-
Protein Science : a Publication of the... Jun 2024De novo protein design expands the protein universe by creating new sequences to accomplish tailor-made enzymes in the future. A promising topology to implement diverse...
De novo protein design expands the protein universe by creating new sequences to accomplish tailor-made enzymes in the future. A promising topology to implement diverse enzyme functions is the ubiquitous TIM-barrel fold. Since the initial de novo design of an idealized four-fold symmetric TIM barrel, the family of de novo TIM barrels is expanding rapidly. Despite this and in contrast to natural TIM barrels, these novel proteins lack cavities and structural elements essential for the incorporation of binding sites or enzymatic functions. In this work, we diversified a de novo TIM barrel by extending multiple βα-loops using constrained hallucination. Experimentally tested designs were found to be soluble upon expression in Escherichia coli and well-behaved. Biochemical characterization and crystal structures revealed successful extensions with defined α-helical structures. These diversified de novo TIM barrels provide a framework to explore a broad spectrum of functions based on the potential of natural TIM barrels.
Topics: Models, Molecular; Escherichia coli; Crystallography, X-Ray; Protein Folding; Protein Engineering; Proteins
PubMed: 38723111
DOI: 10.1002/pro.5001 -
BioRxiv : the Preprint Server For... Dec 2023Transmembrane β-barrels (TMBs) are widely used for single molecule DNA and RNA sequencing and have considerable potential for a broad range of sensing and sequencing...
Transmembrane β-barrels (TMBs) are widely used for single molecule DNA and RNA sequencing and have considerable potential for a broad range of sensing and sequencing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels such as CsgG, which have evolved to carry out functions very different from sensing, and hence provide sub-optimal starting points. In contrast, protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to the design of transmembrane β-barrel pores with different diameter and pore geometry. NMR and crystallographic characterization shows that the designs are stably folded with structures close to the design models. We report the first examples of designed TMBs with 10, 12 and 14 stranded β-barrels. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 pS (~0.5 nm pore diameter) to 430 pS (~1.1 nm pore diameter), and can be converted into sensitive small-molecule sensors with high signal to noise ratio. The capability to generate on demand β-barrel pores of defined geometry opens up fundamentally new opportunities for custom engineering of sequencing and sensing technologies.
PubMed: 38187764
DOI: 10.1101/2023.12.20.572500 -
Frontiers in Zoology Jun 2024Many questions in biology benefit greatly from the use of a variety of model systems. High-throughput sequencing methods have been a triumph in the democratization of... (Review)
Review
Many questions in biology benefit greatly from the use of a variety of model systems. High-throughput sequencing methods have been a triumph in the democratization of diverse model systems. They allow for the economical sequencing of an entire genome or transcriptome of interest, and with technical variations can even provide insight into genome organization and the expression and regulation of genes. The analysis and biological interpretation of such large datasets can present significant challenges that depend on the 'scientific status' of the model system. While high-quality genome and transcriptome references are readily available for well-established model systems, the establishment of such references for an emerging model system often requires extensive resources such as finances, expertise and computation capabilities. The de novo assembly of a transcriptome represents an excellent entry point for genetic and molecular studies in emerging model systems as it can efficiently assess gene content while also serving as a reference for differential gene expression studies. However, the process of de novo transcriptome assembly is non-trivial, and as a rule must be empirically optimized for every dataset. For the researcher working with an emerging model system, and with little to no experience with assembling and quantifying short-read data from the Illumina platform, these processes can be daunting. In this guide we outline the major challenges faced when establishing a reference transcriptome de novo and we provide advice on how to approach such an endeavor. We describe the major experimental and bioinformatic steps, provide some broad recommendations and cautions for the newcomer to de novo transcriptome assembly and differential gene expression analyses. Moreover, we provide an initial selection of tools that can assist in the journey from raw short-read data to assembled transcriptome and lists of differentially expressed genes.
PubMed: 38902827
DOI: 10.1186/s12983-024-00538-y -
Human Reproduction (Oxford, England) Sep 2023What are the differences in gene expression of cumulus cells (CCs) between young women with diminished ovarian reserve (DOR) and those of similar age with normal ovarian...
STUDY QUESTION
What are the differences in gene expression of cumulus cells (CCs) between young women with diminished ovarian reserve (DOR) and those of similar age with normal ovarian reserve (NOR)?
SUMMARY ANSWER
Gene expression and metabolome profiling analysis demonstrate that the de novo serine synthesis pathway (SSP) is increased in the CCs of young women with DOR.
WHAT IS KNOWN ALREADY
The incidence of DOR has risen, tending to present at younger ages. Its mechanisms and aetiologies are still poorly understood. Abnormal metabolism is present in luteinized CCs of patients with DOR. Previous studies have revealed that mitochondrial dysfunction and impaired oxidative phosphorylation in CCs are related to DOR in women of advanced age. The pathogenic mechanisms likely differ between young women with DOR and cases associated with advanced maternal age. Several studies have examined amino acid metabolism in the follicle, with a focus on embryo development, but less information is available about CCs. The physiological significance of de novo serine synthesis in follicles and oocytes remains largely unknown.
STUDY DESIGN, SIZE, DURATION
CC samples were obtained from 107 young infertile women (age <38 years) undergoing ICSI, from July 2017 to June 2019, including 54 patients with DOR and 53 patients with NOR.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Oocyte development data were analysed retrospectively. Comprehensive genome-wide transcriptomics of CCs was performed. Differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to categorize the functions of the DEGs and identify significantly enriched pathways. The transcript and protein levels of key enzymes involved in serine synthesis were verified in additional samples using quantitative real-time PCR (qRT-PCR) (n = 10) and capillary western blotting (n = 36). Targeted metabolomics of amino acids in CC extracts was performed by ultrahigh-performance liquid MS (UHPLC-MS/MS).
MAIN RESULTS AND THE ROLE OF CHANCE
The number of oocytes (2.4 ± 2.2 versus 12.1 ± 5.3) and metaphase II oocytes (2.1 ± 2.0 versus 9.9 ± 4.9) retrieved was significantly decreased in the DOR versus the NOR group, respectively (P < 0.0001). The rates of fertilization (80.7% versus 78.8%), viable embryos (73.7% versus 72.5%), and high-quality embryos (42.8% versus 49.0%) did not differ between the DOR and NOR groups, respectively (P > 0.05). A total of 95 DEGs were found by transcriptome sequencing. GO and KEGG analyses demonstrated that the DEGs were linked to amino acid metabolism and suggested significantly higher activity of the de novo SSP in the CCs of young women with DOR. Further qRT-PCR and capillary western blotting revealed that key enzymes (PHGDH, PSAT1, PSPH, and SHMT2) involved in de novo serine synthesis were upregulated, and UHPLC-MS/MS analysis showed increases in serine and glycine (a downstream product of serine) levels in the CCs of young patients with DOR. Our data clearly demonstrate that the de novo SSP, which diverts 3-phosphoglycerate from glycolysis to serine synthesis, was upregulated in young DOR CCs.
LARGE SCALE DATA
N/A.
LIMITATIONS, REASONS FOR CAUTION
Regarding the reproductive capacity of young patients DOR, the pregnancy outcomes were not analysed. The sample size was limited, and only women undergoing ICSI were examined since this was a prerequisite for the acquisition of CCs, which may cause selection bias. The exact mechanisms by which the SSP in CCs regulates ovarian reserve still require further study.
WIDER IMPLICATIONS OF THE FINDINGS
Our research presents new evidence that alterations of the SSP in CCs of young infertile women are associated with DOR. We believe this is a significant contribution to the field, which should be key for understanding the cause and mechanisms of ovarian hypofunction in young women.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by grants from the Ministry of Science and Technology of China (2018YFC1005001) and National Natural Science Foundation of China (31601197). There were no competing interests.
TRIAL REGISTRATION NUMBER
N/A.
Topics: Pregnancy; Humans; Female; Infertility, Female; Cumulus Cells; Retrospective Studies; Ovarian Reserve; Serine; Tandem Mass Spectrometry; Oocytes; Ovarian Diseases
PubMed: 37533289
DOI: 10.1093/humrep/dead155 -
International Journal of Molecular... Feb 2024The worldwide agricultural system confronts a significant challenge represented by the increasing demand for food in the face of a growing global population. This... (Review)
Review
The worldwide agricultural system confronts a significant challenge represented by the increasing demand for food in the face of a growing global population. This challenge is exacerbated by a reduction in cultivable land and the adverse effects of climate change on crop yield quantity and quality. Breeders actively embrace cutting-edge omics technologies to pursue resilient genotypes in response to these pressing issues. In this global context, new breeding techniques (NBTs) are emerging as the future of agriculture, offering a solution to introduce resilient crops that can ensure food security, particularly against challenging climate events. Indeed, the search for domestication genes as well as the genetic modification of these loci in wild species using genome editing tools are crucial steps in carrying out de novo domestication of wild plants without compromising their genetic background. Current knowledge allows us to take different paths from those taken by early Neolithic farmers, where crop domestication has opposed natural selection. In this process traits and alleles negatively correlated with high resource environment performance are probably eradicated through artificial selection, while others may have been lost randomly due to domestication and genetic bottlenecks. Thus, domestication led to highly productive plants with little genetic diversity, owing to the loss of valuable alleles that had evolved to tolerate biotic and abiotic stresses. Recent technological advances have increased the feasibility of de novo domestication of wild plants as a promising approach for crafting optimal crops while ensuring food security and using a more sustainable, low-input agriculture. Here, we explore what crucial domestication genes are, coupled with the advancement of technologies enabling the precise manipulation of target sequences, pointing out de novo domestication as a promising application for future crop development.
Topics: Domestication; Plant Breeding; Crops, Agricultural; Agriculture; Gene Editing
PubMed: 38397047
DOI: 10.3390/ijms25042374 -
Oncology Reports Sep 2023Systemic therapy has been viewed as the mainstay for metastatic breast cancer (dnMBC). However, as dnMBC is highly heterogeneous both biologically and clinically, and... (Randomized Controlled Trial)
Randomized Controlled Trial
Systemic therapy has been viewed as the mainstay for metastatic breast cancer (dnMBC). However, as dnMBC is highly heterogeneous both biologically and clinically, and with ever-improving systemic strategies, it has been implied that the local therapy of the primary tumor (PT) may be beneficial for certain patients with dnMBC. However, the results from retrospective studies have been questioned due to their selection bias and retrospective nature. To the best of our knowledge, there are two published randomized clinical trials addressing this issue with conflicting conclusions: i) TATA study from India indicated no overall survival (OS) superiority with early local radiotherapy (LRT); and ii) MF07-01 indicated a 5-year OS rate improvement of 17% with upfront LRT. The updated results of a randomized phase III ECOG-ACRIN E2108 trial released in the 2020 American Society of Clinical Oncology (ASCO) meeting reported a negative survival effect of early LRT treatment in patients with dnMBC responding to initial systemic treatment, despite LRT significantly reducing the locoregional failure. Thus, a number of issues, such as the exact value of LRT, the optimal means of LRT (surgery and/or RT to the PT), the ideal timing of LRT and the population most likely to benefit from LRT, warrant further investigation. Herein, the related studies focusing on these aspects were comprehensively reviewed and a decision algorithm was proposed to select suitable patients with dnMBC for reasonable LRT. Generally, upfront systemic therapy is recommended. For good respondents and a subgroup of favorable profiles (young age, good general condition, low tumor burden, hormone receptor-positive and so on), radical LRT including PT surgery followed by RT and the resection of distant metastases is recommended. LRT should also be administered if the PT is still symptomatic. LRT may benefit patients with dnMBC due to the following reasons: Control of the PT decreases tumor burden, eliminates the source of dissemination, enhances the sensitivity to therapy and exerts positive immunomodulation. Therefore, the treatment paradigm for dnMBC may change from 'palliative LRT' into 'curative LRT' in a highly selected entity with careful evaluation.
Topics: Humans; Female; Breast Neoplasms; Retrospective Studies; Combined Modality Therapy
PubMed: 37449542
DOI: 10.3892/or.2023.8600 -
Bioresources and Bioprocessing Oct 2023Nervonic acid, a natural fatty acid compound and also a core component of nerve fibers and nerve cells, has been widely used to prevent and treat related diseases of the...
Nervonic acid, a natural fatty acid compound and also a core component of nerve fibers and nerve cells, has been widely used to prevent and treat related diseases of the brain nervous system. At present, fatty acids and their derivatives are mainly obtained by natural extraction or chemical synthesis which are limited by natural resources and production costs. In this study, the de novo synthetic pathway of nervonic acid was constructed in Yarrowia lipolytica by means of synthetic biology, and the yield of nervonic acid was further improved by metabolic engineering and fermentation optimization. Specially, heterologous elongases and desaturases derived from different organism were successfully expressed and evaluated for their potential for the production of nervonic acid in Y. lipolytica. Meanwhile, we overexpressed the genes involved in the lipid metabolism to increase the nervonic acid titer to 111.6 mg/L. In addition, the potential of adding oil as auxiliary carbon sources for nervonic acid production by the engineered Y. lipolytica was analyzed. The results indicated that supplementation with colleseed oil as an auxiliary carbon source can be beneficial for the nervonic acid productivity, which led to the highest concentration of 185.0 mg/L in this work. To summarize, this study describes that the Y. lipolytica can be used as a promising platform for the production of nervonic acid and other very long-chain fatty acids.
PubMed: 38647797
DOI: 10.1186/s40643-023-00689-6 -
International Journal of Molecular... Jan 2024The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly increasing worldwide at an alarming pace, due to an increase in obesity,... (Review)
Review
The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly increasing worldwide at an alarming pace, due to an increase in obesity, sedentary and unhealthy lifestyles, and unbalanced dietary habits. MASLD is a unique, multi-factorial condition with several phases of progression including steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Sterol element binding protein 1c (SREBP1c) is the main transcription factor involved in regulating hepatic de novo lipogenesis. This transcription factor is synthesized as an inactive precursor, and its proteolytic maturation is initiated in the membrane of the endoplasmic reticulum upon stimulation by insulin. SREBP cleavage activating protein (SCAP) is required as a chaperon protein to escort SREBP from the endoplasmic reticulum and to facilitate the proteolytic release of the N-terminal domain of SREBP into the Golgi. SCAP inhibition prevents activation of SREBP and inhibits the expression of genes involved in triglyceride and fatty acid synthesis, resulting in the inhibition of de novo lipogenesis. In line, previous studies have shown that SCAP inhibition can resolve hepatic steatosis in animal models and intensive research is going on to understand the effects of SCAP in the pathogenesis of human disease. This review focuses on the versatile roles of SCAP/SREBP regulation in de novo lipogenesis and the structure and molecular features of SCAP/SREBP in the progression of hepatic steatosis. In addition, recent studies that attempt to target the SCAP/SREBP axis as a therapeutic option to interfere with MASLD are discussed.
Topics: Animals; Humans; Fatty Liver; Lipid Metabolism; Lipogenesis; Liver Neoplasms; Sterol Regulatory Element Binding Protein 1; Intracellular Signaling Peptides and Proteins; Membrane Proteins
PubMed: 38256181
DOI: 10.3390/ijms25021109 -
BioRxiv : the Preprint Server For... Aug 2023The liver coordinates the systemic response to nutrient deprivation and availability by producing glucose from gluconeogenesis during fasting and synthesizing lipids via...
The liver coordinates the systemic response to nutrient deprivation and availability by producing glucose from gluconeogenesis during fasting and synthesizing lipids via lipogenesis (DNL) when carbohydrates are abundant. Mitochondrial pyruvate metabolism is thought to play important roles in both gluconeogenesis and DNL. We examined the effects of hepatocyte-specific mitochondrial pyruvate carrier (MPC) deletion on the fasting-refeeding response. Rates of DNL during refeeding were impaired by liver MPC deletion, but this did not reduce intrahepatic lipid content. During fasting, glycerol is converted to glucose by two pathways; a direct cytosolic pathway essentially reversing glycolysis and an indirect mitochondrial pathway requiring the MPC. MPC deletion reduced the incorporation of C-glycerol into TCA cycle metabolites but not into newly synthesized glucose. However, suppression of glycerol metabolism did not affect glucose concentrations in fasted hepatocyte-specific MPC-deficient mice. Thus, glucose production by kidney and intestine may compensate for MPC deficiency in hepatocytes.
PubMed: 36824879
DOI: 10.1101/2023.02.17.528992 -
Frontiers in Genetics 2023Intellectual disability (ID) is defined by cognitive and social adaptation defects. Variants in the gene, which encodes the brain-specific cytoplasmic protein SYNGAP1,...
Intellectual disability (ID) is defined by cognitive and social adaptation defects. Variants in the gene, which encodes the brain-specific cytoplasmic protein SYNGAP1, are commonly associated with ID. The aim of this study was to identify novel gene variants in Chinese individuals with ID and evaluate the pathogenicity of the detected variants. Whole exome sequencing (WES) was performed on 113 patients diagnosed with ID. In the study, two variants in were identified. Sanger sequencing was used to confirm these variants. Minigene assays were used to verify whether the intronic variant in influenced the normal splicing of mRNA. Two heterozygous pathogenic variants in c.333del and c.664-2A>G, were identified in two ID patients separately. The c.333del variant has been reported previously as a finding in a child with ID, while the c.664-2A>G variant was novel intronic variant, which has not been reported in the literature. Functional studies showed that c.664-2A>G could cause aberrant splicing, resulting in exon 7 skipping and a 16bp deletion within exon 7. We identified two pathogenic heterozygous variants in in two patients with ID, among which the c.664-2A>G variant was a novel pathogenic variant. Our findings further enrich the variant spectrum of the gene and provide a research basis for the genetic diagnosis of ID.
PubMed: 37928246
DOI: 10.3389/fgene.2023.1270175