percentages of smokers and individuals with a family history of cancer were significantly higher in the patient group than in the control group. Analysis of the G/C polymorphism frequency among the patients and the healthy controls showed that the frequencies of the CC genotype and the CC+GC genotype were significantly related to CRC, but no significant difference in these frequencies was found between genders. The analysis of the relationship between the 5-year survival rate and different genotypes showed that in the total patient group, regardless of gender, the 5-year survival rate was significantly associated with the Asp1104His polymorphism of ERCC5. CONCLUSIONS: The Asp1104His polymorphism of ERCC5 was associated with the risk and 5-year survival rate of CRC as well as treatment sensitivity to oxaliplatin.

Effective gene editing by high-fidelity base editor 2 in mouse zygotes

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.

Enzyme activity and thermostability of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica by site-directed mutagenesis

Background: To identify the critical amino acid residues that contribute to the high enzyme activity and good thermostability of Yersinia enterocolitica subsp. palearctica (Y. NSN), 15 mutants of Y. NSN were obtained by site-directed mutagenesis in this study. And their enzyme activity and thermostability were assayed. Effect of several factors on the enzyme activity and thermostability of Y. NSN, was also investigated. Results: The results showed that the I203F and D264E mutants retained approximately 75% and 70% enzyme activity, respectively, compared to the wild-type enzyme. In addition to the I203F and D264E mutants, the mutant E202A had an obvious influence on the thermostability of Y. NSN. According to the analysis of enzyme activity and thermostability of Y. NSN, we found that Glu202, Ile203 and Asp264 might be the key residues for its high enzyme activity and good thermostability. Conclusions: Among all factors affecting enzyme activity and thermostability of Y. NSN, they failed to explain the experimental results well. One reason might be that the enzyme activity and thermostability of Y. NSN were affected not only by a single factor but also by the entire environment.

Secretory expression and characterization of heat sensitive nuclease in Pichia pastoris / 生物工程学报

Nucleases is an important enzyme widely used in biotechnology. A codon optimized nuclease gene (SNU) from Northern Shrimps was inserted into pPICZα A vector, and expressed extracellularly in strain SMD1168H. On the basis of multi-copy recombinant strain, we further optimized the expression condition and characterized SNU. SNU was highly expressed and stable after 1% methanol induction for 72 h, yield reached 1.4×10⁵ U/mL. SDS-PAGE electrophoresis demonstrated that this is a N-linked glycoprotein of 50 kDa. It was purified by one step DEAE Sephadex chromatography to the purity of about 15 mg/L with a specific activity of 6.291×10⁶ U/mg. Functional analysis on the nuclease activity indicated that it was stimulated by bivalent iron, such as Ca²⁺, Mn²⁺, Co²⁺ and Mg²⁺, but inhibited by Zn²⁺, Cu²⁺ and high salt. Meanwhile, it was irreversibly inactivated at 70 ℃ for 10 min.

Excision Repair Cross-complementation Group 1 is a Prognostic Biomarker in Patients with Colorectal Cancer Receiving Chemotherapy / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Conflicting results about the association between expression level of excision repair cross-complementation group 1 (ERCC1) and clinical outcome in patients with colorectal cancer (CRC) receiving chemotherapy have been reported. Thus, we searched the available articles and performed the meta-analysis to elucidate the prognostic role of ERCC1 expression in patients with CRC.</p><p><b>METHODS</b>A thorough literature search using PubMed (Medline), Embase, Cochrane Library, Web of Science databases, and Chinese Science Citation Database was conducted to obtain the relevant studies. Pooled hazard ratios (HR s) or odds ratios (OR s) with 95% confidence intervals (CI s) were calculated to estimate the results.</p><p><b>RESULTS</b>A total of 11 studies were finally enrolled in this meta-analysis. Compared with patients with lower ERCC1 expression, patients with higher ERCC1 expression tended to have unfavorable overall survival (OS) (HR = 2.325, 95% CI: 1.720-3.143, P < 0.001), progression-free survival (PFS) (HR = 1.917, 95% CI: 1.366-2.691, P < 0.001) and poor response to chemotherapy (OR = 0.491, 95% CI: 0.243-0.990, P = 0.047). Subgroup analyses by treatment setting, ethnicity, HR extraction, detection methods, survival analysis, and study design demonstrated that our results were robust.</p><p><b>CONCLUSIONS</b>ERCC1 expression may be taken as an effective prognostic factor predicting the response to chemotherapy, OS, and PFS. Further studies with better study design and longer follow-up are warranted in order to gain a deeper understanding of ERCC1's prognostic value.</p>

Markerless DNA deletion based on Red recombination and in vivo I-Sec I endonuclease cleavage in Escherichia coli chromosome / 生物工程学报

Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.

Expression of XPG Gene in Forensic Age Estimation / 法医学杂志

OBJECTIVES@#To explore the expression of xeroderma pigmentosum complementation group G （XPG） gene in healthy Han population of different ages and to analysis the relationship between the mRNA and protein expression levels of XPG and age, which may provide a new molecular-biological indicator for forensic age determination.@*METHODS@#Total 150 samples of peripheral blood were collected from healthy Han population of different ages. Total RNA of peripheral blood mononuclear cell （PBMC） were extracted by TRIzol method, and the relative expression of XPG mRNA in PBMC was detected by quantitative real-time PCR, and the protein expression levels of XPG in plasma were detected by ELISA.@*RESULTS@#The mRNA and protein expression levels of XPG in ≤18 years old group were significantly different from 19-45 years old group and ≥46 years old group （P<0.05）, while there was no significant difference between 19-45 years old group and ≥46 years old group （P>0.05）. No significant sex differences were observed in mRNA and protein expression levels of XPG （P>0.05）.@*CONCLUSIONS@#The relative expression level of XPG mRNA in PBMC declines with the increase of age in younger age, while the protein expression level in plasma increases with age, and XPG gene can be used as one of new markers for forensic age estimation.

Benefits of maltodextrin intake 2 hours before cholecystectomy by laparotomy in respiratory function and functional capacity: a prospective randomized clinical trial / Benefícios na função respiratória e capacidade funcional com ingesta de maltodextrina 2 horas antes de colecistectomia por laparotomia: ensaio clínico prospectivo e randomizado

ABSTRACT Objective: To evaluate the change in respiratory function and functional capacity according to the type of preoperative fasting. Methods: Randomized prospective clinical trial, with 92 female patients undergoing cholecystectomy by laparotomy with conventional or 2 hours shortened fasting. The variables measured were the peak expiratory flow, forced expiratory volume in the first second, forced vital capacity, dominant handgrip strength, and non-dominant handgrip strength. Evaluations were performed 2 hours before induction of anesthesia and 24 hours after the operation. Results: The two groups were similar in preoperative evaluations regarding demographic and clinical characteristics, as well as for all variables. However, postoperatively the group with shortened fasting had higher values than the group with conventional fasting for lung function tests peak expiratory flow (128.7±62.5 versus 115.7±59.9; p=0.040), forced expiratory volume in the first second (1.5±0.6 versus 1.2±0.5; p=0.040), forced vital capacity (2.3±1.1 versus 1.8±0.9; p=0.021), and for muscle function tests dominant handgrip strength (24.9±6.8 versus 18.4±7.7; p=0.001) and non-dominant handgrip strength (22.9±6.3 versus 17.0±7.8; p=0.0002). In the intragroup evaluation, there was a decrease in preoperative compared with postoperative values, except for dominant handgrip strength (25.2±6.7 versus 24.9±6.8; p=0.692), in the shortened fasting group. Conclusion: Abbreviation of preoperative fasting time with ingestion of maltodextrin solution is beneficial to pulmonary function and preserves dominant handgrip strength. .

RESUMO Objetivo: Avaliar a alteração da função respiratória e da capacidade funcional, conforme o tipo de jejum pré-operatório. Métodos: Ensaio clínico prospectivo randomizado, com 92 pacientes do sexo feminino, submetidas à colecistectomia por laparotomia, observando jejum convencional ou abreviado de 2 horas com maltodextrina. As variáveis foram: pico de fluxo expiratório, volume expiratório no primeiro segundo, capacidade vital forçada, força de preensão palmar dominante e força de preensão palmar não dominante. As avaliações foram realizadas 2 horas antes da indução anestésica e 24 horas após a operação. Resultados: Os dois grupos foram semelhantes quanto às características demográficas, clínicas e em todas as variáveis estudadas, quando avaliadas no pré-operatório. No entanto, no pós-operatório, o grupo abreviado apresentou valores maiores que o grupo convencional para pico de fluxo expiratório (128,7±62,5 versus 115,7±59,9; p=0,040), volume expiratório no primeiro segundo (1,5±0,6 versus 1,2±0,5; p=0,040), capacidade vital forçada (2,3±1,1 versus 1,8±0,9; p=0,021), força de preensão palmar dominante (24,9±6,8 versus 18,4±7,7; p=0,001) e força de preensão palmar não dominante (22,9±6,3 versus 17,0±7,8; p=0,0002). Na avaliação intragrupo, houve diminuição nas variáveis ao se compararem os valores do pré-operatório em relação ao pós-operatório, exceto para força de preensão palmar dominante (25,2±6,7 versus 24,9±6,8; p=0,692) no grupo de jejum abreviado. Conclusão: A abreviação do tempo de jejum pré-operatório com solução contendo maltodextrina beneficia a função pulmonar e preserva a força de preensão palmar dominante. .