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CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.
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Eliminación de Gen , Tecnología de Genética Dirigida , Animales , Proteína 9 Asociada a CRISPR/metabolismo , Cromosomas/genética , Drosophila melanogaster/genética , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Patrón de Herencia/genética , Mutagénesis/genética , ARN Guía de Kinetoplastida/genética , TransgenesRESUMEN
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity1-4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.
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Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Conversión Génica , Tecnología de Genética Dirigida/métodos , Mutación de Línea Germinal/genética , Heterocigoto , Homocigoto , Alelos , Animales , Cruzamiento , Proteína 9 Asociada a CRISPR/genética , Cromosomas de los Mamíferos/genética , Roturas del ADN de Doble Cadena , Modelos Animales de Enfermedad , Embrión de Mamíferos/enzimología , Embrión de Mamíferos/metabolismo , Femenino , Integrasas/genética , Integrasas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Monofenol Monooxigenasa/genética , ARN Guía de Kinetoplastida/genética , Transgenes/genéticaRESUMEN
OBJECTIVE: To evaluate the efficacy and safety of Huashi Baidu Granules (HSBD) in treating patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant. METHODS: A single-center retrospective cohort study was conducted during COVID-19 Omicron epidemic in the Mobile Cabin Hospital of Shanghai New International Expo Center from April 1st to May 23rd, 2022. All COVID-19 patients with asymptomatic or mild infection were assigned to the treatment group (HSBD users) and the control group (non-HSBD users). After propensity score matching in a 1:1 ratio, 496 HSBD users of treatment group were matched by propensity score to 496 non-HSBD users. Patients in the treatment group were administrated HSBD (5 g/bag) orally for 1 bag twice a day for 7 consecutive days. Patients in the control group received standard care and routine treatment. The primary outcomes were the negative conversion time of nucleic acid and negative conversion rate at day 7. Secondary outcomes included the hospitalized days, the time of the first nucleic acid negative conversion, and new-onset symptoms in asymptomatic patients. Adverse events (AEs) that occurred during the study were recorded. Further subgroup analysis was conducted in vaccinated (378 HSBD users and 390 non-HSBD users) and unvaccinated patients (118 HSBD users and 106 non-HSBD users). RESULTS: The median negative conversion time of nucleic acid in the treatment group was significantly shortened than the control group [3 days (IQR: 2-5 days) vs. 5 days (IQR: 4-6 days); P<0.01]. The negative conversion rate of nucleic acid in the treatment group were significantly higher than those in the control group at day 7 (91.73% vs. 86.90%, P=0.014). Compared with the control group, the hospitalized days in the treatment group were significantly reduced [10 days (IQR: 8-11 days) vs. 11 days (IQR: 10.25-12 days); P<0.01]. The time of the first nucleic acid negative conversion had significant differences between the treatment and control groups [3 days (IQR: 2-4 days) vs. 5 days (IQR: 4-6 days); P<0.01]. The incidence of new-onset symptoms including cough, pharyngalgia, expectoration and fever in the treatment group were lower than the control group (P<0.05 or P<0.01). In the vaccinated patients, the median negative conversion time and hospitalized days were significantly shorter than the control group after HSDB treatment [3 days (IQR: 2-5 days) vs. 5 days (IQR: 4-6 days), P<0.01; 10 days (IQR: 8-11 days) vs. 11 days (IQR: 10-12 days), P<0.01]. In the unvaccinated patients, HSBD treatment efficiently shorten the median negative conversion time and hospitalized days [4 days (IQR: 2-6 days) vs. 5 days (IQR: 4-7 days), P<0.01; 10.5 days (IQR: 8.75-11 days) vs. 11.0 days (IQR: 10.75-13 days); P<0.01]. No serious AEs were reported during the study. CONCLUSION: HSBD treatment significantly shortened the negative conversion time of nuclear acid, the length of hospitalization, and the time of the first nucleic acid negative conversion in patients infected with SARS-COV-2 Omicron variant (Trial registry No. ChiCTR2200060472).
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COVID-19 , Medicamentos Herbarios Chinos , Ácidos Nucleicos , Humanos , SARS-CoV-2 , Estudios Retrospectivos , ChinaRESUMEN
Candida auris is an emerging, multidrug-resistant fungal pathogen that poses a significant public health threat in healthcare settings. Despite yearly clinical cases rapidly increasing from 77 to 8,131 in the last decade, surveillance data on its distribution and prevalence remain limited. We implemented a novel assay for C. auris detection on a nationwide scale prospectively from September 2023 to March 2024, analyzing a total of 13,842 samples from 190 wastewater treatment plants across 41 U.S. states. Assays were extensively validated through comparison to other known assays and internal controls. Of these 190 wastewater treatment plants, C. auris was detected in the wastewater solids of 65 of them (34.2%) with 1.45% of all samples having detectable levels of C. auris nucleic-acids. Detections varied seasonally, with 2.00% of samples positive in autumn vs 1.01% in winter (P < 0.0001). The frequency of detection in wastewater was significantly associated with states having older populations (P < 0.001), sewersheds containing more hospitals (P < 0.0001), and sewersheds containing more nursing homes (P < 0.001). These associations are in agreement with known C. auris epidemiology. This nationwide study demonstrates the viability of wastewater surveillance for C. auris surveillance and further highlights the value of wastewater surveillance when clinical testing is constrained. IMPORTANCE: This study highlights the viability of wastewater surveillance when dealing with emerging pathogens. By leveraging an existing framework of wastewater surveillance, we reveal the widespread presence of C. auris in the United States. We further demonstrate that these wastewater detections are consistent with demographic factors relevant to C. auris epidemiology like age and number of hospitals or nursing homes. As C. auris and other pathogens continue to emerge, the low-cost and rapid nature of wastewater surveillance will provide public health officials with the information necessary to enact targeted prevention and control strategies.
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Candida auris , Aguas Residuales , Aguas Residuales/microbiología , Estados Unidos , Estudios Prospectivos , Candida auris/genética , Humanos , Estaciones del Año , Candidiasis/microbiología , Candidiasis/epidemiologíaRESUMEN
Schistosoma japonicum causes schistosomiasis in humans and livestock in the Asia-Pacific region. Knowledge of the genome of this parasite should improve understanding of schistosome-host interactions, biomedical aspects of schistosomiasis and invertebrate evolution. We assigned 43,707 expressed sequence tags (ESTs) derived from adult S. japonicum and their eggs to 13,131 gene clusters. Of these, 35% shared no similarity with known genes and 75% had not been reported previously in schistosomes. Notably, S. japonicum encoded mammalian-like receptors for insulin, progesterone, cytokines and neuropeptides, suggesting that host hormones, or endogenous parasite homologs, could orchestrate schistosome development and maturation and that schistosomes modulate anti-parasite immune responses through inhibitors, molecular mimicry and other evasion strategies.
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ADN de Helmintos/genética , Evolución Molecular , Schistosoma japonicum/genética , Secuencia de Aminoácidos , Animales , ADN Complementario/genética , Genes de Helminto , Interacciones Huésped-Parásitos , Humanos , Mamíferos , Datos de Secuencia Molecular , Filogenia , Schistosoma japonicum/clasificación , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
OBJECTIVE: To assess the effect and safety of Reyanning Mixture (RYN) in treating asymptomatic or mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children and adolescents. METHODS: This is a prospective, open-label, randomized controlled trial. Patients aged 1-17 years and diagnosed with asymptomatic or mild coronavirus disease-2019 (COVID-19) were assigned to an intervention group (RYN plus standard care) and a control group (standard care) according to a randomization list. The primary outcomes were SARS-CoV-2 negative conversion time. Secondary outcomes included negative conversion rate on days 3 and 7, hospital length of stay, symptom relief rate, new-onset symptoms of asymptomatic infected patients, and progressive disease rate. The cycle threshold (Ct) values of ORF1ab or N genes were also tested. RESULTS: A total of 214 patients in the intervention group and 217 in the control group were analyzed. The SARS-CoV-2 negative conversion time was significantly shortened in the intervention group [5 days (interquartile range (IQR): 5-6) vs. 7 days (IQR: 6-7), P<0.01]. By days 3 and 7, the negative conversion rates were significantly higher in the intervention group (day 3: 32.7% vs. 21.2%, P=0.007; day 7: 75.2% vs. 60.8%, P=0.001). Ct values significantly increase on day 2 [ORF1ab gene: 35.62 (IQR: 29.17-45.00) vs. 34.22 (IQR: 28.41-39.41), P=0.03; N gene: 34.97 (IQR: 28.50-45.00) vs. 33.51 (IQR: 27.70-38.25), P=0.024] and day 3 [ORF1ab gene: 38.00 (IQR: 32.72-45.00) vs. 35.81 (IQR: 29.96-45.00), P=0.003; N gene: 37.16 (IQR: 32.01-45.00) vs. 35.26 (IQR: 29.09-45.00), P=0.01]. No significant difference was found in hospital length of stay between the two groups (P>0.05). Symptoms of cough were significantly improved (82.2% vs. 70.0%, P=0.02) and wheezing was significantly reduced (0.7% vs. 12.9%, P<0.01) in the intervention group compared with the control group. During the trial, no disease progression or serious adverse events were reported. CONCLUSION: Adding RYN to standard care may be a safe and effective treatment for children with asymptomatic and mild SARS-CoV-2 infection. (Registration No. ChiCTR2200060292).
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Long-term fertilization causes the differences in water, heat, nutrients and microbial activities between topsoil and deep soil, with consequences on the decomposition and turnover of straw carbon (C) in soils. At a long-term positioning experimental station in Shenyang Agricultural University, we mixed the topsoil (0-20 cm) and deep soil (40-60 cm) samples from different fertilization treatments with 13C-labeled straw for in-situ incubation. We analyzed the content of organic C and its δ13C value in soil aggregates, compared the difference in the distribution of straw C between topsoil and deep soil aggregates, and explored the effects of fertilization on the sequestration of straw C in soil aggregates. Compared with fertilization treatments (i.e., single chemical nitrogen fertilizer application and combination of organic manure with nitrogen fertilizer application), the treatment without fertilization increased the content of straw C of <0.053 mm aggregate in the topsoil by 106.7% and that of >0.25 mm aggregate in the deep soil by 34.2%. The contribution percentage of straw C to organic C of >0.053 mm aggregate in the deep soil was about two times of that in the topsoil. About 22.6% and 11.4% of straw C was distributed into the >0.25 mm and <0.25 mm aggregates of topsoil, and about 29.4% and 8.8% of straw C was distributed into the >0.25 mm and <0.25 mm aggregates of deep soil, respectively. In conclusion, straw addition promoted the regeneration and sequestration of carbon in deep soil macroaggregates and increased the carbon sequestration potential of deep soil.
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Carbono , Suelo , Agricultura , Secuestro de Carbono , Fertilización , Fertilizantes , Humanos , Nitrógeno/análisis , Suelo/químicaRESUMEN
OBJECTIVE: To evaluate the effectiveness and safety of Baidu Jieduan Granules (BDJDG) to treat common type coronavirus disease 2019 (COVID-19). METHODS: This multicenter, retrospective, and observational clinical trial included 230 common COVID-19 patients in Leishenshan, Huangshi, and Laohekou Hospitals in Wuhan from January 21 to March 26, 2020. The included patients were further divided into two subgroups according to the use of supplemental oxygen, mild and moderate groups. During the first 14 d of hospitalization, all patients were administered BDJDG combined with conventional Western medicine, and observed for continuous 28 d. Primary outcomes were disease progression rate and discharge rate. Secondary outcomes included negative conversion time of nucleic acid, hospitalization duration, clinical symptom subsidence time, and symptom regression rate. RESULTS: A total of 230 common COVID-19 patients were analyzed (138 in moderate group and 92 in mild group). By day 28, the disease progression rate was 4.3% and the discharge rate was 95.7%. All mild cases recovered and were discharged from hospital. The median negative conversion time of nucleic acid of all 230 COVID-19 patients was 12 d [inter-quartile range (IQR) 3.5-17], the median hospitalization duration was 15 d (IQR 12-20). The median time to fever, cough, and fatigue recovery was 4 d (IQR 2-6), 8 d (IQR 5-12), and 8 d (IQR 5-11). The recovery rate of fever, cough, and fatigue was 94.6%, 90.5%, and 93.5%. The median time to clinical improvement was 12 d (IQR 10-17). Compared with the baseline, total leukocyte counts, neutrophil counts, lymphocyte counts, and platelet counts were increased significantly on days 7 and 14 (P<0.01). C-reactive protein markedly increased on day 3 and significantly decreased on days 7 and 14 (P<0.01). No serious adverse events occurred during treatment. CONCLUSION: BDJDG may be effective and safe for treatment of common type COVID-19. (Registration No. ChiCTR2000030836).
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Tratamiento Farmacológico de COVID-19 , Ácidos Nucleicos , Proteína C-Reactiva , China , Tos/tratamiento farmacológico , Progresión de la Enfermedad , Fatiga , Fiebre , Humanos , Oxígeno , Estudios Retrospectivos , SARS-CoV-2 , Resultado del TratamientoRESUMEN
OBJECTIVE: Severe cases of coronavirus disease 2019 (COVID-19) are expected to have a worse prognosis than mild cases. Shenhuang Granule (SHG) has been shown to be a safe and effective treatment for severe COVID-19 in a previous randomized clinical trial, but the active chemical constituents and underlying mechanisms of action remain unknown. The goal of this study is to explore the chemical basis and mechanisms of SHG in the treatment of severe COVID-19, using network pharmacology. METHODS: Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry was employed to screen chemical constituents of SHG. Putative therapeutic targets were predicted by searching traditional Chinese medicine system pharmacology database and analysis platform, SwissTargetPrediction, and Gene Expression Omnibus (GEO) databases. The target protein-protein interaction network and enrichment analysis were performed to investigate the hub genes and presumptive mechanisms. Molecular docking and molecular dynamics simulations were used to verify the stability and interaction between the key chemical constituents of SHG and COVID-19 protein targets. RESULTS: Forty-five chemical constituents of SHG were identified along with 131 corresponding therapeutic targets, including hub genes such as HSP90AA1, MMP9, CXCL8, PTGS2, IFNG, DNMT1, TYMS, MDM2, HDAC3 and ABCB1. Functional enrichment analysis indicated that SHG mainly acted on the neuroactive ligand-receptor interaction, calcium signaling pathway and cAMP signaling pathway. Molecular docking showed that the key constituents had a good affinity with the severe acute respiratory syndrome coronavirus 2 protein targets. Molecular dynamics simulations indicated that ginsenoside Rg4 formed a stable protein-ligand complex with helicase. CONCLUSION: Multiple components of SHG regulated multiple targets to inhibit virus invasion and cytokine storm through several signaling pathways; this provides a scientific basis for clinical applications and further experiments.
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Tratamiento Farmacológico de COVID-19 , Medicamentos Herbarios Chinos , Humanos , Simulación del Acoplamiento Molecular , Ligandos , Farmacología en Red , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Medicamentos Herbarios Chinos/química , Medicina Tradicional ChinaRESUMEN
RATIONALE: Atherosclerotic cardiovascular disease (ASCVD), including coronary heart disease (CHD), atherosclerotic stroke and peripheral vascular disease, has become the most deadly chronic noncommunicable disease throughout the world in recent decades, while plaque regression could reduce the occurrence of ASCVD. Traditional Chinese Medicine (TCM) has been widely used for prevention and treatment of these diseases. In the perspective of TCM, phlegm and blood stasis are considered to be leading pathogenesis for CHD. Hence, activating blood circulation and dissipating phlegm, which is of great benefit to regress plaque, have been regarded as general principles in treatment. PATIENT CONCERNS: A 36-year-old man presented with a 3-month history of intermittent exertional chest pain. Coronary angiography revealed 60% stenosis of the proximal left anterior descending coronary artery. Liver function showed: alanine transaminase (ALT):627U/L, aspartate transaminase (AST):243U/L. DIAGNOSES: CHD and hepatitis B with severe liver dysfunction. INTERVENTIONS: The patient should have been treated with high-intensity statin therapy. Actually, due to severe liver dysfunction, Huazhirougan granule instead of statins was administered. In addition, he was treated with TCM according to syndrome differentiation for two and a half years. OUTCOMES: The chest pain disappeared and other symptoms alleviated as well after treatment. Coronary computed tomographic angiography revealed no stenosis in the proximal left anterior descending coronary artery. ALT and AST level returned to normal (ALT:45U/L,AST:24U/L). LESSONS: For patients with CHD and severe hepatic dysfunction, antilipidemic drugs such as statins are not recommended. This case suggested that TCM might fill a gap in lipid-lowering therapy. Thus, we could see that statins were not the only drug for plaque regression and the effect of TCM in treating coronary artery disease cannot be ignored.
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Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Hepatitis B/diagnóstico , Hepatitis B/tratamiento farmacológico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Medicina Tradicional China , Adulto , Diagnóstico Diferencial , Humanos , Pruebas de Función Hepática , MasculinoRESUMEN
Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. We also characterize a phenomenon we refer to as lethal-mosaicism that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process we refer to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox.
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Alelos , Reparación del ADN por Unión de Extremidades/genética , Drosophila/genética , Tecnología de Genética Dirigida/métodos , Agricultura/métodos , Animales , Animales Modificados Genéticamente/genética , Sistemas CRISPR-Cas/genética , Análisis Mutacional de ADN , Femenino , Edición Génica/métodos , Patrón de Herencia/genética , Masculino , Mosaicismo , ARN Guía de Kinetoplastida/genéticaRESUMEN
RATIONALE: Sinus bradycardia refers to a sinus heart rate <60âbpm. Cardiac sinus arrests refer to the omission of atrial activation caused by transient cessation of impulse generation at the sinoatrial node. Normally, drugs such as atropine, isoproterenol, dopamine, dobutamine, or epinephrine can be used for the acute treatment of bradycardia. Temporary pacing is used for treating severe symptomatic bradycardia due to a reversible cause. Permanent cardiac pacing is used for chronic therapy of bradycardia. However, for traditional Chinese medicine (TCM), benefiting qi and nourishing yin and activating blood circulation is the general principle in treatment and show remarkable curative effects. PATIENT CONCERNS: A 32-year-old man was found to have 1-degree atrioventricular block and sinus bradycardia during a physical examination. He reported suffering from palpitation and shortness of breath occasionally. An ambulatory electrocardiogram showed sinus arrhythmia, sinus bradycardia, and significant sinus arrhythmia. The minimum heart rate was 33âbpm (beats per minute). The number of sinus arrest was 42 and the maximum RR interval was 2216âms. DIAGNOSES: The patient was diagnosed with bradyarrhythmia in Western medicine and "palpitation" in TCM. INTERVENTIONS: The patient was treated with methods of benefiting qi and nourishing yin and activating blood circulation along with warming yan for nearly 5 months. CPM (Chinese patent medicine) such as Yixinshu capsule, Bingdouling oral liquid, Zhenyuan capsule, Zhibaidihuang pills were used for treatment. At the same time, he was suggested to change his lifestyles including falling asleep before 10:00 PM and abandoning spicy diets. OUTCOMES: The symptoms of palpitation and shortness of breath disappeared. The minimum heart rate increased from 33 to 42âbpm and sinus arrests did not occur. The maximum RR interval decreased from 2216 to 1650âms and the remarkable sinus arrhythmia had improved obviously. LESSONS: This case report shows that TCM can be an effective alternative therapy for sinus bradycardia and cardiac sinus arrests. CPM may have been a successful intervention in arrhythmias.
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Bradicardia/tratamiento farmacológico , Medicamentos Herbarios Chinos/uso terapéutico , Medicina Tradicional China , Paro Sinusal Cardíaco/tratamiento farmacológico , Adulto , Humanos , MasculinoRESUMEN
The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these 'in locus' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis.
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Sistemas CRISPR-Cas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/genética , Alas de Animales/crecimiento & desarrollo , Animales , Drosophila melanogaster/clasificación , Drosophila melanogaster/crecimiento & desarrollo , Especificidad de la Especie , Alas de Animales/fisiologíaRESUMEN
The characteristics and changes of soil organic carbon (SOC) and total nitrogen (TN) in different size particles of soil under different agricultural practices are the basis for better understanding soil carbon sequestration of mollisols. Based on a 31-year long-term field experiment located at the Heilongjiang Academy of Agricultural Sciences (Harbin) , soil samples under six treatments were separated by size-fractionation method to explore changes and distribution of SOC and TN in coarse sand, fine sand, silt and clay from the top layer (0-20 cm) and subsurface layer (20-40 cm). Results showed that long-term application of manure (M) increased the percentages of SOC and TN in coarse sand and clay size fractions. In the top layer, application of nitrogen, phosphorus and potassium fertilizers combined with manure (NPKM) increased the percentages of SOC and TN in coarse sand by 191.3% and 179.3% compared with the control (CK), whereas M application increased the percentages of SOC and TN in clay by 45% and 47% respectively. For subsurface layers, the increase rates of SOC and TN in corresponding parts were lower than that in top layer. In the surface and subsurface layers, the percentages of SOC storage in silt size fraction accounted for 42%-63% and 48%-54%, TN storage accounted for 34%-59% and 41%-47%, respectively. The enrichment factors of SOC and TN in coarse sand and clay fractions of surface layers increased significantly under the treatments with manure. The SOC and TN enrichment factors were highest in the NPKM, being 2.30 and 1.88, respectively, while that in the clay fraction changed little in the subsurface layer.
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Agricultura , Carbono/química , Fertilizantes , Nitrógeno/química , Suelo/química , Silicatos de Aluminio , Secuestro de Carbono , Arcilla , Estiércol , Tamaño de la Partícula , Fósforo , Potasio , Dióxido de SilicioRESUMEN
AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a "gene switch" system, which can induce gene expression by administration of tetracycline (Tet) derivatives such as doxcycline (Dox). Using this system, we established 293 cells to an artificial cell line secreting insulin in response to stimulation by Dox. METHODS: The mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by the polymerase chain reaction (PCR), and was inserted downstream from the promoter on the expression vector pTRE2, to construct a recombined expression vector pTRE2mINS. The promoter on pTRE2 consists of the tetracycline-response element and the CMV minimal promoter and is thus activated by the reverse tetracycline-controlled transactivator (rtTA) when Dox is administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin were co-transfected in the tet293 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Dox was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of Dox in culture medium on the expression of proinsulin gene was estimated respectively with Northern blot, RT-PCR, and radioimmunoassay. RESULTS: From the 28 hygromycin-resistant cell strains, we selected one cell strain (tet293/Ins6) secreting insulin not only automatically, but in response to stimulation by Dox. The amount on insulin secretion was dependent on the Dox dose (0,10,100,200,400,800 and 1000 microg.L(-1)), the level of insulin secreted by the cells treated with Dox (1000 microg.L(-1)) was 241.0pU.d(-1).cell(-1) , which was 25-fold that of 9.7pU.d(-1).cell(-1) without Dox treatment. Northern blot analyses and RT-PCR further confirmed that the transcription of insulin gene had already been up-regulated after exposing tet293/Ins6 cells to Dox for 15 minutes, and was also induced in a dose-dependent manner. However, the concentration of insulin in the media did not increase significantly until 5 hours following the addition of Dox. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability of gene therapy, suggesting that conditional establishment of artificial beta-cells may be a useful approach to develop cellular therapy for diabetes mellitus.