To determine passive haemagglutination (PHA) antibody titer that would protect chicks against Nigerian isolates of the Infectious Bursa Disease Virus (IBDV), five groups of chicks aged 30 days which had different anti...To determine passive haemagglutination (PHA) antibody titer that would protect chicks against Nigerian isolates of the Infectious Bursa Disease Virus (IBDV), five groups of chicks aged 30 days which had different antibody titers were challenged with a Nigerian isolate of virulent IBDV. Mortality rates of the different groups were plotted against their respective mean PHA antibody titers. A group with zero antibody titer had a mortality rate of 75% while those with PHA antibody titers of 185.6, 243.2, 256 and 307.2 had mortality rates of 40%, zero, zero and zero respectively. Linear equation generated for a line of best fit of the graph of mortality rates of the chicks on their IBD antibody titers gave antibody titer (X) at which mortality (Y) would be zero as 300. A mortality of 75% and the high antibody level needed to protect chicks suggest that the isolate may be a hypervirulent strain.展开更多
Candidatus Liberibacter asiaticus （CaLas）, an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing （HLB） in citrus. CaLas resides in phloem sieve tubes and has been shown to be unequ...Candidatus Liberibacter asiaticus （CaLas）, an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing （HLB） in citrus. CaLas resides in phloem sieve tubes and has been shown to be unequally distributed in different tissues. Although HLB is a disease of citrus plants, it has been demonstrated that periwinkle can serve as an experimental host of CaLas, which can be transmitted from citrus to periwinkle via the parasitic plant dodder （Cuscuta spp.）. To investigate the distribution of CaLas in various periwinkle tissues, the bacteria were transmitted from an infected periwinkle plant to healthy periwinkles by top-grafting. The movement of the inoculum and associated titer changes were observed over time in various tissues. CaLas could be detected in the leaves, main stems, and roots of infected periwinkle by conventional PCR, and in all three tissues a clear time-dependent change in CaLas titer was observed, with titer increasing soon after inoculation and then decreasing as disease symptoms became severe. The highest titer was found at 25, 35 and 35 days after inoculation in leaves, main stems and roots, respectively. The titer in leaves was much higher than in the main stems and roots at the same time point, and the spatial distribution of CaLas in the leaves, main stems and roots of infected periwinkle was uneven, similar to what has been shown in citrus. The results provide guidance for selecting the proper periwinkle tissues and sampling times for early detection of CaLas.展开更多
[ Objective] To study the correlation between HI titer and protection against IBV challenge in SPF layers. [ Method ] SPF layers were randomly divided into four groups, namely group A1, A2, B1 and B2. The group A1 was...[ Objective] To study the correlation between HI titer and protection against IBV challenge in SPF layers. [ Method ] SPF layers were randomly divided into four groups, namely group A1, A2, B1 and B2. The group A1 was immunized with H120 live vaccine. The group A2 was first immunized with H120 live vaccine and later boosted with ND-IB-EDS trivalent inactivated vaccine. The group B1 was used as unimmunized chal- lenge control. The group B2 was kept as unimmunized unchallenged control. The blood samples were taken prior and post-vaccination at intervals and HI tests were conducted. At the laying peak, the group A1, A2 and B1 were challenged with IBV M4t virulent strain. The clinical features and egg production of layers were monitored and recorded. [Result] After 30 d post vaccination with H120 live vaccine, the HI titer reached 4.45 log2; after 30 days boosting with ND-IB-EDS trivalent inactivated vaccine, the HI titer reached to 7.35 log2. Before challenge, HI antibody titer in group A1, A2, B1 and B2 were respectively 4.24 log2, 7.40 Iog2, 2.10 log2 and 2.10 log2. After challenge, chickens in unimmunized challenge control group B1 showed respiratory symptoms, egg production dropped by 30.9%, and they produced more soft-shelled, no-shelled or abnormal eggs. In the group A1, some chickens had light respiratory symptoms and egg production dropped by 11.7%. In the group A2, the egg production of all chickens was as normal as the group B2. [ Conclusion] When the HI titer was over 6 log2, challenge by virulent virus had no impact on egg produc- tion; when the HI titer was 5 log2, 4 log2 and less 3 log2, egg production dropped by 6.0%, 11.3% and 29.6%, respectively. Thus, the HI anti- body level in chickens has close correlation with protection against IBV challenge.展开更多
Brucellosis is an important re-emerging zoonotic disease caused by Brucella organisms. In the absence of a Differentiation of Infected from Vaccinated Animal (DIVA) assay for bovine Brucellosis, it becomes difficult t...Brucellosis is an important re-emerging zoonotic disease caused by Brucella organisms. In the absence of a Differentiation of Infected from Vaccinated Animal (DIVA) assay for bovine Brucellosis, it becomes difficult to assess whether the anti-Brucella antibody response in an animal is due to vaccination or infection. We compared the anti-Brucella antibody titers of naturally Brucellosis affected unvaccinated cows, previously vaccinated infected cows, normal healthy vaccinated cows and healthy unvaccinated calves. The titers of anti-Brucella antibodies were estimated by indirect ELISA. The mean titer (log10) was found to be 1.518 ± 0.005 in case of naturally Brucellosis affected cattle which had been vaccinated during calf hood. The mean titer in case of naturally infected cattle which had never been vaccinated was 1.5441 ± 0.005. The mean titer in healthy unaffected cattle vaccinated during calf hood was 1.504 ± 0.002 and that of unvaccinated healthy calves was 0.560 ± 0.016. It was interesting to find that the antibody titers in naturally affected cattle which had never been vaccinated were very significantly (p < 0.01) higher than those of Brucellosis affected cows which had been vaccinated during calf hood. The titer in vaccinated infected cattle was very significantly (p < 0.01) higher than that of uninfected vaccinated cows.展开更多
Cell disruption focuses on obtaining a desired bioproduct within a cell, and it is the cell wall that must be disrupted to allow access to the contents of the cell. In animal cells, the plasma membrane is the only bar...Cell disruption focuses on obtaining a desired bioproduct within a cell, and it is the cell wall that must be disrupted to allow access to the contents of the cell. In animal cells, the plasma membrane is the only barrier separating cell contents from the environment. Sound waves from sonication, a mechanical technique for cell disintegration, have been used to disrupt as well as to aggregate cells as a step towards purification of a desired bioproduct. In the present study, an improved sonication process for the high yield of Newcastle disease virus （NDV） propagated in tissue culture was described. DF-I cell was cultured in 25cm^2 T flask. When cells were about 80% confluent, a lentogenic strain of NDV （F strain） was used to infect the cell monolayer. With evident cytopathic effect, cells were subjected to cycles of freeze-thaw before sonicating with varying combinations of amplitude, temperature and time. Cells were sonicated using a water bath Sonicator, Jac Ultrasonic 1505 JEIO TECH 4 KHz. From ANOVA analysis, a significant interaction between sonication time and amplitude was observed. This also corresponds to the highest F value observed.展开更多
Objective: to study the correlation between the preeclampsia IgG antibody A(B) titer and hemolytic disease of the newborn (HDN). Methods: from February 2018 to February 2020, the clinical data of 36 pregnant women wit...Objective: to study the correlation between the preeclampsia IgG antibody A(B) titer and hemolytic disease of the newborn (HDN). Methods: from February 2018 to February 2020, the clinical data of 36 pregnant women with blood type not blood O in our hospital were retrospectively analyzed, and the IgG anti-A (B) titer was measured and the relationship with HDN was observed. Results: in 36 cases of blood O pregnant women, IgG antibody A(B) titer less than 1:64 was 41.67%, HDN production rate was 6.67%;IgG antibody A(B) titer equal to 1:64 was 19.44%, and HDN production rate was 28.57%. IgG antibody A(B) titer = 1:128 was 13.89%, and HDN production rate was 40.00%. The titer of IgG against A(B) was 1:256, which was 16.67%, and the HDN production rate was 50.00%. The titer of IgG anti-A (B) was more than or equal to 1:552, 8.33%, and the HDN production rate was 66.67%. The titer of IgG anti-A (B) was positively correlated with the HDN production rate, and the difference was very significant (P < 0.05). Conclusion: the pregnant female IgG anti-A (B) titer of blood O is closely related to HDN. The higher the IgG anti-A (B) titer is, the more people suffer from HDN. Clinical should be to IgG anti-a (B) titer high pregnant women and fetus, newborn increased observation, as early as possible to take intervention measures.展开更多
Aphids are sap-feeding plant pests that depend on their symbiotic relationships with the primary endosymbiont Buchnera aphidicola to adapt to impoverished diets. However, how the host plant affects the aphid primary s...Aphids are sap-feeding plant pests that depend on their symbiotic relationships with the primary endosymbiont Buchnera aphidicola to adapt to impoverished diets. However, how the host plant affects the aphid primary symbiont and aphid adaptation to host plant transfer are poorly known. In this study, aphid symbiont screening and genotype identification were used to establish 2 aphid strains (Rhopalosiphum maidis [Rm] and Rhopalosiphum padi [Rp] strains) containing only Buchnera without any secondary symbionts for both wheat aphid species (R. maidis and R. padi). Aphid fitness and Buchnera titers were unstable on some of these host plants after transferring to novel host plants (G1–G5), which were influenced by host plant species and generations;however, they stabilized after prolonged feeding on the same plants for 10 generations. The electropenetrography (EPG) records showed that the allocation of aphid feeding time was significantly distinct in the 6 host plants;aphids had more intracellular punctures and spent more nonprobing time on green bristlegrass which was not conducive to its growth compared with other plants. The content of soluble sugar, soluble protein, and amino acid in the leaves of the 6 host plants were also clearly separated. The correlation coefficient analysis showed that the nutrient contents of host plants had significant correlations with aphid feeding behaviors, fitness, and Buchnera titers. In the meantime, aphid fitness, and Buchnera titers were also affected by aphid feeding behaviors. Also, Buchnera titers of aphid natural populations on 6 host plants showed a visible difference. Our study deepened our understanding of the interaction among aphids, endosymbionts, and host plants, indicating that the host plant nutrient content is a predominant factor affecting aphid adaptation to their diet, initially affecting aphid feeding behaviors, and further affecting aphid fitness and Buchnera titers, which would further contribute to exploiting new available strategies for aphid control.展开更多
Base editors(BEs)are a promising tool for precise base conversion in human cells and animals,while the adeno-associated virus(AAV)is the major vector for human gene therapy.However,the size of the DNA cassette require...Base editors(BEs)are a promising tool for precise base conversion in human cells and animals,while the adeno-associated virus(AAV)is the major vector for human gene therapy.However,the size of the DNA cassette required for BE expression exceeds the 4.7 kb packing capacity of the AAV vector,making dual-AAV approaches based on trans-splicing intein necessary.Even with this approach,current split DNA cassettes are still larger than the AAV packing limit,posing a challenge for cellular production of AAV.Moreover,some split strategies yield variable editing results and target coverage.To address these limitations,25 different split sets for BE4max and A3A-BE4max were tested at two target sites respectively,with splitting sites ranging from 493rd to 517th amino acids on the Cas9 peptide.Fortunately,the best Cas9 split site was identified between His511 and Ser512 and the arrangement of the AAV expression cassette was further manipulated to create evenly distributed CBE and ABE intein systems within 4.7 kb.These novel dual-AAV systems,designated 4.6AAV-CBE and 4.7AAV-ABE,were found to have base editing efficiencies similar to wild-type BEs,with a narrower editing window than the current 573 split system.Notably,4.6AAV-CBE yield a higher AAV production titer,up to 2.1-fold in AAV-N and 1.5-fold in AAV-C,compared to the split-573BE system,likely due to the reduction of DNA cassette size within the AAV packaging capacity.Moreover,after packaging and infecting cells with AAV-N and AAV-C at the same volume and number of cells,the multiplicities of infection(MOI)and editing efficiency of 4.6 AAV-CBE were both higher than those of the split-573BE system.This study present advanced dual-AAV systems for ABE and CBE delivery,establishing a basis for safe and efficient BE therapies.展开更多
Dramatic decrease of sugar uptake is a general phenomenon in Streptomyces at stationary phase,when antibiotics are extensively produced.Milbemycins produced by Streptomyces bingchenggensis are a group of valuable macr...Dramatic decrease of sugar uptake is a general phenomenon in Streptomyces at stationary phase,when antibiotics are extensively produced.Milbemycins produced by Streptomyces bingchenggensis are a group of valuable macrolide biopesticides,while the low yield and titer impede their broad applications in agricultural field.Considering that inadequate sugar uptake generally hinders titer improvement of desired products,we mined the underlying sugar uptake systems and fine-tuned their expression in this work.First,we screened the candidates at both genomic and transcriptomic level in S.bingchenggensis.Then,two ATP-binding cassette transporters named TP2 and TP5 were characterized to improve milbemycin titer and yield significantly.Next,the appropriate native temporal promoters were selected and used to tune the expression of TP2 and TP5,resulting in a maximal milbemycin A3/A4 titer increase by 36.9%to 3321 mg/L.Finally,TP2 and TP5 were broadly finetuned in another two macrolide biopesticide producers Streptomyces avermitilis and Streptomyces cyaneogriseus,leading to a maximal titer improvement of 34.1%and 52.6%for avermectin B1a and nemadectin,respectively.This work provides useful transporter tools and corresponding engineering strategy for Streptomyces.展开更多
Juvenile hormone plays an important role in controlling the processes of metamorphosis and reproduction of most insects. There has been a growing amount of research works on the determination of JH titers at various p...Juvenile hormone plays an important role in controlling the processes of metamorphosis and reproduction of most insects. There has been a growing amount of research works on the determination of JH titers at various physiological levels in a variety of insect species, yet our knowledge concerning JH titer lags behind that of ecdysteroid titer. The reasons are: (ⅰ) JH titers in most insects seem to be generally quite low, and (ⅱ) it is difficult to separate JH from the lipids that are present in rather large quantities and have similarities in chemical and physical properties.展开更多
T. Ogata reported that the so-called antiserum titer varied with the absolute volume of the diluted solution as well as with the concentration of erythrocytes or bacteria. If these factors were not strictly determined...T. Ogata reported that the so-called antiserum titer varied with the absolute volume of the diluted solution as well as with the concentration of erythrocytes or bacteria. If these factors were not strictly determined, it would be impossible to展开更多
Objective To explore whether prophylactic anti-viral therapy is needed for hepatitis B virus(HBV) infection resolvers with high titer of hepatitis B surface antibody(anti-HBs) receiving rituximab treatment.
The study investigated the enhanced production of 2-hydroxybutyric acid(2-HBA)from threonine using a two-step whole-cell bioconversion by recombinant Escherichia coli BL21(DE3)overexpressing threonine dehydratase and ...The study investigated the enhanced production of 2-hydroxybutyric acid(2-HBA)from threonine using a two-step whole-cell bioconversion by recombinant Escherichia coli BL21(DE3)overexpressing threonine dehydratase and keto-reductase.To address the rate-limiting step posed by NADH regeneration for the keto-reductase reaction converting 2-ketobutyric acid(2-KBA)to 2-HBA,formate dehydrogenase from Candida boidinii was overexpressed under the T7 promoter,resulting in a high titer of 1015 mM and a yield of 0.70 mol/mol.Furthermore,the yield was improved by disrupting three enzymes responsible for the degradation of the intermediate(2-KBA),pyruvate-formate lyase(PflB),pyruvate oxidase(PoxB),and pyruvate dehydrogenase complex(PDHc),leading to an impressive yield of 0.99 mol/mol,closely approaching the theo-retical maximum of 1.00 mol/mol.The triple mutant,designed to prevent 2-KBA degradation,achieved a remarkable titer of 1,400 mM and volumetric productivity of 58 mmol/L/h.To the best of our knowledge,this achievement represents the highest reported titer and yield for 2-HBA production to date.展开更多
文摘To determine passive haemagglutination (PHA) antibody titer that would protect chicks against Nigerian isolates of the Infectious Bursa Disease Virus (IBDV), five groups of chicks aged 30 days which had different antibody titers were challenged with a Nigerian isolate of virulent IBDV. Mortality rates of the different groups were plotted against their respective mean PHA antibody titers. A group with zero antibody titer had a mortality rate of 75% while those with PHA antibody titers of 185.6, 243.2, 256 and 307.2 had mortality rates of 40%, zero, zero and zero respectively. Linear equation generated for a line of best fit of the graph of mortality rates of the chicks on their IBD antibody titers gave antibody titer (X) at which mortality (Y) would be zero as 300. A mortality of 75% and the high antibody level needed to protect chicks suggest that the isolate may be a hypervirulent strain.
基金supported by the earmarked fund for China Agriculture Research System (CARS-27)the Special Fund for Agro-Scientific Research in the Public Interest, China (2010003067)
文摘Candidatus Liberibacter asiaticus （CaLas）, an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing （HLB） in citrus. CaLas resides in phloem sieve tubes and has been shown to be unequally distributed in different tissues. Although HLB is a disease of citrus plants, it has been demonstrated that periwinkle can serve as an experimental host of CaLas, which can be transmitted from citrus to periwinkle via the parasitic plant dodder （Cuscuta spp.）. To investigate the distribution of CaLas in various periwinkle tissues, the bacteria were transmitted from an infected periwinkle plant to healthy periwinkles by top-grafting. The movement of the inoculum and associated titer changes were observed over time in various tissues. CaLas could be detected in the leaves, main stems, and roots of infected periwinkle by conventional PCR, and in all three tissues a clear time-dependent change in CaLas titer was observed, with titer increasing soon after inoculation and then decreasing as disease symptoms became severe. The highest titer was found at 25, 35 and 35 days after inoculation in leaves, main stems and roots, respectively. The titer in leaves was much higher than in the main stems and roots at the same time point, and the spatial distribution of CaLas in the leaves, main stems and roots of infected periwinkle was uneven, similar to what has been shown in citrus. The results provide guidance for selecting the proper periwinkle tissues and sampling times for early detection of CaLas.
文摘[ Objective] To study the correlation between HI titer and protection against IBV challenge in SPF layers. [ Method ] SPF layers were randomly divided into four groups, namely group A1, A2, B1 and B2. The group A1 was immunized with H120 live vaccine. The group A2 was first immunized with H120 live vaccine and later boosted with ND-IB-EDS trivalent inactivated vaccine. The group B1 was used as unimmunized chal- lenge control. The group B2 was kept as unimmunized unchallenged control. The blood samples were taken prior and post-vaccination at intervals and HI tests were conducted. At the laying peak, the group A1, A2 and B1 were challenged with IBV M4t virulent strain. The clinical features and egg production of layers were monitored and recorded. [Result] After 30 d post vaccination with H120 live vaccine, the HI titer reached 4.45 log2; after 30 days boosting with ND-IB-EDS trivalent inactivated vaccine, the HI titer reached to 7.35 log2. Before challenge, HI antibody titer in group A1, A2, B1 and B2 were respectively 4.24 log2, 7.40 Iog2, 2.10 log2 and 2.10 log2. After challenge, chickens in unimmunized challenge control group B1 showed respiratory symptoms, egg production dropped by 30.9%, and they produced more soft-shelled, no-shelled or abnormal eggs. In the group A1, some chickens had light respiratory symptoms and egg production dropped by 11.7%. In the group A2, the egg production of all chickens was as normal as the group B2. [ Conclusion] When the HI titer was over 6 log2, challenge by virulent virus had no impact on egg produc- tion; when the HI titer was 5 log2, 4 log2 and less 3 log2, egg production dropped by 6.0%, 11.3% and 29.6%, respectively. Thus, the HI anti- body level in chickens has close correlation with protection against IBV challenge.
文摘Brucellosis is an important re-emerging zoonotic disease caused by Brucella organisms. In the absence of a Differentiation of Infected from Vaccinated Animal (DIVA) assay for bovine Brucellosis, it becomes difficult to assess whether the anti-Brucella antibody response in an animal is due to vaccination or infection. We compared the anti-Brucella antibody titers of naturally Brucellosis affected unvaccinated cows, previously vaccinated infected cows, normal healthy vaccinated cows and healthy unvaccinated calves. The titers of anti-Brucella antibodies were estimated by indirect ELISA. The mean titer (log10) was found to be 1.518 ± 0.005 in case of naturally Brucellosis affected cattle which had been vaccinated during calf hood. The mean titer in case of naturally infected cattle which had never been vaccinated was 1.5441 ± 0.005. The mean titer in healthy unaffected cattle vaccinated during calf hood was 1.504 ± 0.002 and that of unvaccinated healthy calves was 0.560 ± 0.016. It was interesting to find that the antibody titers in naturally affected cattle which had never been vaccinated were very significantly (p < 0.01) higher than those of Brucellosis affected cows which had been vaccinated during calf hood. The titer in vaccinated infected cattle was very significantly (p < 0.01) higher than that of uninfected vaccinated cows.
文摘Cell disruption focuses on obtaining a desired bioproduct within a cell, and it is the cell wall that must be disrupted to allow access to the contents of the cell. In animal cells, the plasma membrane is the only barrier separating cell contents from the environment. Sound waves from sonication, a mechanical technique for cell disintegration, have been used to disrupt as well as to aggregate cells as a step towards purification of a desired bioproduct. In the present study, an improved sonication process for the high yield of Newcastle disease virus （NDV） propagated in tissue culture was described. DF-I cell was cultured in 25cm^2 T flask. When cells were about 80% confluent, a lentogenic strain of NDV （F strain） was used to infect the cell monolayer. With evident cytopathic effect, cells were subjected to cycles of freeze-thaw before sonicating with varying combinations of amplitude, temperature and time. Cells were sonicated using a water bath Sonicator, Jac Ultrasonic 1505 JEIO TECH 4 KHz. From ANOVA analysis, a significant interaction between sonication time and amplitude was observed. This also corresponds to the highest F value observed.
文摘Objective: to study the correlation between the preeclampsia IgG antibody A(B) titer and hemolytic disease of the newborn (HDN). Methods: from February 2018 to February 2020, the clinical data of 36 pregnant women with blood type not blood O in our hospital were retrospectively analyzed, and the IgG anti-A (B) titer was measured and the relationship with HDN was observed. Results: in 36 cases of blood O pregnant women, IgG antibody A(B) titer less than 1:64 was 41.67%, HDN production rate was 6.67%;IgG antibody A(B) titer equal to 1:64 was 19.44%, and HDN production rate was 28.57%. IgG antibody A(B) titer = 1:128 was 13.89%, and HDN production rate was 40.00%. The titer of IgG against A(B) was 1:256, which was 16.67%, and the HDN production rate was 50.00%. The titer of IgG anti-A (B) was more than or equal to 1:552, 8.33%, and the HDN production rate was 66.67%. The titer of IgG anti-A (B) was positively correlated with the HDN production rate, and the difference was very significant (P < 0.05). Conclusion: the pregnant female IgG anti-A (B) titer of blood O is closely related to HDN. The higher the IgG anti-A (B) titer is, the more people suffer from HDN. Clinical should be to IgG anti-a (B) titer high pregnant women and fetus, newborn increased observation, as early as possible to take intervention measures.
基金funded by the programs of the China Agriculture Research System of MOF and MARA(CARS-02)the Agricultural Science and Technology Innovation Program(ASTIP)of CAAS.
文摘Aphids are sap-feeding plant pests that depend on their symbiotic relationships with the primary endosymbiont Buchnera aphidicola to adapt to impoverished diets. However, how the host plant affects the aphid primary symbiont and aphid adaptation to host plant transfer are poorly known. In this study, aphid symbiont screening and genotype identification were used to establish 2 aphid strains (Rhopalosiphum maidis [Rm] and Rhopalosiphum padi [Rp] strains) containing only Buchnera without any secondary symbionts for both wheat aphid species (R. maidis and R. padi). Aphid fitness and Buchnera titers were unstable on some of these host plants after transferring to novel host plants (G1–G5), which were influenced by host plant species and generations;however, they stabilized after prolonged feeding on the same plants for 10 generations. The electropenetrography (EPG) records showed that the allocation of aphid feeding time was significantly distinct in the 6 host plants;aphids had more intracellular punctures and spent more nonprobing time on green bristlegrass which was not conducive to its growth compared with other plants. The content of soluble sugar, soluble protein, and amino acid in the leaves of the 6 host plants were also clearly separated. The correlation coefficient analysis showed that the nutrient contents of host plants had significant correlations with aphid feeding behaviors, fitness, and Buchnera titers. In the meantime, aphid fitness, and Buchnera titers were also affected by aphid feeding behaviors. Also, Buchnera titers of aphid natural populations on 6 host plants showed a visible difference. Our study deepened our understanding of the interaction among aphids, endosymbionts, and host plants, indicating that the host plant nutrient content is a predominant factor affecting aphid adaptation to their diet, initially affecting aphid feeding behaviors, and further affecting aphid fitness and Buchnera titers, which would further contribute to exploiting new available strategies for aphid control.
基金financially supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDC0110200)National Natural Science Foundation of China(31522002,32171449,3240120454)China Postdoctoral Science Foundation(2024M763495).
文摘Base editors(BEs)are a promising tool for precise base conversion in human cells and animals,while the adeno-associated virus(AAV)is the major vector for human gene therapy.However,the size of the DNA cassette required for BE expression exceeds the 4.7 kb packing capacity of the AAV vector,making dual-AAV approaches based on trans-splicing intein necessary.Even with this approach,current split DNA cassettes are still larger than the AAV packing limit,posing a challenge for cellular production of AAV.Moreover,some split strategies yield variable editing results and target coverage.To address these limitations,25 different split sets for BE4max and A3A-BE4max were tested at two target sites respectively,with splitting sites ranging from 493rd to 517th amino acids on the Cas9 peptide.Fortunately,the best Cas9 split site was identified between His511 and Ser512 and the arrangement of the AAV expression cassette was further manipulated to create evenly distributed CBE and ABE intein systems within 4.7 kb.These novel dual-AAV systems,designated 4.6AAV-CBE and 4.7AAV-ABE,were found to have base editing efficiencies similar to wild-type BEs,with a narrower editing window than the current 573 split system.Notably,4.6AAV-CBE yield a higher AAV production titer,up to 2.1-fold in AAV-N and 1.5-fold in AAV-C,compared to the split-573BE system,likely due to the reduction of DNA cassette size within the AAV packaging capacity.Moreover,after packaging and infecting cells with AAV-N and AAV-C at the same volume and number of cells,the multiplicities of infection(MOI)and editing efficiency of 4.6 AAV-CBE were both higher than those of the split-573BE system.This study present advanced dual-AAV systems for ABE and CBE delivery,establishing a basis for safe and efficient BE therapies.
基金This work was financially supported by National Natural Science Foundation of China(Grant Nos:31772242,31972348,and 31672092).
文摘Dramatic decrease of sugar uptake is a general phenomenon in Streptomyces at stationary phase,when antibiotics are extensively produced.Milbemycins produced by Streptomyces bingchenggensis are a group of valuable macrolide biopesticides,while the low yield and titer impede their broad applications in agricultural field.Considering that inadequate sugar uptake generally hinders titer improvement of desired products,we mined the underlying sugar uptake systems and fine-tuned their expression in this work.First,we screened the candidates at both genomic and transcriptomic level in S.bingchenggensis.Then,two ATP-binding cassette transporters named TP2 and TP5 were characterized to improve milbemycin titer and yield significantly.Next,the appropriate native temporal promoters were selected and used to tune the expression of TP2 and TP5,resulting in a maximal milbemycin A3/A4 titer increase by 36.9%to 3321 mg/L.Finally,TP2 and TP5 were broadly finetuned in another two macrolide biopesticide producers Streptomyces avermitilis and Streptomyces cyaneogriseus,leading to a maximal titer improvement of 34.1%and 52.6%for avermectin B1a and nemadectin,respectively.This work provides useful transporter tools and corresponding engineering strategy for Streptomyces.
基金Project supported by the National Natural Science Foundation of China.
文摘Juvenile hormone plays an important role in controlling the processes of metamorphosis and reproduction of most insects. There has been a growing amount of research works on the determination of JH titers at various physiological levels in a variety of insect species, yet our knowledge concerning JH titer lags behind that of ecdysteroid titer. The reasons are: (ⅰ) JH titers in most insects seem to be generally quite low, and (ⅱ) it is difficult to separate JH from the lipids that are present in rather large quantities and have similarities in chemical and physical properties.
文摘T. Ogata reported that the so-called antiserum titer varied with the absolute volume of the diluted solution as well as with the concentration of erythrocytes or bacteria. If these factors were not strictly determined, it would be impossible to
文摘Objective To explore whether prophylactic anti-viral therapy is needed for hepatitis B virus(HBV) infection resolvers with high titer of hepatitis B surface antibody(anti-HBs) receiving rituximab treatment.
基金supported by the National Research Foundation of Korea(NRF)grant funded by the Korean government(MSIT)(No.NRF-2020R1A5A1019631).
文摘The study investigated the enhanced production of 2-hydroxybutyric acid(2-HBA)from threonine using a two-step whole-cell bioconversion by recombinant Escherichia coli BL21(DE3)overexpressing threonine dehydratase and keto-reductase.To address the rate-limiting step posed by NADH regeneration for the keto-reductase reaction converting 2-ketobutyric acid(2-KBA)to 2-HBA,formate dehydrogenase from Candida boidinii was overexpressed under the T7 promoter,resulting in a high titer of 1015 mM and a yield of 0.70 mol/mol.Furthermore,the yield was improved by disrupting three enzymes responsible for the degradation of the intermediate(2-KBA),pyruvate-formate lyase(PflB),pyruvate oxidase(PoxB),and pyruvate dehydrogenase complex(PDHc),leading to an impressive yield of 0.99 mol/mol,closely approaching the theo-retical maximum of 1.00 mol/mol.The triple mutant,designed to prevent 2-KBA degradation,achieved a remarkable titer of 1,400 mM and volumetric productivity of 58 mmol/L/h.To the best of our knowledge,this achievement represents the highest reported titer and yield for 2-HBA production to date.
