High Titers Research Articles

Targeted isolation of extracellular vesicles from cell culture supernatant using immuno-affinity chromatography

Extracellular vesicles (EVs) have emerged as promising therapeutics with broad clinical applications as diagnostic biomarkers and therapeutic drug delivery systems. Yet, these biopharmaceuticals pose a challenge in terms of manufacturing due to their complexity and heterogeneity. Despite advancements in the field, current purification technologies lack scalability and/or selectivity. Affinity chromatography (AC) − coupling unmatched specificity and scalability − could be used to simplify purification processing and generate clinical-grade EVs with higher titers and purity.In the present work, we report the implementation of an immuno-AC resin to capture and purify EVs directly from clarified cellular feedstocks. Firstly, to guide and support marker selection, vesicle phenotype characterization was conducted using single particle interferometric reflectance image sensing (SP-IRIS) coupled with immunofluorescence. CD81 was the marker which shown to be more present and more likely to have the other markers (CD63 and CD9). Thus, anti-CD81 VHH ligand was generated and evaluated towards recombinant CD81 protein and CD81 bearing EV particles using surface plasmon resonance (SPR). Different chromatographic studies with Anti-CD81 ligand immobilized onto agarose beads resin were conducted to optimize the process parameters (residence time, dynamic binding capacity and impurity clearance). At residence time of 2 min, on average 40 % of pure triple tetraspanin-positive EV fraction was recovered. The enrichment in EV particles herein obtained, based on scale-up calculations, it would be possible to produce 1 × 1013 EVs from a 1L cell culture, while meeting impurity requirements in a single-step purification process (impurity removal over 2 log reduction value). A single-step purification process is possible, enabling the successful isolation of homogeneous EVs population, counting with a final HCP titer of 60 ng/mL and 9 ng/mL of dsDNA impurities. EV’s morphological integrity and internalization ability were also demonstrated, showcasing elution’s efficiency under mild conditions.Overall, this work contributes to the development of a novel, highly specific, AC technology using a camelid-derived affinity ligand which, bridging the scalability requirements demanded of large-scale production, could potentiate the advent of EV-based therapies.

Fluxomic, Metabolomic, and Transcriptomic Analyses Reveal Metabolic Responses to Phenazine-1-carboxamide Synthesized in Pseudomonas chlororaphis.

Phenazine-1-carboxamide (PCN) has been exploited as a successful biopesticide due to its broad-spectrum antifungal activity. We engineered a PCN-overproducing Pseudomonas chlororaphis strain through overexpressing shikimate pathway genes (aroB, aroQ, aroE, and phzC) and deleting negative regulatory genes (relA, fleQ, and pykF). The optimized strain produced 1.92 g/L PCN with a yield of 0.11 g/g glycerol, the highest titer ever reported by using minimal media. To gain deeper insights into the underlying regulatory network, the final strain and the parental strain were examined using three distinct omic data sets. 13C-metabolic flux analysis revealed a substantial flux reconfiguration in the optimized strain, including the activation of the EDEMP cycle, the PP pathway, the glyoxylate shunt, and the shikimate pathway. Metabolomic results indicated that central carbon was rerouted to the shikimate pathway. Transcriptomic data identified global gene expression changes. This study forms the basis for further engineering of strains to achieve outstanding performance.

Broad immunogenicity to prior SARS-CoV-2 strains and JN.1 variant elicited by XBB.1.5 vaccination in nursing home residents.

SARS-CoV-2 vaccination has reduced hospitalization and mortality for nursing home residents (NHRs) but emerging variants and waning immunity challenge vaccine effectiveness. This study assesses the immunogenicity of the most recent XBB.1.5 monovalent vaccine to variant strains among NHRs. Participants were subset of a longitudinal study of consented NHRs and Healthcare workers (HCWs) who have received serial blood draws to assess immunogenicity with each SARS-CoV-2 mRNA vaccine dose. We report data on participants who received the XBB.1.5 monovalent vaccine post-FDA approval in Fall 2023. NHRs were categorized by whether they had an interval SARS-CoV-2 infection between their first bivalent vaccine dose and their XBB.1.5 monovalent vaccination. The sample included 61 NHRs [median age 76 (IQR 68-86), 51% female] and 28 HCWs [median age 45 (IQR 31-58), 46% female). After XBB.1.5 vaccination, a robust geometric mean fold rise (GMFR) in XBB.1.5-specific neutralizing antibody titers was observed:17.3 (95% confidence interval [CI] 9.3, 32.4) and NHRs with interval infection and 11.3 (95% CI 5, 25.4) in those without and 13.6 (95% CI 8.4,22) in HCWs. For JN.1-specific titers, GMFRs were 14.9 (95% CI 7.9, 28) and 6.5 (95% CI 3.3, 13.1) in NHRs with and without interval infection, and 11.4 (95% CI 6.2, 20.9) in HCWs. NHRs with interval SARS-CoV-2 infection had higher titers across all analyzed strains analyzed. The XBB.1.5 vaccine significantly elevates Omicron-specific neutralizing antibody titers to XBB.1.5 and JN.1 strains in both NHRs and HCWs with more pronounced in those previously infected with SARS-CoV-2 since bivalent vaccination.

B-103 Correlation of Novel Markers, Anti-Sa (Citrullinated Vimentin) and Anti-CarP (Carbamylated Protein), with High Vectra® DA Disease Activity Scores in Rheumatoid Arthritis (RA)

Abstract Background Citrullination and carbamylation are post-translational modifications where autoantibodies against citrullinated and carbamylated proteins in RA have been associated with more aggressive disease. At the same time, Vectra® DA is blood test that yields a RA disease activity score from 1 to 100. Here, 120 patient samples were analyzed to assess the relationship of disease activity as measured by Vectra® score and the presence of Anti-Sa (Citrullinated Vimentin) and Anti-CarP (Carbamylated Protein). Methods Serum samples were analyzed for Vectra® DA (which is validated for adults with RA) where 12 biomarker measurements (VCAM-1, EGF, VEGF-A, IL-6, TNF-RI, MMP-1, YKL-40, Leptin, Resistin, SAA, CRP) together with patient age, gender and adiposity are used to generate a score where established categories are low for 1 to 29, moderate for 30 to 44, and high for 45 to 100. Anti-Sa and Anti-CarP were by ELISA lab-developed tests (LDT) with a cut-off for positive of >20 Units (U). Results Anti-Sa and Anti-CarP for 40 samples from each Vectra® DA category are reported. Patients with the highest Vectra® scores (mean 58.2, range 46.8 - 87.5) had highest rates of positivity for Anti-Sa (52.5%) and Anti-CarP (35%). In contrast, the group with the lowest Vectra® scores (mean 11.8, range 3.8 - 18.1) were infrequently positive for Anti-Sa (17.5%) and Anti-CarP (0%). Frequencies with moderate Vectra® scores (mean 37.1, range 32.4 - 41.8) were mid-range: Anti-Sa (25%) and 7.5% for Anti-CarP (7.5%). Of note, positivity of Anti-Sa was analytically stronger, i.e. higher titer, in patients with moderate or high Vectra® (mean 90.0U or 81.7U), compared to values seen in those with low Vectra® (mean 60.4 U). Conclusions Our analysis of 120 patient samples demonstrates that high disease activity as determined by Vectra® DA corresponds to higher positivity of novel autoantibody markers, Anti-Sa and Anti-CarP. High Vectra® DA scores (>44) are associated with a 20% 1-year risk of radiographic progression while low scores (<30) carry only a 1% risk. Here, we found that patients with high Vectra® scores were 3 times more likely to test positive for Anti-Sa than those with low disease activity scores. At the same time, Anti-CarP occurred in high frequency (35%) with high Vectra® in contrast to zero positivity observed with low Vectra® scores. Thus, our findings support Anti-CarP’s reported association with more active disease with higher risk of developing joint erosions. Also of note, patients with high Vectra® scores had particularly high Anti-Sa positivity - both a 52.5% positivity rate as well as higher Anti-Sa levels (titers) than the group with lower Vectra® scores. These data corroborate other studies where Anti-Sa not only predicted more aggressive, rapid disease progression, but its titers correlated with clinical measures of disease activity. Taken together, our data support the consistency and clinical usefulness of the Vectra® score along with novel autoantibody markers, Anti- Anti-Sa and Anti-CarP, in order to assess RA severity, prognosis, and patient-specific treatment strategies.

Metabolic engineering of rapidly growing Synechococcus elongatus strains for phototrophic production of alkanes.

Alkanes are high-energy hydrocarbons that are foreseen as next generation biofuels. Cyanobacteria are known to naturally synthesize C15-C19 alkanes; however, the titers are too low to make this a commercially viable process. Therefore, to leverage these photosynthetic platforms for improved alkane production, here we engineered three novel isolates of Synechococcus elongatus PCC 11801, PCC 11802, and IITB6. The two gene AAR-ADO alkane biosynthesis pathway was constructed by cloning the genes for acyl-ACP reductase (aar) and aldehyde deformylating oxygenase (ado) from S. elongatus PCC 7942 under the regulation of PrbcL promoter from PCC 7942 and native promoters from PCC 11801 such as PcpcB300, PpsbAI, and PpsbAIII. The genes were separately cloned under two different promoters, creating a library of the engineered strains. The results indicated that the engineered strains of novel S. elongatus isolates produced significantly higher amounts of alkanes than the model strain PCC 7942. The highest alkane yield achieved was 4.1 mg/gDCW in BG-11, while the highest titer was 31.5 mg/L in 5X BG-11, with an engineered IITB6 strain (PcpcB300:aar:TrrnB::PrbcL:ado:TLac). Overall, the study highlights the potential of newly isolated S. elongatus strains as efficient alkane production platforms.

Cellular and humoral response to SARS-CoV-2 vaccine BNT162b2 in adults with Chronic Kidney Disease G4/5.

The mRNA vaccines have proven to be very effective in preventing severe disease and death from SARS-CoV-2 in the general population. However, in patients with chronic kidney disease (CKD) in dialysis or with kidney transplants (KT) the vaccine responses vary, with severe breakthrough infections as a consequence. In this intervention study we investigated the magnitude and quality of the responses to mRNA vaccination administered prior to kidney replacement therapy (KRT). Twenty patients with CKD G4/5 and nine healthy controls were followed for 12 months after receiving two doses of BNT162b2 four weeks apart and a booster dose after 3–6 months. Induction of anti-Spike and anti-RBD IgG in plasma followed the same kinetics in CKD patients and controls, with a trend towards higher titers in controls. In accordance, there was no differences in the establishment of Spike-specific memory B-cells between groups. In contrast, the CKD patients showed lower levels of anti-Spike IgG in saliva and Spike-specific CD8+ T-cells in blood, possibly influencing the capacity of viral clearance which can contribute to an elevated risk of severe breakthrough infections. In conclusion, we found that CKD patients, despite having a reduced mucosal and cytotoxic immunity to BNT162b2, demonstrated a serological response in plasma similar to healthy controls. This suggests that immunization prior to RRT is efficient and motivated. (EudraCT-nr 2021-000988-68).

Plasmid-Stabilizing Strains for Antibiotic-Free Chemical Fermentation.

Plasmid-mediated antibiotic-free fermentation holds significant industrial potential. However, the requirements for host elements and energy during plasmid inheritance often cause cell burden, leading to plasmid loss and reduced production. The stable maintenance of plasmids is primarily achieved through a complex mechanism, making it challenging to rationally design plasmid-stabilizing strains and characterize the associated genetic factors. In this study, we introduced a fluorescence-based high-throughput method and successfully screened plasmid-stabilizing strains from the genomic fragment-deletion strains of Escherichia coli MG1655 and Bacillus subtilis 168. The application of EcΔ50 in antibiotic-free fermentation increased the alanine titer 2.9 times. The enhanced plasmid stability in EcΔ50 was attributed to the coordinated deletion of genes involved in plasmid segregation and replication control, leading to improved plasmid maintenance and increased plasmid copy number. The increased plasmid stability of BsΔ44 was due to the deletion of the phage SPP1 surface receptor gene yueB, resulting in minimized sporulation, improved plasmid segregational stability and host adaptation. Antibiotic-free fermentation results showed that strain BsΔyueB exhibited a 61.99% higher acetoin titer compared to strain Bs168, reaching 3.96 g/L. When used for the fermentation of the downstream product, 2,3-butanediol, strain BsΔyueB achieved an 80.63% higher titer than Bs168, reaching 14.94 g/L using rich carbon and nitrogen feedstocks. Overall, our work provided a plasmid-stabilizing chassis for E. coli and B. subtilis, highlighting their potential for antibiotic-free fermentation of valuable products and metabolic engineering applications.

Factors affecting the outcome of primary rabies vaccination in young cats

Limited data exist on the factors affecting feline rabies vaccination outcomes during primary immunization. This study aimed to assess if specific factors (signalment, vaccination count, vaccine brand, and time since last vaccination) correlated with meeting global antibody titer standards and absolute titers in young cats given monovalent inactivated rabies vaccines. Analyzing a dataset from cats tested before their first annual booster using the FAVN test, logistic and linear regression models were applied. Among 379 cats, 94.2 % achieved titers meeting or exceeding the standard threshold (≥0.5 IU/ml). Time since last vaccination proved to be the primary predictor of vaccination success. Cats receiving two vaccinations tended toward higher titers. Age, sex, breed, and vaccine type showed no impact on outcomes. The present study indicates that vaccination failure in young cats is uncommon, and that the time interval from the latest vaccination is the single most important predictor of successful rabies vaccination.

Effects of producing high levels of hyperthermophile-specific C25,C25-archaeal membrane lipids in Escherichia coli

A hyperthermophilic archaeon, Aeropyrum pernix, synthesizes C25,C25-archaeal membrane lipids, or extended archaeal membrane lipids, which contain two C25 isoprenoid chains that are linked to glycerol-1-phosphate via ether bonds and are longer than the usual C20,C20-archaeal membrane lipids. The C25,C25-archaeal membrane lipids are believed to allow the archaeon to survive under harsh conditions, because they are able to form lipid membranes that are impermeable at temperatures approaching the boiling point. The effect that C25,C25-archaeal membrane lipids exert on living cells, however, remains unproven along with an explanation for why the hyperthermophilic archaeon synthesizes these specific lipids instead of the more common C20,C20-archaeal lipids or double-headed tetraether lipids. To shed light on the effects that these hyperthermophile-specific membrane lipids exert on living cells, we have constructed an E. coli strain that produces C25,C25-archaeal membrane lipids. However, a resultant low level of productivity would not allow us to assess the effects of their production in E. coli cells. Herein, we report an enhancement of the productivity of C25,C25-archaeal membrane lipids in engineered E. coli strains via the introduction of metabolic pathways such as an artificial isoprenol utilization pathway where the precursors of isoprenoids are synthesized via a two-step phosphorylation of prenol and isoprenol supplemented to a growth medium. In the strain with the highest titer, a major component of C25,C25-archaeal membrane lipids reached ∼11 % of total lipids of E. coli. It is noteworthy that the high production of the extended archaeal lipids did not significantly affect the growth of the bacterial cells. The permeability of the cell membrane of the strain became slightly lower in the presence of the exogenous membrane lipids with longer hydrocarbon chains, which demonstrated the possibility to enhance bacterial cell membranes by the hyperthermophile-specific lipids, along with the surprising robustness of the E. coli cell membrane.

Engineering sub-organelles of a diploid Saccharomyces cerevisiae to enhance the production of 7-dehydrocholesterol

7-Dehydrocholesterol (7-DHC) is widely present in various organisms and is an important precursor of vitamin D3. Despite significant improvements in the biosynthesis of 7-DHC, it remains insufficient to meet the industrial demands. In this study, we reported high-level production of 7-DHC in an industrial Saccharomyces cerevisiae leveraging subcellular organelles. Initially, the copy numbers of DHCR24 were increased in combination with sterol transcriptional factor engineering and rebalanced the redox power of the strain. Subsequently, the effects of compartmentalizing the post-squalene pathway in peroxisomes were validated by assembling various pathway modules in this organelle. Furthermore, several peroxisomes engineering was conducted to enhance the production of 7-DHC. Utilizing the peroxisome as a vessel for partial post-squalene pathways, the potential of yeast for 7-dehydrocholesterol production was demonstrated by achieving a 26-fold increase over the initial production level. 7-DHC titer reached 640.77 mg/L in shake flasks and 4.28 g/L in a 10 L bench-top fermentor, the highest titer ever reported. The present work lays solid foundation for large-scale and cost-effective production of 7-DHC for practical applications.

Screening of ent-copalyl diphosphate synthase and metabolic engineering to achieve de novo biosynthesis of ent-copalol in Saccharomyces cerevisiae

The diterpene ent-copalol is an important precursor to the synthesis of andrographolide and is found only in green chiretta (Andrographis paniculata). De novo biosynthesis of ent-copalol has not been reported, because the catalytic activity of ent-copalyl diphosphate synthase (CPS) is very low in microorganisms. In order to achieve the biosynthesis of ent-copalol, Saccharomyces cerevisiae was selected as the chassis strain, because its endogenous mevalonate pathway and dephosphorylases could provide natural promotion for the synthesis of ent-copalol. The strain capable of synthesizing diterpene geranylgeranyl pyrophosphate was constructed by strengthening the mevalonate pathway genes and weakening the competing pathway. Five full-length ApCPSs were screened by transcriptome sequencing of A. paniculata and ApCPS2 had the best activity and produced ent-CPP exclusively. The peak area of ent-copalol was increased after the ApCPS2 saturation mutation and its configuration was determined by NMR and ESI-MS detection. By appropriately optimizing acetyl-CoA supply and fusion-expressing key enzymes, 35.6 mg/L ent-copalol was generated. In this study, de novo biosynthesis and identification of ent-copalol were achieved and the highest titer ever reported. It provides a platform strain for the further pathway analysis of andrographolide and derivatives and provides a reference for the synthesis of other pharmaceutical intermediates.

Influence of donor age, sex and ethnicity on high-titre anti-A and -B: Review of 6 million donations from two national blood providers.

Some blood operators routinely screen blood donations for high-titre (HT) anti-A/B to reduce the risk of a haemolytic transfusion reaction due to out-of-group plasma-rich components. We assessed donor factors associated with an increased likelihood of screening positive and compared routine data between England and Australia. Data were assessed from HT screening during 2018-2020 in Australia and 2018-2021 in England, totalling nearly 6 million blood donations. Screening was performed using a Beckman Coulter PK7300 analyser with a micro-titre plate saline direct agglutination test in both countries, although different reagent red cells were chosen. HT-positive was defined as testing positive at a titre of 128 or above. The likelihood of a donor testing HT-positive was greater for females than males, declined with age and was dependent on the ABO group. However, the proportion of donors testing HT-positive was consistently higher in Australia than in England: overall, 14% of group O donations and 5% of group A donations in England tested HT-positive, compared with 51% and 22%, respectively in Australia. English data also showed that donors from Black, Asian or mixed ethnic backgrounds were more likely to test HT-positive than White donors. These data demonstrate that donor sex, age, ABO group and ethnicity affect the likelihood of testing HT-positive. Differences in testing methods likely had a significant impact on the proportion of donors testing as HT-positive or -negative rather than any differences in donor populations.

Metabolic engineering of Saccharomyces cerevisiae for high-level production of (+)-ambrein from glucose.

(+)-Ambrein is the primary component of ambergris, a rare product found in sperm whales (Physeter microcephalus). Microbial production using sustainable resources is a promising way to replace animal extraction and chemical synthesis. We constructed an engineered yeast strain to produce (+)-ambrein de novo. Squalene is a substrate for the biosynthesis of (+)-ambrein. Firstly, strain LQ2, with a squalene yield of 384.4mg/L was obtained by optimizing the mevalonate pathway. Then we engineered a method for the de novo production of (+)-ambrein using glucose as a carbon source by overexpressing codon-optimized tetraprenyl-β-curcumene cyclase (BmeTC) and its double mutant enzyme (BmeTCY167A/D373C), evaluating different promoters, knocking out GAL80, and fusing the protein with BmeTC and squalene synthase (AtSQS2). Nevertheless, the synthesis of (+)-ambrein is still limited, causing low catalytic activity in BmeTC. We carried out a protein surface amino acid modification of BmeTC. The dominant mutant BmeTCK6A/Q9E/N454A for the first step was obtained to improve its catalytic activity. The yield of (+)-ambrein increased from 35.2 to 59.0mg/L in the shake flask and finally reached 457.4mg/L in the 2 L fermenter, the highest titer currently available for yeast. Efficiently engineered strains and inexpensive fermentation conditions for the industrial production of (+)-ambrein. The metabolic engineering tools provide directions for optimizing the biosynthesis of other high-value triterpenes.

Evaluation of monkeypox‐ and vaccinia‐virus neutralizing antibodies before and after smallpox vaccination: A sero‐epidemiological study

AbstractSince May 2022, several countries outside of Africa experienced multiple clusters of monkeypox virus (MPXV)‐associated disease. In the present study, anti‐MPXV and anti‐vaccinia virus (VACV) neutralizing antibody responses were evaluated in two cohorts of subjects from the general Italian population (one half born before the WHO‐recommended end of smallpox vaccination in 1980, the other half born after). Higher titers (either against MPXV or VACV) were observed in the cohort of individuals born before the interruption of VACV vaccination. An association between VACV and MPXV antibody levels was observed, suggesting that the smallpox vaccination may confer some degree of cross‐protection against MPXV infection. Results from this study highlight low levels of immunity toward the assessed Orthopoxviruses, especially in young adults, advocating the introduction of a VACV‐ or MPXV‐specific vaccine in case of resurgence of monkeypox disease outbreaks.

Combinatorial metabolic engineering of Streptomyces sp. CB03234-S for the enhanced production of anthraquinone-fused enediyne tiancimycins

BackgroundAnthraquinone-fused enediynes (AFEs) are excellent payloads for antibody-drug conjugates (ADCs). The yields of AFEs in the original bacterial hosts are extremely low. Multiple traditional methods had been adopted to enhance the production of the AFEs. Despite these efforts, the production titers of these compounds are still low, presenting a practical challenge for their development. Tiancimycins (TNMs) are a class of AFEs produced by Streptomyces sp. CB03234. One of their salient features is that they exhibit rapid and complete cell killing ability against various cancer cell lines.ResultsIn this study, a combinatorial metabolic engineering strategy guided by the CB03234-S genome and transcriptome was employed to improve the titers of TNMs. First, re-sequencing of CB03234-S (Ribosome engineered mutant strains) genome revealed the deletion of a 583-kb DNA fragment, accounting for about 7.5% of its genome. Second, by individual or combined inactivation of seven potential precursor competitive biosynthetic gene clusters (BGCs) in CB03234-S, a double-BGC inactivation mutant, S1009, was identified with an improved TNMs titer of 28.2 ± 0.8 mg/L. Third, overexpression of five essential biosynthetic genes, including two post-modification genes, and three self-resistance auxiliary genes, was also conducted, through which we discovered that mutants carrying the core genes, tnmE or tnmE10, exhibited enhanced TNMs production. The average TNMs yield reached 43.5 ± 2.4 mg/L in a 30-L fermenter, representing an approximately 360% increase over CB03234-S and the highest titer among all AFEs to date. Moreover, the resulting mutant produced TNM-W, a unique TNM derivative with a double bond instead of a common ethylene oxide moiety. Preliminary studies suggested that TNM-W was probably converted from TNM-A by both TnmE and TnmE10.ConclusionsBased on the genome and transcriptome analyses, we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a high-yield strain of TNMs based on CB03234-S. Our study establishes a solid basis for the clinical development of AFE-based ADCs.

Escalating Dose Immunization Regimen in Non-Human Primates to Improve Immunogen-Specific Responses

Abstract HIV-1 remains a major health threat, highlighting the need for a vaccine that induces broadly neutralizing antibodies. Epitope-based vaccine designs that focus humoral responses to vulnerable sites on the HIV-1 Env trimer protein are promising approaches, including those targeting the HIV-1 fusion peptide (FP). Escalating dose (ED) immunizations are a strategy to increase immunogen uptake into germinal centers and boost affinity maturation of the antiviral antibody response. In this study, we tested the impact of an ED immunization scheme on enhancing the FP-directed humoral response. Five groups of non-human primates were immunized: four received an ED immunization of either FP or a cocktail of FP+trimer, and one control group was dosed with a bolus FP+trimer cocktail. Two ED groups were adjuvanted with SMNP. The remaining groups used 3M052/alum. Fourteen weeks post-immunization, all ED groups had higher anti-FP IgG titers compared to the control, with the cocktail ED group adjuvanted with 3M052/alum characterized by 14.5-fold higher titers than the bolus control. While the ED cocktail and bolus groups had similar anti-trimer IgG titers, the ED groups had a higher non-base trimer response, shown by an 8.6-fold IgG increase between the 3M052/alum cocktail ED group and its bolus control. ED prime seems promising in raising anti-immunogen titers and lowering responses to the artificial trimer base, and further boosts will be administered to elicit serum neutralizing activity.

Antibody Response after Homologous and Heterologous Prime-Boost COVID-19 Vaccination in a Bangladeshi Residential University Cohort.

COVID-19 vaccination strategies, including heterologous prime-boost regimens and additional booster doses, aim to optimize immune responses. However, seroepidemiological studies on immune responses to different COVID-19 vaccine types and schedules remain limited. This study investigated antibody levels following homologous and heterologous prime-and-boost COVID-19 vaccination in Bangladesh. In a cohort of 606 participants who received first/second/booster doses of vaccines (AstraZeneca, Moderna, Pfizer-BioNTech, and Sinopharm), anti-spike IgG and anti-nucleocapsid IgG levels were measured. Antibody titer variations with respect to age, gender, intervals between doses, and prior infection status were analyzed. mRNA vaccines elicited the highest antibody levels after homologous and heterologous boosting. The AstraZeneca booster resulted in a sharp titer decline rate of ~0.04 units per day. Second or booster vaccine doses significantly increased antibody levels, especially in males (p < 0.05). Older age correlated with higher titers, likely reflecting previous infection, which was further confirmed by the elevation of anti-nucleocapsid IgG levels. About 95.5% of non-Sinopharm recipients were anti-nucleocapsid IgG positive, suggesting prior exposure exceeding self-reported infections (12.5%). mRNA and heterologous COVID-19 boosting enhances humoral immunity over homologous prime-boost vector/inactivated vaccination. However, waning immunity merits further investigation across vaccine platforms.

Understanding the dewatering of fermentation-based 1,3-propanediol with acetone before cyclohexanone synthesis

Acetone can be produced by microbial fermentation using Clostridium acetobutylicum (acetone-n-butanol-ethanol fermentation, ABE fermentation), but it provides a mixture of acetone, 1-butanol, and ethanol. N-butanol and ethanol can be used as biofuels, but high-value applications of acetone generally require upgrading. Bio-based 1,3-propanediol is more readily produced by fermentation and separated at higher titers and has been used industrially. Therefore, the coupled upgrading of the two fermentation products can provide another avenue for high-value utilization of acetone. In this paper, salting-out extraction of 1,3-propanediol with acetone was carried out, and 1,3-propanediol and acetone would spontaneously form an organic phase, thus reducing the energy consumption in the 1,3-propanediol separation process. As the double alkylation reaction of acetone with equivalent 1,3-propanediol would generate cyclohexanone, the extraction of 1,3-propanediol with acetone can provide new insight into the process intensification (removing water, salt and water concentrated in the aqueous phase) for the 1,3-propanediol separation and conversion.

Efficient Conversion of Stevioside to Rebaudioside M in Saccharomyces cerevisiae by a Engineering Hydrolase System and Prolonging the Growth Cycle.

Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in Stevia rebaudiana and low catalytic capacity of the glycosyltransferases in heterologous microorganisms limit its production. In order to improve the catalytic efficiency of the conversion of stevioside to Reb M by Saccharomyces cerevisiae, several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase SCW2 was knocked out in S. cerevisiae. The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from S. rebaudiana was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing UGP1, and co-expressing UGT91D2 and UGT76G1 achieved efficient conversion of stevioside to Reb M. In order to extend the catalytic process, the silencing information regulator 2 (SIR2) which can prolong the growth cycle of S. cerevisiae was introduced. Finally, combining these modifications produced 12.5 g/L Reb M and the yield reached 77.9% in a 5 L bioreactor with 10.0 g/L stevioside, the highest titer from steviol glycosides to Reb M reported to date. The engineered strain could facilitate the industrial production of Reb M, and the strategies provide references for the production of steviol glycosides.

Seasonal Variation in Grapevine Red Blotch Virus Titer in Relation to Disease Symptom Expression in Vineyards

Grapevine red blotch virus (GRBV) is the causative agent of red blotch disease. Limited information is available on the seasonal variation of GRBV titer in relation to disease symptom expression in vineyards across the United States. In this study, no statistically significant difference in GRBV titer was found among asymptomatic infected vines in June ( P = 0.451) and among symptomatic infected vines in October ( P = 0.068) in a diseased ‘Cabernet franc’ vineyard in California, regardless of the years symptomatic, one to seven, as shown by qPCR. Similarly, there were no statistically significant differences in GRBV titer as it related to isolates of the two phylogenetic clades in asymptomatic infected ‘Cabernet franc’ and ‘Cabernet Sauvignon’ vines in June ( P = 0.138 and P = 0.778, respectively) and in symptomatic infected vines in October ( P = 0.806 and P = 0.490, respectively). GRBV titer differed among cultivars in diseased California vineyards ( P &lt; 0.001) and increased over the course of the growing season in infected ‘Merlot’ and ‘Cabernet franc’ vines, but not in ‘Cabernet Sauvignon’ vines. Patterns observed in California were consistent in New York and Georgia vineyards. In a Geneva double curtain-trellised ‘Cabernet Sauvignon’ vineyard in Georgia, GRBV distribution was uneven between cordons, and virus titer was variable within the vine canopy in June ( P = 0.017) but not in October ( P = 0.107). This work revealed consistent patterns of GRBV titer during a growing season in different vineyards across the United States. It also highlighted relatively high virus titer in symptomless grapevines in June, when Spissistilus festinus-mediated GRBV transmission is documented in northern California.