Prevention and control of antimicrobial resistance using CRISPR-Cas system: a review / 生物工程学报

Bacterial multi-drug resistance (MDR) is a global challenge in the fields of medicine and health, agriculture and fishery, ecology and environment. The cross-region spread of antibiotic resistance genes (ARGs) among different species is one of the main cause of bacterial MDR. However, there is no effective strategies for addressing the intensifying bacterial MDR. The CRISPR-Cas system, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins, can targetedly degrade exogenous nucleic acids, thus exhibiting high application potential in preventing and controlling bacterial MDR caused by ARGs. This review briefly introduced the working mechanism of CRISPR-Cas systems, followed by discussing recent advances in reducing ARGs by CRISPR-Cas systems delivered through mediators (e.g. plasmids, bacteriophages and nanoparticle). Moreover, the trends of this research field were envisioned, providing a new perspective on preventing and controlling MDR.

Lentivirus vectors fail to deliver transgenes into bovine zygotes after co-incubation with sperm during in vitro fertilization / [Lentivírus falham na entrega de transgenes em zigotos bovinos após coincubação com espermatozoide, durante fertilização in vitro]

As vantagens dos animais transgênicos têm sido demonstradas em diferentes aplicações, entretanto muitas metodologias usadas para gerar animais geneticamente modificados (GM) apresentam baixas taxas de eficiência. O objetivo deste estudo foi avaliar a entrega dos vetores lentivirais (VLs) em zigotos durante a fertilização in vitro (FIV), para gerar embriões GM, com o gene da proteína verde fluorescente (GFP) ou do fator IX de coagulação humana (FIX). Vetores lentivirais com os genes GFP (pLGW-GFP-LV) ou FIX (pLWE2-FIX-LV) foram utilizados na FIV ou na cultura de embriões in vitro (CIV). A coincubação de pLWE2-FIX-LV com espermatozoides e complexos oócitos-células do cumulus (COCs) durante a FIV diminuiu (P<0,05) as taxas de clivagem e de blastocistos, enquanto com pLGW-GFP-LV diminuiu (P<0,05) a taxa de blastocisto quando se comparou ao controle sem VL. A coincubação de pLWE2-FIX-LV e pLGW-GFP-LV com presumíveis zigotos durante a CIV não afetou (P>0,05) o desenvolvimento embrionário. A expressão da proteína GFP não foi detectada em embriões após a coincubação de FIV ou CIV, embora as células do cumulus expressassem a proteína até o dia oito de cultivo in vitro. Reações em cadeia da polimerase (PCR) não detectaram os genes GFP ou FIX em embriões, mas ambos foram detectados em células do cumulus. Assim, a coincubação de VL com espermatozoide bovino e COCs não é eficaz para produzir embriões geneticamente modificados por meio de FIV.(AU)

Analysis of indigenous plasmid sequences of A. baumannii DS002 reveals the existence of lateral mobility and extensive genetic recombination among Acinetobacter plasmids

Genome sequence of Acinetobacter baumannii DS002 revealed the existence of seven contigs with features of indigenous plasmids. Of the seven contigs, three of them have shown size and sequence identity. They appeared to have been generated due to the unique recombination events leading to a large-scale recombination and sequence inversions. The rest of the indigenous plasmids have shown significant size variations and contained the genetic repertoire required for the detoxification of formaldehyde and biosynthesis of exopolysaccharides. Genetic modules encoding novel toxin–antitoxin systems were found in most of the plasmids to ensure their survival in the host. In some instances, the toxin and antitoxin coding sequences were found on two different plasmids promoting the cosegregation of these two plasmids into the daughter cells

A soil bacterial catabolic pathway on the move: Transfer of nicotine catabolic genes between Arthrobacter genus megaplasmids and invasion by mobile elements

The 165,137 bp plasmid pAO1 of Paenarthrobacter nicotinovorans carries the genes of a nicotine catabolicpathway. The genes are organized into several gene modules responsible for the catabolism of L- and D-nicotineto nicotine blue, a-ketoglutarate and succinate. Various modules of these genes have been shown to be presentin gram-positive (Gram?) soil bacteria. The presence of the identical pAO1 nic-genes on the 288,370 bpplasmid pZXY21 of Arthrobacter sp. ZXY2 (96% to 100% at the nucleotide level) permitted the identificationof the limits of this DNA fragment. At the 50 end of the nic-genes are located the ORFs of two predictedintegrases of the tyrosine recombinase family with conserved R, H, R and Y catalytic residues and that of asmall transposase with a predicted leucine zipper motive. They are related to Tn554A, Tn554B and Tn554C ofStaphylococcus aureus and suggest that the entire nic-genes DNA fragment represents a large catabolictransposon. Surprisingly the nic-genes on pZXY21 were found to be interspersed by mobile elements encodingtransposases of various IS families. Insertion of these IS elements disrupts nicotine degradation and divide thenic-genes DNA into potentially new transposons. This finding may illustrate how nicotine catabolic genes canbe mobilized and spread by horizontal gene transfer to other soil bacteria.

Elimination of a disulfide bond in the light chain of coagulation factor VIII improves secretion of a BDD-FVIII variant with an engineered inter-chain disulfide / 药学学报

The coagulation VIII factor (FVIII) contains eight pairs of disulfide bonds, which are involved in maintaining its structure and function. It has been demonstrated that the disulfide bond between Cys1899/Cys1903 of the A3 domain in the light chain impedes secretion. In our previous work, an engineered inter-chain disulfide in the B domain-deleted FVIII (BDD-FVIII) promoted heterodimer assembly and secretion of separately expressed heavy and light chains. In this study, we constructed two BDD-FVIII variants, one of which contains an engineered inter-chain disulfide bond (F8C) between Met662 > Cys and Asp1828 > Cys mutations and another contains an endogenous A3 domain with a disrupted disulfide bond from F8C (F8CG) by replacement of Cys1899 and Cys1903 with Gly in F8C. We explored their function and secretion. By transducing F8C and F8CG into HEK293 and COS-7 cells, the formation of disulfide bonds and the secretion and coagulation activity of the two variants in the culture media and their binding affinity for von Willebrand factor (vWF) could be observed. The results show that variants F8C and F8CG are mainly the disulfide bonded heavy and light chain dimer, while the wild type BDD-FVIII (F8) is dominated by the easily dissociated heavy and light chain dimer. The secretion and activity of F8C was significantly higher than that of F8, while the secretion and activity of F8CG was significantly higher than that of F8C. The vWF binding of the two variants is similar to F8. This indicates that the BDD-FVIII variant F8CG may be attractive molecule for protein replacement and as a transgene in gene-therapy strategies. These findings are encouraging for future studies targeting disulfide bond elimination for further enhancement of FVIII secretion.

Direct reprogramming hepatocytes into islet-like cells by efficiently targeting and activating the endogenous genes / 中国组织工程研究

BACKGROUND: Islet cell transplantation is one of the most effective methods to treat diabetes. However, the shortage of transplanted cells has limited its clinical application. Direct reprogramming of hepatocytes to islet β cells in vitro is a new idea to solve this problem, but differentiation of hepatocytes to islet β cells is a complicated process. OBJECTIVE: Direct reprogramming hepatocytes into islet-like cells by efficient targeting and activating the endogenous genes of hepatocytes with Casilio system (constructed CRISPR/ Cas9-Pumilio system) through modifying guide RNA sequence combined with the transcriptional activator PUFa-P65-HSF1. METHODS: The endogenous PNM (Pdx1, Ngn3, MafA) genes of hepatocytes were targeted and activated by using the Casilio system, which was transfected to the HEK293T cell line by liposome transfection. The expression of endogenous PNM was detected by real-time fluorescence quantitative PCR and immunofluorescence. The Ins-EGFP cell line with stable expression of enhanced green fluorescent protein was constructed by the lentivirus carrying Ins-promoter-EGFP. The Ins-EGFP-HepG2-Cas9-PUFa-p65-HSF1 cell line (referred to as stable translocated cell lines) with stable expression of dCas9 and PUFa-P65-HSF1 in Ins-EGFP-HepG2 cell line was constructed by the PiggyBac(PB) transposon system. By using liposome, the gRNAs were transfected to stable translocated cell line, and the expression level of endogenous gene PNM was detected by real-time fluorescence quantitative PCR and immunofluorescence. Simultaneously, reprogramming efficiency was observed. RESULTS AND CONCLUSION: (1) The activation of endogenous genes by Casilio system was verified in 293T cell line. (2) The expression of EGFP, dCas9 and PUFa-P65-HSF1 in stable cell line was detected by RT-PCR, western blot assay and immunofluorescence. (3) The real-time fluorescence quantitative PCR results confirmed that this new Casilio system could target and activate the PNM which led to the up-regulation of the endogenous PNM gene expression. The efficiency of direct reprogramming was 10%-15%. (4) Based on CRISPR/dCas9, the new Casilio system can efficiently activate the endogenous PNM gene in hepatocytes, enabling the hepatocyte line to be directly reprogrammed as islet-like cells.

Productive infection and transduction by bacteriophage P1 in the species Salmonella bongori

Background: Horizontal gene transfer (HGT) is the most important mechanism in the evolution of new genetic capabilities in bacteria, including specific degradative pathways, virulence factors, and resistance to antibiotics. Among the processes involved in HGT, transduction is noteworthy. This is a mechanism for gene transmission mediated by a bacteriophage that functions both as a reservoir and as a vector of exogenous genes, which remain protected from environmental effects in the bacteriophage's capsid. Within this context, this investigation aimed to evaluate the ability of the generalized transducing bacteriophage P1 to productively infect and transduce in the bacterial species Salmonella bongori. Results: We could establish that a derivative of bacteriophage P1, P1Cm, infects strains of S. bongori with frequencies of lysogenization in the order of ~10−2 lysogens/UFP. Through thermal induction, infective viral progeny was obtained, and we could show that P1Cm readily formed plaques on S. bongori lawns, a phenomenon thus far not reported for other members of the genus Salmonella. Finally, we showed P1Cm-mediated transduction of the model plasmid RP4 at frequencies of ~10−7 transductants/donor. Conclusion: Therefore, bacteriophage P1 can be used as a tool for the genetic manipulation in the species S. bongori.

Lateral transfer of organophosphate degradation (opdopd) genes among soil bacteria: mode of transfer and contributions to organismal fitness

Genes encoding structurally independent phosphotriesterases (PTEs) are identified in soil bacteria. These pte genes, often identified on mobilizable and self-transmissible plasmids are organized as mobile genetic elements. Their dissemination through lateral gene transfer is evident due to the detection of identical organophosphate degradation genes among soil bacteria with little orno taxonomic relationship. Convergent evolution of PTEs provided selective advantages to the bacterial strain as they convert toxic phosphotriesters (PTs) into a source of phosphate. The residues of organophosphate (OP) compounds that accumulate in a soil are proposed to contribute to the evolution of PTEs through substrate-assisted gain-of-function. This review provides comprehensiveinformation on lateral transfer of pte genes and critically examines proposed hypotheses on their evolution in the light of the short half-life of OPs in the environment. The review also proposes alternate factors that have possibly contributed to the evolution and lateral mobility of PTEs by taking into account their biology and analyses of pte genes in genomic and metagenomic databases.

Plasmid profiles among some ESKAPE pathogens in a tertiary care centre in south India

Background & objectives: Plasmid has led to increase in resistant bacterial pathogens through the exchange of antimicrobial resistance (AMR) genetic determinants through horizontal gene transfer. Baseline data on the occurrence of plasmids carrying AMR genes are lacking in India. This study was aimed to identify the plasmids associated with AMR genetic determinants in ESKAPE pathogens. Methods: A total of 112 ESKAPE isolates including Escherichia coli (n=37), Klebsiella pneumoniae (n=48, including 7 pan-drug susceptible isolates), Acinetobacter baumannii (n=8), Pseudomonas aeruginosa (n=1) and Staphylococcus aureus (n=18) were analyzed in the study. Isolates were screened for antimicrobial susceptibility and whole genome sequencing of isolates was performed using Ion Torrent (PGM) sequencer. Downstream data analysis was done using PATRIC, ResFinder, PlasmidFinder and MLSTFinder databases. All 88 whole genome sequences (WGS) were deposited at GenBank. Results: Most of the study isolates showed resistant phenotypes. As analyzed from WGS, the isolates included both known and unknown sequence types. The plasmid analysis revealed the presence of single or multiple plasmids in the isolates. Plasmid types such as IncHI1B(pNDM-MAR), IncFII(pRSB107), IncFIB(Mar), IncFIB(pQil), IncFIA, IncFII(K), IncR, ColKP3 and ColpVC were present in K. pneumoniae. In E. coli, IncFIA, IncFII, IncFIB, Col(BS512), IncL1, IncX3 and IncH were present along with other types. S. aureus harboured seven different plasmid groups pMW2 (rep 5), pSAS1 (rep 7), pDLK1 (rep 10), pUB110 (rep US12), Saa6159 (rep 16), pKH12 (rep 21) and pSA1308 (rep 21). The overall incidence of IncF type plasmids was 56.5 per cent followed by Col type plasmids 18.3 per cent and IncX 5.3 per cent. Other plasmid types identified were <5 per cent. Interpretation & conclusions: Results from the study may serve as a baseline data for the occurrence of AMR genes and plasmids in India. Information on the association between phenotypic and genotypic expression of AMR was deciphered from the data. Further studies on the mechanism of antibiotic resistance dissemination are essential for enhancing clinical lifetime of antibiotics.

Antibiotic Treatment Drives the Diversification of the Human Gut Resistome / 基因组蛋白质组与生物信息学报·英文版

Despite the documented antibiotic-induced disruption of the gut microbiota, the impact of antibiotic intake on strain-level dynamics, evolution of resistance genes, and factors influencing resistance dissemination potential remains poorly understood. To address this gap we analyzed public metagenomic datasets from 24 antibiotic treated subjects and controls, combined with an in-depth prospective functional study with two subjects investigating the bacterial community dynamics based on cultivation-dependent and independent methods. We observed that short-term antibiotic treatment shifted and diversified the resistome composition, increased the average copy number of antibiotic resistance genes, and altered the dominant strain genotypes in an individual-specific manner. More than 30% of the resistance genes underwent strong differentiation at the single nucleotide level during antibiotic treatment. We found that the increased potential for horizontal gene transfer, due to antibiotic administration, was ∼3-fold stronger in the differentiated resistance genes than the non-differentiated ones. This study highlights how antibiotic treatment has individualized impacts on the resistome and strain level composition, and drives the adaptive evolution of the gut microbiota.

Angiotensin-Converting Enzyme 2 in the Rostral Ventrolateral Medulla Regulates Cholinergic Signaling and Cardiovascular and Sympathetic Responses in Hypertensive Rats / 神经科学通报·英文版

The rostral ventrolateral medulla (RVLM) is a key region in cardiovascular regulation. It has been demonstrated that cholinergic synaptic transmission in the RVLM is enhanced in hypertensive rats. Angiotensin-converting enzyme 2 (ACE2) in the brain plays beneficial roles in cardiovascular function in hypertension. The purpose of this study was to determine the effect of ACE2 overexpression in the RVLM on cholinergic synaptic transmission in spontaneously hypertensive rats (SHRs). Four weeks after injecting lentiviral particles containing enhanced green fluorescent protein and ACE2 bilaterally into the RVLM, the blood pressure and heart rate were notably decreased. ACE2 overexpression significantly reduced the concentration of acetylcholine in microdialysis fluid from the RVLM and blunted the decrease in blood pressure evoked by bilateral injection of atropine into the RVLM in SHRs. In conclusion, we suggest that ACE2 overexpression in the RVLM attenuates the enhanced cholinergic synaptic transmission in SHRs.

Antibiotic Resistance: Origins, evolution and healthcare-associated infections / Resistencia a antibióticos: Origen, evolución e infecciones asociadas a la atención en salud

Abstract The increased incidences of Healthcare-associated Infections (HAI) caused by multidrug-resistant bacteria, have led to an enlarged number of morbidity and mortality cases. Besides, other factors that are affected are patients, families and institutions providing health services. Therefore, the permanent study of the subject is necessary to identify possible strategies that contribute to the reduction of the issue. A critical review of the literature based on the origin of antibiotics, the evolution of their respective resistance, and the impact on public health from a historical and current perspective was developed. The search of the literature was carried out in the bibliographic databases: Pubmed, Web of Science, Scopus, SciELO, The Cochrane Library and Lilacs. The reviewed literature showed, from the historical viewpoint, the discovery of antibiotics to the last-generation antibiotics. The rapid coevolution of genes for antibiotics resistance and its subsequent spread to hundreds of species of microorganisms by Horizontal Transfer gene (HTG) was also reviewed. It is also discussed how the expansion in antimicrobial resistance (AMR) generates a series of factors that increase health-care associated infections care (HAI) and their impact on public health. The development of antibiotics from the discovery to recent changes in the behavior and response of the microorganisms with the generation of AMR shortly after, is one of the most fantastic examples of the evolution that exists in nature.

Resumen El aumento en la incidencia de infecciones asociadas a la atención en salud causada por microorganismos multiresistentes a antibióticos, han incrementado la morbilidad, mortalidad y otros factores que afectan a paciente, familias e instituciones prestadoras de servicios de salud; por lo que se ha hecho necesario el estudio permanente del tema, para identificar posibles estrategias que contribuyan a disminuir la situación. Se realizó una revisión de la literatura sobre el origen de los antibióticos, la evolución de su respectiva resistencia, el impacto en la salud pública; desde una perspectiva histórica y actual. La búsqueda de la literatura se realizó en las bases de datos bibliográficas: Pubmed, Web of Science, Scopus, SciELO, The Cochrane Library y Lilacs. El análisis de la literatura mostró desde el punto de vista histórico, el descubrimiento de los antibióticos hasta los últimos antibióticos de última generación, y la rápida coevolución de los genes de resistencia a los antibióticos y su posterior diseminación a cientos de especies de microorganismos mediante la Transferencia Horizontal de Genes (THG). También es discutido como el incremento de la resistencia a los antibióticos (RAM) genera una serie de factores que potencian las infecciones asocia de las a los cuidados de la salud (IACS) y su impacto en la salud pública. La historia desde el descubrimiento, los cambios en el comportamiento de uso de los antibióticos y la respuesta de los microorganismos con la generación de la RAM poco tiempo después, es uno de los ejemplos más fantásticos de coevolución que existe en la naturaleza.

Resistencia microbiana desde una perspectiva metagenómica / Microbial resistance from one metagenomic perspective

Resumen Objetivo. La finalidad de esta revisión es abarcar la temática relacionada con los genes de resistencia a antibióticos, sus orígenes, reservorios y movimientos en los diferentes hábitats mediante la metagenómica funcional que permite aislar, identificar y analizar estos genes, así como el impacto que tienen en salud pública. Durante los últimos años se ha visto un gran avance en la microbiología, una de las grandes limitaciones a las que se venían enfrentado los microbiólogos era no poder acceder a la totalidad de los microorganismos que habitan el planeta. Gracias al desarrollo de diferentes disciplinas como la metagenómica se ha logrado tener el acceso a estos microorganismos. Metodología. La importancia de la metagenómica en la resistencia microbiana radica en que, actualmente, solo el 1 % de los microorganismos que habitan el suelo pueden ser estudiados por técnicas convencionales de microbiología, quedando alrededor del 99 % de estos sin estudiar. Al mitigar este gran inconveniente, la metagenómica permite el estudio de la microbiota del suelo en su totalidad generando nuevo conocimiento e información relevante en diferentes campos científicos. Resultados. Mediante la metagenómica funcional se ha podido determinar que el suelo puede ser un posible reservorio de determinantes de resistencia microbiana, debido a que la microbiota que allí habita contiene en su material genético genes de resistencia a antibióticos que confieren resistencia a un amplio espectro de antibióticos utilizados en terapia humana de forma indiscriminada y además tienen todos los mecanismos de resistencia conocidos, algunos de estos genes son generados por presión selectiva ante diferentes agentes presentes en su medio y otros son genes constitutivos que cumplen con funciones significativas en su hábitat. El gran impacto que tienen estos hallazgos está dado en que pueden representar un posible riesgo en salud pública si se adquieren por los patógenos humanos.

Abstract Objective. The purpose of this review is to cover the issues related to antibiotic resistance genes, their origins, reservoirs and movements in different habitats through functional metagenomics that allows to isolate, identify and analyze these genes, as well as the impact they have on health public. During the last years a great advance in the microbiology has been seen, one of the great limitations to which the microbiologists had been facing was not being able to have access to the totality of the microorganisms that inhabit the planet. Thanks to the development of different disciplines such as metagenomics, access to these microorganisms has been achieved. Method. The importance of metagenomics in microbial resistance lies in the fact that currently only 1 % of the microorganisms that inhabit the soil can be studied by conventional microbiology techniques, leaving about 99 % of these without studying, the metagenomics by mitigating this great disadvantage allows the study of the soil microbiota in its entirety generating new knowledge and relevant information in different scientific fields. Results. Through functional metagenomics it has been possible to determine that the soil can be a possible reservoir of determinants of microbial resistance, because the microbiota that live there contain in their genetic material antibiotic resistance genes that confer resistance to a broad spectrum of antibiotics used in human therapy indiscriminately and also have all known mechanisms of resistance, some of these genes are generated by selective pressure against different agents present in their environment and others are constitutive genes that fulfill significant functions in their habitat. The great impact of these findings is that they can represent a possible public health risk if they were acquired by human pathogens.

Overview of Staphylococcus aureus mobile genetic elements and horizontal gene transfer of antimicrobial resistance genes / 中华预防医学杂志

The mechanism of antimicrobial resistance transmission mediated by mobile genetic elements (MGEs) in Staphylococcus aureus is highly complicated, leading a significant challenge for controlling the spread of the resistant Staphylococcus aureus strains. Based on the latest literature acquired in this work, we have overviewed the transmission mechanism of antimicrobial resistance encoding MGEs. It is notably that there are a number of MGEs, which may encode different antimicrobial resistance determinants and possess specific transmission mechanism. In spite of this specificity of the strains to their host (human or animal), some Staphylococcus aureus strains can be transmitted from animals to humans or vice versa. This ability of cross staphylococci transfer is an additional means to acquire new genetic material encoded by MGE. It was suggested in this review that study on transmission mechanism of MGEs mediated antimicrobial resistance genes could provide important biological information of their spreading and effectively help prevent and control of the resistant strains and/or resistance genes among human, animals and ecologies.

Changes of plasma miR-1470 expression in patients with diabetic retinopathy and its possible mechanism / 中华眼底病杂志

Objective To investigate the expression and mechanism of miR-1470 in plasma of diabetic retinopathy (DR) patients.Methods Thirty patients with DR (DR group),30 patients with diabetes (DM group) and 30 normal healthy subjects (normal group) were enrolled in the study.Three groups of subjects were taken 5 ml of venous blood,and total plasma RNA was extracted and purified.The differentially expressed miRNAs in the plasma of DR patients were screened by gene chip,and the results of gene chip detection were verified by reverse transcription polymerase chain reaction (RT-PCR).Bioinformatics was used to predict potential target genes for miRNA regulation,and miR-1470 and its target gene epidermal growth factor receptor (EGFR) were screened.Human retinal microvascular endothelial cells (hREC) were divided into normal group (sugar concentration 5.5 mmol/L) and high glucose group (sugar concentration 25.0 mmol/L).hREC was transfected into miR-1470 mimics to establish a miR-1470 high expression cell model,which was divided into blank control group,high expression group and negative control group.The expression of miR-1470 was detected by RT-PCR.The expression of EGFR protein was detected by Western blot.The measurement data of the two groups were compared using the independent sample t test.The comparison of the measurement data between the two groups was analyzed by ANOVA.The comparison between the measurement data of the groups was compared by multiple comparisons.Results The results of RT-PCR were consistent with those of the gene chip.The expression of miR-1470 in the plasma of the DR group,the DM group and the normal group was statistically significant (F=63.486,P=0.049).Compared with the DM group and the normal group,the expression of miR-1470 in the DR group was significantly decreased,and the difference was statistically significant (q=l 11.2,73.9;P＜0.05).The expression of miR-1470 in hREC in the high glucose group was significantly lower than that in the normal group (t=42.082,P=0.015).The expression of EGFR protein in hREC of high glucose group was significantly higher than that of normal group (t=-39.939,P=0.016).The expression of miR-1470 (F=637.069,P=0.000) and EGFR (F=122.908,P=0.000) protein expression in hREC of blank control group,negative control group and high expression group were statistically significant.Compared with the blank control group and the negative control group,the expression of miR-1470 in hREC of high expression group was significantly increased (q=329.7,328.8;P＜0.05),and the expression of EGFR protein was significantly decreased (q=242.5,234.6;P＜0.05).There was no significant difference in the expression of miR-1470 and EGFR protein in hREC between the negative control group and the blank control group (q=1.5,7.9;P＞0.05).Conclusion The expression ofmiR-1470 in the plasma of patients with DR is significantly down-regulated,and the increase of EGFR expression may be related to it.

Perspectiva histórica del origen evolutivo de la resistencia a antibióticos / Evolutionary origin of antibiotic resistance, a historical perspective

Resumen La resistencia a antimicrobianos representa un aspecto natural de evolución bacteriana, que puede resultar de mutaciones o por adquisición de genes foráneos. Hay diferentes posturas sobre el origen de ésta resistencia que explican la habilidad de estos microorganismos de adquirir nuevas características. Las teorías de la evolución de Lamarck y Darwin, han dado pie a experimentos diseñados para explorar el origen de la variación bacteriana y surgimiento de nuevas características. Estos estudios muestran que la resistencia está relacionada con mutaciones en genes cromosomales y/o la transferencia de elementos genéticos extracromosomales, que se expresan según la presión antibiótica ejercida. Está revisión recopila los principales experimentos y las conclusiones derivadas para explicar el fenómeno de resistencia a antibióticos.

Abstract Antimicrobial resistance is a natural aspect of bacterial evolution that can result from mutations or acquisition of foreign genes. Various views on the origin of this resistance explain the ability of these organisms to acquire new features. Lamarck and Darwin's theories of evolution have led to experiments designed to explore the origin of bacterial variation and the emergence of new features. These experiments show that antimicrobial resistance is related to mutations in chromosomal genes and/or transfer of extrachromosomal genetic elements that can be expressed based on the antibiotic pressure exerted. The main experiments and findings that seek to explain the phenomenon of antibiotic resistance are reviewed here in.

Introduction of a synthetic Thermococcus-derived α-amlyase gene into barley genome for increased enzyme thermostability in grains

Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.

Gene therapy: advances, challenges and perspectives / Terapia gênica: avanços, desafios e perspectivas

ABSTRACT The ability to make site-specific modifications to the human genome has been an objective in medicine since the recognition of the gene as the basic unit of heredity. Thus, gene therapy is understood as the ability of genetic improvement through the correction of altered (mutated) genes or site-specific modifications that target therapeutic treatment. This therapy became possible through the advances of genetics and bioengineering that enabled manipulating vectors for delivery of extrachromosomal material to target cells. One of the main focuses of this technique is the optimization of delivery vehicles (vectors) that are mostly plasmids, nanostructured or viruses. The viruses are more often investigated due to their excellence of invading cells and inserting their genetic material. However, there is great concern regarding exacerbated immune responses and genome manipulation, especially in germ line cells. In vivo studies in in somatic cell showed satisfactory results with approved protocols in clinical trials. These trials have been conducted in the United States, Europe, Australia and China. Recent biotechnological advances, such as induced pluripotent stem cells in patients with liver diseases, chimeric antigen receptor T-cell immunotherapy, and genomic editing by CRISPR/Cas9, are addressed in this review.

RESUMO A habilidade de fazer modificações pontuais no genoma humano tem sido o objetivo da medicina desde o conhecimento do DNA como unidade básica da hereditariedade. Entende-se terapia gênica como a capacidade do melhoramento genético por meio da correção de genes alterados (mutados) ou modificações sítio-específicas, que tenham como alvo o tratamento terapêutico. Este tipo de procedimento tornou-se possível por conta dos avanços da genética e da bioengenharia, que permitiram a manipulação de vetores para a entrega do material extracromossomal em células-alvo. Um dos principais focos desta técnica é a otimização dos veículos de entrega (vetores) que, em sua maioria, são plasmídeos, nanoestruturados ou vírus − sendo estes últimos os mais estudados, devido à sua excelência em invadir as células e inserir seu material genético. No entanto, existe grande preocupação referente às respostas imunes exacerbadas e à manipulação do genoma, principalmente em linhagens germinativas. Estudos em células somáticas in vivo apresentaram resultados satisfatórios, e já existem protocolos aprovados para uso clínico. Os principais trials têm sido conduzidos nos Estados Unidos, Europa, Austrália e China. Recentes avanços biotecnológicos empregados para o aprimoramento da terapia gênica, como células-tronco pluripotentes induzidas em pacientes portadores de doenças hepáticas, imunoterapia com células T do receptor do antígeno quimera e edição genômica pelos sistema CRISPR/Cas9, são abordados nesta revisão.

Molecular characterization genetic diversity of extended-spectrum β-lactamases-harboring conjugative plasmids identified in multi-drug resistant Escherichia coli isolated from food in China / 中华预防医学杂志

Objective@#The purpose of this study was to investigate the molecular characteristics of ESBL-encoding conjugative plasmid identified in muti-drug resistant Escherichia coli isolated from food.@*Methods@#465 Escherichia coli isolates were collected from national foodborne disease surveillance net from 2013 to 2014 (salad, n=159; meat, n=102; processed meat, n=95; cakes/rice, n=46; cooked dish, n=63). ESBLs strain was detected by Mueller-Hinton agar plate, and then its drug resistance was tested by agar dilution method. Polymerase chain reaction (PCR) and DNA sequencing were performed to identify the corresponding ESBL genes. Plasmids were typed by PCR-based replicon typing and their characteristics were determined by S1-nuclease pulsed-field gel electrophoresis method. Broth mating assays were carried out for all isolates to determine whether the ESBL marker could be transferred by conjugation.@*Results@#12 E. coli were found to be resistant to cefotaxime, and all of which were confirmed as ESBLs. The 12 isolates all carried different types of CTX-M genes resistant to drug, and 7 of which carried TEM type as well. All 12 isolates contained at least one plasmid and some had four plasmids, with size ranging from 47-to 220-kb by S1-PFGE anaylsis. Seven isolates demonstrated the ability to transfer their cefotaxime resistance marker to the recotper strain J53 by only one plasmid.@*Conclusion@#This study highlights the diversity of the multi-drug resistant E. coli and also the diversity of ESBL genes in China. Plasmids carrying these genes poses a serious threat to food safety in China.

Preparation and characterization of a gene-activated matrix mimicking extracellular matrix / 药学学报

This study aims to establish a novel gene-activated matrix that mimics the structure and function of extracellular matrix (ECM-m-GAM). The structure, mechanical property and release profile were also characterized. Firstly, the liposome/DNA lipoplex (LPD) was modified with cell penetrating peptide TAT. The obtained TAT-LPD was then mixed with RGD grafting hyaluronic acid solution. After addition of the matrix metalloproteinase (MMPs) sensitive crosslinker (HS-MMP-SH), hyaluronic acid was crosslinked and TAT-LPD was encapsulated in the subsequently formed hydrogel. As a result, the cell adhesion factor RGD, MMPs sensitive substrate and the efficient gene transfer vector TAT-LPD were all integrated in the hyaluronic acid hydrogel, which was named as ECM-m-GAM. The release profile of DNA from ECM-m-GAM in different release medium was evaluated with PicoGreen kits. The results suggested that the mean diameter of the spherical TAT-LPD was (263.0 ±4.30) nm. TAT-LPD was successfully encapsulated in ECM-m-GAM, which had the typical porous network structure of hydrogels. The mechanical strength of GAM was enhanced with the increasing of hyaluronic acid content. When the content was 4%, the elastic modulus of GAM reached 1 600 Pa. The highly elastic GAM may be suitable for implantation and tissue regeneration. The DNA release showed significant MMPs sensitive property. Especially, the released DNA still existed in form of nanoparticles. Bone marrow mesenchymal stem cells (BMSCs) were successfully transfected with GAM and the green fluorescent protein was expressed. The results have laid a solid foundation for future study of the cell transfection and tissue regeneration.