Hasil untuk "Biotechnology"

Isabella Frighetto Bomfiglio, Isabelli Seiler de Medeiros Mendes, Diego Bonatto

DNA cloning methods are fundamental tools in molecular biology, synthetic biology, and genetic engineering that enable precise DNA manipulation for various scientific and biotechnological applications. This review systematically summarizes the major restriction-free overlapping sequence cloning (RFOSC) techniques currently used in synthetic biology and examines their development, efficiency, practicality, and specific applications. In vitro methods, including Gibson Assembly, Circular Polymerase Extension Cloning (CPEC), Polymerase Incomplete Primer Extension (PIPE), Overlap Extension Cloning (OEC), Flap Endonuclease Cloning (FEN-Cloning), and commercially available techniques such as TOPO and In-Fusion, have been discussed alongside hybrid approaches such as Ligation-Independent Cloning (LIC), Sequence-Independent Cloning (SLIC), and T5 Exonuclease-Dependent Assembly (TEDA). Additionally, in vivo methods leveraging host recombination machinery, including Yeast Homologous Recombination (YHR), In Vivo Assembly (IVA), Transformation-Associated Recombination (TAR), and innovative approaches such as Multiple-Round In Vivo Site-Specific Assembly (MISSA) and Phage Enzyme-Assisted Direct Assembly (PEDA), are critically evaluated. The review highlights that method selection should consider the scale, complexity, cost, and specific needs of individual research projects, noting that no single technique is universally optimal. Future trends suggest the increased integration of enzymatic efficiency, host versatility, and automation, broadening the accessibility and capabilities of DNA assembly technologies.

Natalia Flechas Manrique, Alberto Martínez, Elena López-Martínez et al.

Epitopes are short antigenic peptide sequences which are recognized by antibodies or immune cell receptors. These are central to the development of immunotherapies, vaccines, and diagnostics. However, the rational design of synthetic epitope libraries is challenging due to the large combinatorial sequence space, $20^n$ combinations for linear epitopes of n amino acids, making screening and testing unfeasible, even with high throughput experimental techniques. In this study, we present a large language model, epiGPTope, pre-trained on protein data and specifically fine-tuned on linear epitopes, which for the first time can directly generate novel epitope-like sequences, which are found to possess statistical properties analogous to the ones of known epitopes. This generative approach can be used to prepare libraries of epitope candidate sequences. We further train statistical classifiers to predict whether an epitope sequence is of bacterial or viral origin, thus narrowing the candidate library and increasing the likelihood of identifying specific epitopes. We propose that such combination of generative and predictive models can be of assistance in epitope discovery. The approach uses only primary amino acid sequences of linear epitopes, bypassing the need for a geometric framework or hand-crafted features of the sequences. By developing a method to create biologically feasible sequences, we anticipate faster and more cost-effective generation and screening of synthetic epitopes, with relevant applications in the development of new biotechnologies.

Sebastián Espinel-Ríos, José L. Avalos, Ehecatl Antonio del Rio Chanona et al.

Efficient and robust bioprocess control is essential for maximizing performance and adaptability in advanced biotechnological systems. In this work, we present a reinforcement-learning framework for multi-setpoint and multi-trajectory tracking. Tracking multiple setpoints and time-varying trajectories in reinforcement learning is challenging due to the complexity of balancing multiple objectives, a difficulty further exacerbated by system uncertainties such as uncertain initial conditions and stochastic dynamics. This challenge is relevant, e.g., in bioprocesses involving microbial consortia, where precise control over population compositions is required. We introduce a novel return function based on multiplicative reciprocal saturation functions, which explicitly couples reward gains to the simultaneous satisfaction of multiple references. Through a case study involving light-mediated cybergenetic growth control in microbial consortia, we demonstrate via computational experiments that our approach achieves faster convergence, improved stability, and superior control compliance compared to conventional quadratic-cost-based return functions. Moreover, our method enables tuning of the saturation function's parameters, shaping the learning process and policy updates. By incorporating system uncertainties, our framework also demonstrates robustness, a key requirement in industrial bioprocessing. Overall, this work advances reinforcement-learning-based control strategies in bioprocess engineering, with implications in the broader field of process and systems engineering.

Zheng Gong, Ziyi Jiang, Weihao Gao et al.

The mRNA optimization is critical for therapeutic and biotechnological applications, since sequence features directly govern protein expression levels and efficacy. However, current methods face significant challenges in simultaneously achieving three key objectives: (1) fidelity (preventing unintended amino acid changes), (2) computational efficiency (speed and scalability), and (3) the scope of optimization variables considered (multi-objective capability). Furthermore, existing methods often fall short of comprehensively incorporating the factors related to the mRNA lifecycle and translation process, including intrinsic mRNA sequence properties, secondary structure, translation elongation kinetics, and tRNA availability. To address these limitations, we introduce \textbf{RNop}, a novel deep learning-based method for mRNA optimization. We collect a large-scale dataset containing over 3 million sequences and design four specialized loss functions, the GPLoss, CAILoss, tAILoss, and MFELoss, which simultaneously enable explicit control over sequence fidelity while optimizing species-specific codon adaptation, tRNA availability, and desirable mRNA secondary structure features. Then, we demonstrate RNop's effectiveness through extensive in silico and in vivo experiments. RNop ensures high sequence fidelity, achieves significant computational throughput up to 47.32 sequences/s, and yields optimized mRNA sequences resulting in a significant increase in protein expression for functional proteins compared to controls. RNop surpasses current methodologies in both quantitative metrics and experimental validation, enlightening a new dawn for efficient and effective mRNA design. Code and models will be available at https://github.com/HudenJear/RPLoss.

Agata Kryczyk-Poprawa, Wojciech Baran, Katarzyna Sułkowska-Ziaja et al.

Oxybenzone, a common sunscreen ingredient, has been widely detected in various environmental matrices, posing significant ecological and health risks. The present study demonstrates, for the first time, the capacity of <i>Pleurotus djamor</i> to degrade oxybenzone in in vitro cultures. After 14 days of mycelial incubation, oxybenzone concentrations in the medium decreased from 25 mg to 1.5394 ± 0.095 mg. The final amount of oxybenzone in the mycelium after lyophilization was 6.2067 ± 0.2459 mg. Furthermore, oxybenzone addition significantly reduced biomass growth from 2.510 ± 0.6230 g to 1.4697 ± 0.0465 g. The transformation products in the dry mycelium and medium were assessed and identified using UPLC-Q-tof based on monoisotopic molecular mass and fragmentation spectra. In processes initiated by <i>P. djamor</i>, mainly acylated derivatives of oxybenzone were formed. Additionally, compounds with thiol and amino groups were identified. Alterations in antioxidant profiles (L-tryptophan, 6-methyl-D,L-tryptophan, p-hydroxybenzoic acid, ergosterol, lovastatin, L-phenylalanine, and ergothioneine) in response to oxybenzone exposure were observed. Our findings reveal significant changes in the antioxidant levels and biomass growth inhibition, underscoring the potential toxicological risks associated with oxybenzone. The observed reduction in oxybenzone concentration highlights the potential of <i>P. djamor</i> as an effective and environmentally friendly strategy for mitigating this pollutant.

Tam Dinh Le Vo, Thu Huynh, Thuy Thi Le et al.

Baby clam (Corbiculidae sp.) meat is served as a traditional dish in Vietnam, and the antioxidant activity of its protein extract has been discovered. This study evaluates baby clam meat protein hydrolysate's iron-binding capacity (IBC) and antidiabetic activity. Initially, an analysis of the basic chemical composition of the meat was conducted. Subsequently, Alcalase was employed for hydrolysis. The highest IBC and α-amylase inhibition activity were targets for obtaining the best hydrolysis condition, including the clam meat-to-water ratio, enzyme-to-substrate (E:S) ratio, and time. Under the best condition, the hydrolysates demonstrated the IBC of 1246.20 ± 44.00 µg Fe2+/g protein and α-amylase inhibition activity of 48.33 ± 1.44%, approaching three-quarters of the activity of ethylenediaminetetraacetic acid (EDTA) sodium salt and acarbose, respectively. These results served as preliminary data for the development of the protein hydrolysates as a natural iron chelator or α-amylase inhibitor, which could support the treatment of iron deficiency and diabetes.

STEVEN J. ZWEIG

Gianmarco Callegher, Thomas Kneib, Johannes Söding et al.

In structured additive distributional regression, the conditional distribution of the response variables given the covariate information and the vector of model parameters is modelled using a P-parametric probability density function where each parameter is modelled through a linear predictor and a bijective response function that maps the domain of the predictor into the domain of the parameter. We present a method to perform inference in structured additive distributional regression using stochastic variational inference. We propose two strategies for constructing a multivariate Gaussian variational distribution to estimate the posterior distribution of the regression coefficients. The first strategy leverages covariate information and hyperparameters to learn both the location vector and the precision matrix. The second strategy tackles the complexity challenges of the first by initially assuming independence among all smooth terms and then introducing correlations through an additional set of variational parameters. Furthermore, we present two approaches for estimating the smoothing parameters. The first treats them as free parameters and provides point estimates, while the second accounts for uncertainty by applying a variational approximation to the posterior distribution. Our model was benchmarked against state-of-the-art competitors in logistic and gamma regression simulation studies. Finally, we validated our approach by comparing its posterior estimates to those obtained using Markov Chain Monte Carlo on a dataset of patents from the biotechnology/pharmaceutics and semiconductor/computer sectors.

Houjun Zhou, Xueqin Song, Meng-Zhu Lu

IntroductionHybrid poplars are industrial trees in China. An understanding of the molecular mechanism underlying wood formation in hybrid poplars is necessary for molecular breeding. Although the division and differentiation of vascular cambial cells is important for secondary growth and wood formation, the regulation of this process is largely unclear.MethodsIn this study, mPagGRF15 OE and PagGRF15-SRDX transgenic poplars were generated to investigate the function of PagGRF15. RNA-seq and qRT-PCR were conducted to analyze genome-wide gene expression, while ChIP‒seq and ChIP-PCR were used to identified the downstream genes regulated by PagGRF15.Results and discussionWe report that PagGRF15 from hybrid poplar (Populus alba × P. glandulosa), a growth-regulating factor, plays a critical role in the regulation of vascular cambium activity. PagGRF15 was expressed predominantly in the cambial zone of vascular tissue. Overexpression of mPagGRF15 (the mutated version of GRF15 in the miR396 target sequence) in Populus led to decreased plant height and internode number. Further stem cross sections showed that the mPagGRF15 OE plants exhibited significant changes in vascular pattern with an increase in xylem and a reduction in phloem. In addition, cambium cell files were decreased in the mPagGRF15 OE plants. However, dominant suppression of the downstream genes of PagGRF15 using PagGRF15-SRDX showed an opposite phenotype. Based on the RNA-seq and ChIP-seq results, combining qRT-PCR and ChIP-PCR analysis, candidate genes, such as WOX4b, PXY and GID1.3, were obtained and found to be mainly involved in cambial activity and xylem differentiation. Accordingly, we speculated that PagGRF15 functions as a positive regulator mediating xylem differentiation by repressing the expression of the WOX4a and PXY genes to set the pace of cambial activity. In contrast, PagGRF15 mediated the GA signaling pathway by upregulating GID1.3 expression to stimulate xylem differentiation. This study provides valuable information for further studies on vascular cambium differentiation mechanisms and genetic improvement of the specific gravity of wood in hybrid poplars.

Steven J. Zweig

Steven J. Zweig

Nosheen Masood, San-Gang Wu

Zhiwei Jiang, Na Li, Qin Shao et al.

Abstract Cell aggregates that mimic in vivo cell–cell interactions are promising and powerful tools for tissue engineering. This study isolated a new, easily obtained, population of mesenchymal stem cells (MSCs) from rat hard palates named hard palatal‐derived mesenchymal stem cells (PMSCs). The PMSCs were positive for CD90, CD44, and CD29 and negative for CD34, CD45, and CD146. They exhibited clonogenicity, self‐renewal, migration, and multipotent differentiation capacities. Furthermore, this study fabricated scaffold‐free 3D aggregates using light‐controlled cell sheet technology and a serum‐free method. PMSC aggregates were successfully constructed with good viability. Transplantation of the PMSC aggregates and the PMSC aggregate‐implant complexes significantly enhanced bone formation and implant osseointegration in vivo, respectively. This new cell resource is easy to obtain and provides an alternative strategy for tissue engineering and regenerative medicine.

Renan dos Reis, Luciano da F. Costa

There is a crescent use of enzymes in multiple industries and sciences, ranging from materials and fuel synthesis to pharmaceutical and food production. Their applicability in this variety of fields depends not only on their biochemical function but also on their physicochemical properties. In the present work, we describe how the coincidence methodology can be employed to construct similarity networks of seventy well-studied enzymes of the Glycoside Hydrolase Family 13 and to identify communities of physicochemically related enzymes. More specifically, each of the selected enzymes is mapped into a network node, while the links between pairs of enzymes are determined by the coincidence similarity between selected physicochemical features of interest. The obtained networks have modularity and number of isolated nodes optimized respectively to two parameters involved in the coincidence methodology, resulting in highly modular networks. In order to investigate the effect of the considered physicochemical features on the enzymes relationships, the coincidence-based method also is applied to create a meta-network, in which the enzymes similarity networks obtained by the combination of every possible feature becomes nodes of a feature combination network, and the coincidence similarity between those networks defines the respective links. The obtained feature combination network systematically and comprehensively indicates the impact of the selected physicochemical features on enzyme similarity. Several interesting results are reported and discussed, including the identification of subgroups of enzymes with similar physicochemical features within catalytical classes, providing important information for the selection and design of enzymes for targeted biotechnological applications.

Halie M. Rando, Ronan Lordan, Likhitha Kolla et al.

In the 21st century, several emergent viruses have posed a global threat. Each pathogen has emphasized the value of rapid and scalable vaccine development programs. The ongoing SARS-CoV-2 pandemic has made the importance of such efforts especially clear. New biotechnological advances in vaccinology allow for recent advances that provide only the nucleic acid building blocks of an antigen, eliminating many safety concerns. During the COVID-19 pandemic, these DNA and RNA vaccines have facilitated the development and deployment of vaccines at an unprecedented pace. This success was attributable at least in part to broader shifts in scientific research relative to prior epidemics; the genome of SARS-CoV-2 was available as early as January 2020, facilitating global efforts in the development of DNA and RNA vaccines within two weeks of the international community becoming aware of the new viral threat. Additionally, these technologies that were previously only theoretical are not only safe but also highly efficacious. Although historically a slow process, the rapid development of vaccines during the COVID-19 crisis reveals a major shift in vaccine technologies. Here, we provide historical context for the emergence of these paradigm-shifting vaccines. We describe several DNA and RNA vaccines and in terms of their efficacy, safety, and approval status. We also discuss patterns in worldwide distribution. The advances made since early 2020 provide an exceptional illustration of how rapidly vaccine development technology has advanced in the last two decades in particular and suggest a new era in vaccines against emerging pathogens.

Zhi Li, Minyu Zhou, Yao Ruan et al.

The Pacific white shrimp Litopenaeus vannamei is an economically important penaeid species worldwide. Under farming conditions, the full ovarian maturation of L. vannamei generally depends on the combination of artificial ablation of unilateral eyestalk and feeding high-quality natural diets, suggesting that nutrient accumulation is important to ovarian maturation. In this study, we aimed to investigate the gene expression related to nutritional accumulation in L. vannamei during ovarian development by transcriptomic analysis. A total of 52.45 Gb of high-quality transcriptome data were obtained from 8 samples from the hepatopancreas and ovaries from shrimp in gonadal developmental stages I-IV. A total of 23,149 expressed genes were detected, of which 19,852 were known genes and 3,297 were novel genes. Our study found that genes related to amino acid, carbohydrate and lipid metabolism were expressed at higher levels in the hepatopancreas than in the ovary. The genes for most lipoproteins and their receptors were predominantly expressed in the hepatopancreas, while vitellogenin receptor (VgR) was specifically expressed in the ovary. Moreover, two vitellogenin (Vg) genes were identified, in which one was specifically expressed in the hepatopancreas and defined as Hp-Vg, and the other was specifically expressed in the ovary and defined as Ov-Vg. In addition, genes related to carotenoid metabolism were enriched in the hepatopancreas. This study provides solid evidence that the hepatopancreas is the major exogenous vitellogenesis site for ovarian maturation of L. vannamei. Two distinct Vg genes perform exogenous and endogenous vitellogenesis in the hepatopancreas and ovary in L. vannamei, respectively. The results of this study also presented some new insights for understanding the nutritional dynamics from the hepatopancreas to ovary during ovarian maturation in penaeids.

Guangming Shao, Tianliang He, Yinnan Mu et al.

Summary: How organisms cope with coldness and high pressure in the hadal zone remains poorly understood. Here, we sequenced and assembled the genome of hadal sea cucumber Paelopatides sp. Yap with high quality and explored its potential mechanisms for deep-sea adaptation. First, the expansion of ACOX1 for rate-limiting enzyme in the DHA synthesis pathway, increased DHA content in the phospholipid bilayer, and positive selection of EPT1 may maintain cell membrane fluidity. Second, three genes for translation initiation factors and two for ribosomal proteins underwent expansion, and three ribosomal protein genes were positively selected, which may ameliorate the protein synthesis inhibition or ribosome dissociation in the hadal zone. Third, expansion and positive selection of genes associated with stalled replication fork recovery and DNA repair suggest improvements in DNA protection. This is the first genome sequence of a hadal invertebrate. Our results provide insights into the genetic adaptations used by invertebrate in deep oceans.

I. A. Kirillov, A. P. Pirozhkov, V. V. Rubtsov et al.

In November 2020, the Armed Forces of the Russian Federation began mass immunisation of the personnel with Gam-COVID-Vac (Sputnik V), the first Russia vaccine against the new coronavirus infection (COVID-19). Thus, it became necessary to assess post-vaccination antibody levels and the duration and intensity of humoral immunity to COVID-19.The aim of the study was to investigate the immunogenicity and efficacy of Gam-COVID-Vac in military medical staff after vaccination.Materials and methods: the authors determined the presence of specific antibodies in the serum of individuals immunised with Gam-COVID-Vac (477 volunteers) and COVID-19 convalescents (73 patients), using virus neutralisation (VN), enzyme-linked immunosorbent assay (ELISA) with reagent kits by several manufacturers, and immunoblotting. The results of the study were evaluated using analysis of variance.Results: VN detected virus neutralising antibodies in 90.7% of vaccinated subjects; ELISA, in 95.4%. Both VN and ELISA showed lower antibody levels in the vaccinated over 50 years of age. ELISA demonstrated a significantly higher concentration of anti-SARS-CoV-2 spike IgG in the Gam-COVID-Vac group than in the COVID-19 convalescent group. The correlation between antibody detection results by VN and ELISA was the strongest when the authors used their experimental reagent kit for quantitative detection of virus neutralising antibodies by competitive ELISA with the recombinant human ACE2 receptor. Having analysed the time course of neutralising antibody titres, the authors noted a significant, more than two-fold decrease in geometric means of the titres three months after administration of the second vaccine component.Conclusions: the subjects vaccinated with Gam-COVID-Vac gain effective humoral immunity to COVID-19. The decrease in titres indicates the need for revaccination in 6 months.

Toko Tokunaga, Koji Hagihara, Michiaki Yamasaki et al.

The variation in the mechanical properties with the volume fraction of the long-period stacking ordered (LPSO) phase in directionally solidified (DS) Mg/LPSO two-phase alloys was examined. Unexpectedly, the yield stress of the DS alloys increases non-monotonically with an increase in the volume fraction of the LPSO phase. The LPSO phase is considered an effective strengthening phase in Mg alloys, when the stress is applied parallel to the growth direction. Nevertheless, the highest strength was obtained in alloys with 61–86 vol.% of the LPSO phase, which was considerably higher than that in the LPSO single-phase alloy. It was clarified that this complicated variation in the yield stress was generated from the change in the formation stress of kink bands, which varied with the thickness of the LPSO-phase grains. Furthermore, the coexistence of Mg in the LPSO phase alloy induced the homogeneous formation of kink bands in the alloys, leading to the enhancement of the ‘kink-band strengthening’. The results demonstrated that microstructural control is significantly important in Mg/LPSO two-phase alloys, in which both phases exhibit strong plastic anisotropy, to realize the maximum mechanical properties.