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Triple-negative breast cancer (TNBC) doesn't have well-defined molecular targets making it unable to treat with chemotherapy also have faster metastatic rate and worse survival rate. In the current study we aim to target TNBC through eco-friendly green synthesized silver nanoparticles having anti-cancer phytoconstituents from the traditional anti-cancer medicinal plant Eclipta alba. Green synthesized silver nanoparticles (AgNPs) are agglomerates of spherical shaped 40-60 nm sized showing characteristic light absorption at 437 nm, banding pattern at 1479, 1285, 1036, and 471 showing and further X-ray diffraction pattern confirm face-centered cubic crystal silver structure exist in the green synthesized silver nanoparticle preparation. Further in vitro anti-oxidant analysis results revealed that green synthesized AgNPs showed 2.6-fold higher anti-oxidant potential (IC50 15.70 g/ml) than that of aqueous plant leaf extract (IC50 39.80 g/ml). In MTT cytotoxicity analysis Eclipta alba plant extract and AgNPs both display dose-dependent cytotoxicity against triple-negative breast cancer cells (MDA-MB-231), although their IC 50 values differ substantially, at 105.80 µg/ml and 77.20 g/ml, respectively. Finally, AgNPs from Eclipta alba tested for anti-leishmanial activity and it showed 91.36 ± 1.05 for promastigotes and 76.62 ± 0.95 for amastigotes at the highest dose of 400 g/ml. Altogether present data showed that Eclipta alba leaf extract actively bonded with silver nanoparticles suppresses the MDA-MB-231 cells growth through high antioxidant characters and anti-leishmanial activity. From together we confirm that Eclipta alba was recommended to a future therapeutic drug and agent to control breast cancer in the clinical level., Competing Interests: Declarations Conflict of interest All authors declare that they have no conflict of interest., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Astaxanthin, a lipophilic carotenoid renowned for its strong antioxidant activity, holds significant commercial value across industries such as feed, food, and cosmetics. Although astaxanthin can be synthesized through chemical methods, it may contain toxic by-products in the synthesized astaxanthin, limiting its application in medicine or functional food. Natural astaxanthin can be extracted from algae, however, the cultivation cycle of algae is relatively longer compared to microorganisms. With the advancement of synthetic biology and metabolic engineering, the method of microbial fermentation has emerged as a promising strategy for the large-scale production of astaxanthin. This article provides a comprehensive overview of the research progress in astaxanthin biosynthesis, highlighting the use of the natural host Xanthophyllomyces dendrorhous, and the heterologous hosts Yarrowia lipolytica and Saccharomyces cerevisiae. Additionally, future research prospects are also discussed., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The reconstruction and analysis of the protein-protein interaction (PPI) network is a powerful approach to understand the complex biological and molecular functions in normal and disease states of the cell. The interactome of most organisms is largely unidentified except some model organisms. The current study focused on the construction of PPI network for the human pathogen Mycobacterium tuberculosis (MTB)-resistant strain XDR1219 using computational methods. In this work, a bioinformatics approach was employed to reveal potential drug targets. The pipeline adopted the combination of an extensive integrated network analysis that led to identify 22 key proteins involved in drug resistance, resistant metabolic pathways, virulence, pathogenesis and persistency of the infection. The MTB XDR1219 interactome consists of 11,383 non-redundant PPIs among 1499 proteins covering 38% of the entire MTB XDR1219 proteome. The overall quality of the network was assessed and topological parameters of the PPI were calculated. The predicted interactions were functionally annotated and their relevance was assessed with the functional similarity. The study attempts to present the interactome of previously unidentified MTB XDR1219 and revealed potential drug targets that can be further explored by scientific community., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Salvia hispanica (chia) is the highest reported terrestrial plant source of alpha-linolenic acid (ALA, ~ 65%), an ω-3 polyunsaturated fatty acid with numerous health benefits. The molecular basis of high ALA accumulation in chia is yet to be understood. We have identified lysophosphatidylcholine acyltransferase (LPCAT) gene from the developing seed transcriptome data of chia and carried out its biochemical characterization through heterologous expression in Saccharomyces cerevisiae. Expression profiling showed that the enzyme was active throughout the seed development, indicating a pivotal role in oil biosynthesis. The enzyme could utilize both saturated and unsaturated lysophosphatidylcholine substrates at the same rate, to synthesize phosphatidylcholine (PC). The enzyme also exhibited lysophosphatidic acid acyltransferase (LPAAT) activity, by preferring lysophosphatidic acid substrate. Substrate specificity studies showed that the enzyme preferred both monounsaturated and polyunsaturated fatty acyl CoAs over saturated CoAs. This activity may play a key role in enriching the PC fraction with polyunsaturated fatty acids (PUFAs). PUFAs present on PC can be transferred to oil through the action of other acyltransferases. Our results describe a new LPCAT enzyme that can be used to biotechnologically alter oilseed crops to incorporate more PUFA in its seed oil., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The utilization of heterologous 5' untranslated regions (5'UTRs) for expressing foreign proteins in the chloroplast of Chlamydomonas reinhardtii (C. reinhardtii) has posed a persistent challenge over the years. This challenge stems from the lack of a defined and comprehensive set of translational cis-elements responsible for stability, ribosome binding, and translation initiation, which are mediated by trans-acting factors native to C. reinhardtii. In the current study, we aimed to address this bottleneck by employing the 5'UTR from gene 10 of the T7 bacteriophage (T7g10 5'UTR), fused to the promoter of C. reinhardtii small subunit ribosomal RNA (rrnS), to facilitate the translation of a reporter gene, YFP. Using a chimeric construct, the YFP mRNA was efficiently translated utilizing the heterologous T7g10 5'UTR. Furthermore, the accumulation of YFP protein under the control of the T7g10 5'UTR was approximately one third of that observed under the control of the endogenous psaA promoter/5'UTR in the C. reinhardtii chloroplast. The results of computational analyses demonstrated that the T7g10 5'UTR sequence shares common elements with the endogenous 5'UTRs of the chloroplast genes. Moreover, the findings of the current study highlighted the potential of employing bacteriophage 5'UTRs for the foreign protein accumulation from the chloroplast genome of C. reinhardtii., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Heterologous functional expression of the recombinant lipases is typically a bottleneck due to the expression in the insoluble fraction as inclusion bodies (IBs) which are in inactive form. Due to the importance of lipases in various industrial applications, many investigations have been conducted to discover suitable approaches to obtain functional lipase or increase the expressed yield in the soluble fraction. The utilization of the appropriate prokaryotic and eukaryotic expression systems, along with the suitable vectors, promoters, and tags, has been recognized as a practical approach. One of the most powerful strategies to produce bioactive lipases is using the molecular chaperones co-expressed along with the target protein's genes into the expression host to produce the lipase in soluble fraction as a bioactive form. The refolding of expressed lipase from IBs (inactive) is another practical strategy which is usually carried out through chemical and physical methods. Based on recent investigations, the current review simultaneously highlights strategies to express the bioactive lipases and recover the bioactive lipases from the IBs in insoluble form., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Natural peptides have been the source of some important tools to address challenges in protein therapy of diseases. Bypassing cell plasma membrane has been a bottleneck in the intracellular delivery of biomolecules. Among others, cell-penetrating peptides (CPPs) provide an efficient strategy for intracellular delivery of various cargos. Brevinin-2R peptide is an antimicrobial peptide isolated from the skin secretions of marsh frog, Rana ridibunda with semi-selective anticancer properties. Here, we investigated cell-penetrating properties of Brevinin-2R peptide and its ability to deliver functional protein cargos. Bioinformatics studies showed that Brevinin-2R is a cationic peptide with a net charge of + 5 with an alpha-helix structure and a heptameric ring at the carboxylic terminal due to disulfide bond between C19 and C25 amino acids and a hinge region at A10. To evaluate the ability of this peptide as a CPP, β-galactosidase protein and GFP were transfected into HeLa cells. The entry pathway of the peptide/protein complex into the cell was investigated by inhibiting endocytic pathways at 4 °C. It was observed that Brevinin-2R can efficiently transfer β-galactosidase and GFP with 21% and 90% efficacy, respectively. Brevinin-2R opts for endocytosis pathways to enter cells. The cytotoxicity of this peptide against HeLa cells was studied using MTT assay. The results showed that at the concentration of 131.5 μg/ml of Brevinin-2R peptide, the proliferation of 50% of HeLa cells was inhibited. The results of this study suggest that Brevinin-2R peptide can act as a CPP of natural origin and low cytotoxicity., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Research on microbial fatty acid metabolism started in the late 1960s, and till date, various developments have aided in elucidating the fatty acid metabolism in great depth. Over the years, synthesis of microbial fatty acid has drawn industrial attention due to its diverse applications. However, fatty acid overproduction imparts various stresses on its metabolic pathways causing a bottleneck to further increase the fatty acid yields. Numerous strategies to increase fatty acid titres in Escherichia coli by pathway modulation have already been published, but the stress generated during fatty acid overproduction is relatively less studied. Stresses like pH, osmolarity and oxidative stress, not only lower fatty acid titres, but also alter the cell membrane composition, protein expression and membrane fluidity. This review discusses an overview of fatty acid synthesis pathway and presents a panoramic view of various stresses caused due to fatty acid overproduction in E. coli. It also addresses how certain stresses like high temperature and nitrogen limitation can boost fatty acid production. This review paper also highlights the interconnections that exist between these stresses., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The repressor element 1 (RE1) silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) modulates the expression of genes with RE1/neuron-restrictive silencing element (RE1/NRSE) sites by recruiting the switch independent 3 (SIN3) factor and the REST corepressor (COREST) to its N and C-terminal repressor domain, respectively. Both, SIN3 and COREST assemble into protein complexes that are composed of multiple subunits including a druggable histone deacetylase (HDAC) enzyme. The SIN3 core complex comprises the eponymous proteins SIN3A or SIN3B, the catalytically active proteins HDAC1 or HDAC2, the histone chaperone retinoblastoma-associated protein 46/retinoblastoma-binding protein 7 (RBAP46/RBBP7) or RBAP48/RBBP4, the SIN3-associated protein 30 (SAP30), and the suppressor of defective silencing 3 (SDS3). Here, we overcome a bottleneck limiting the molecular characterization of the REST/NRSF-SIN3 transcriptional corepressor complex. To this end, SIN3 genes were amplified from the complementary DNA of neural stem/progenitor cells, and expressed in a baculovirus/insect cell expression system. We show that the isolates bind to DNA harboring RE1/NRSE sites and demonstrate that the histone deacetylase activity is blocked by small-molecule inhibitors. Thus, our isolates open up for future biomedical research on this critical transcriptional repressor complex and are envisioned as tool for drug testing.

The introduction of new genome editing tools such as ZFNs, TALENs and, more recently, the CRISPR/Cas9 system, has greatly expanded the ability to knock-out genes in different animal models, including zebrafish. However, time and costs required for the screening of a huge number of animals, aimed to identify first founder fishes (F0), and then carriers (F1) are still a bottleneck. Currently, high-resolution melting (HRM) analysis is the most efficient technology for large-scale InDels detection, but the very expensive equipment demanded for its application may represent a limitation for research laboratories. Here, we propose a rapid and cheap method for high-throughput genotyping that displays efficiency rate similar to the HRM. In fact, using a common ViiA™7 real-time PCR system and optimizing the parameters of the melting analysis, we demonstrated that it is possible to discriminate between the mutant and the wild type melting curves. Due to its simplicity, rapidity and cheapness, our method can be used as a preliminary one-step approach for massive screening, in order to restrict the scope at a limited number of embryos and to focus merely on them for the next sequencing step, necessary for the exact sequence identification of the induced mutation. Moreover, thanks to its versatility, this simple approach can be readily adapted to the detection of any kind of genome editing approach directed to genes or regulatory regions and can be applied to many other animal models.

Hypermethylated in Cancer-1 (HIC-1) is a novel tumor suppressor plays crucial role in tumor formation through loss of function by hypermethylation. HIC-1 is known as transcriptional factor whereas little known about its structure and function. Requirement felt to clone and express full coding protein and reveal various domains and binding pattern onto promoters conducting biophysical studies which lack in current scenario. Production of sufficient amounts of protein is frequent bottleneck in structural biology. Cloning full-length HIC-1 with >73 % GC content poses a daunting task with sequencing and expression adds more to the challenge. We describe the methodology for specific amplification, cloning, sequencing, and expression of HIC-1 in E. coli. Standardization using 1.5 U pfu polymerase in (NH4)2SO4 containing buffer gave specific amplification with 10 % DMSO and 1.5 mM MgCl2. Sequencing achieved using base analog 7-de aza dGTP (0.2 mM) or denaturant like DMSO (10 %) or betaine (1 M). Expression using strains of E. coli induced by different concentrations of IPTG (0.5-5.0 mM) for time points of 4, 8, 16, 20, and 24 h at different temperatures 25, 30, and 37 °C. Full-length clone successfully expressed in BL21-Codon Plus-RP using 1 mM concentration of IPTG for 8 h at 37 °C gave prominent band of 74 kDa.

Hereditary cancers account for approximately 10 % of breast and ovarian cancers. Mutations of the BRCA1 and BRCA2 genes, encoding two proteins involved in DNA repair, underlie most cases of such hereditary cancers. Women with BRCA mutations develop breast cancer in 50-80 % of cases and ovarian cancer in 10-40 % of cases. Assessing BRCA mutational status is needed to direct the clinical management of women with predisposition to these hereditary cancers. However, BRCA screening constitutes a bottleneck in terms of costs and time to deliver results. We developed a PCR-based assay using 73 primer pairs covering the entire coding regions of BRCA1 and BRCA2. PCR primers, containing at the 5' end the universal M13 primer sequences, were pre-spotted in 96-well plates. Following PCR, direct sequencing was performed using M13 primers, allowing to standardize the conditions. PCR amplification and sequencing were successful for each amplicon. We tested and validated the assay on 10 known gDNAs from patients with Hereditary breast and ovarian cancer (HBOC). Our strategy is a promising time and cost-effective method to detect BRCA mutations in the clinical setting, which is essential to formulate a personalized therapy for patients with HBOC.

The choice of tools that enable efficient targeting of exogenous antigens (Ag) for processing and presentation by professional Ag-presenting cells (APC) remains limited. This represents, indeed, a bottleneck in development of vaccines inducing specific T-cell responses. Here, we describe a novel strategy of Ag delivery into APCs. The Ag of choice is fused to the N- or C-terminus of streptavidin (SA) and tetrameric Ag-SA or SA-Ag fusion proteins are produced in E. coli and purified by 2-Iminobiotin-Agarose affinity chromatography. Alternatively, Ag-SA proteins are purified from urea extracts of E. coli inclusion bodies and refolded in vitro into functional tetramers. Complexes with biotinylated antibodies targeting cell surface receptors are formed and used to deliver the Ags of choice for processing and presentation by APCs and induction of Ag-specific CD4+ and CD8+ T-cell responses in vitro and in vivo.

Recently, we have developed a method of adapter-ligation mediated allele-specific amplification (ALM-ASA) for simultaneously typing multiple single nucleotide polymorphisms (SNPs) at a low cost. We usually use agarose gel-electrophoresis for analyzing PCR products. As the processes of sampling and PCR can be carried out at a format of 96-well or 384-well, the throughput-bottleneck of whole process of ALM-ASA is only the agarose gel-electrophoresis. Here we improved the typing throughput of ALM-ASA by using a microplate array parallel gel electrophoresis (MAPGE) system, with which 96 amplicons can be detected at a time. By coupling with multiplexed preamplification, seven SNPs distributed on four different human genes (IL1A (549C>T), 1L1B (794C>T and 5277C>T), IL10 (2940G>A, 3203C>T, and 3430C>A), and TNFA (1431G>A)) were successfully typed. The optimization of allele-specific primers in ALM-ASA was performed by the software of "SNiPdesigner" which was designed especially for ALM-ASA. We also demonstrated that the specificity of ALM-ASA assay for SNP typing is superior to that of amplification refractory mutation system (ARMS).

The present century has witnessed an unprecedented rise in genome sequences owing to various genome-sequencing programs. However, the same has not been replicated with cDNA or expressed sequence tags (ESTs). Hence, prediction of protein coding sequence of genes from this enormous collection of genomic sequences presents a significant challenge. While robust high throughput methods of cloning and expression could be used to meet protein requirements, lack of intron information creates a bottleneck. Computational programs designed for recognizing intron-exon boundaries for a particular organism or group of organisms have their own limitations. Keeping this in view, we describe here a method for construction of intron-less gene from genomic DNA in the absence of cDNA/EST information and organism-specific gene prediction program. The method outlined is a sequential application of bioinformatics to predict correct intron-exon boundaries and splicing by overlap extension PCR for spliced gene synthesis. The gene construct so obtained can then be cloned for protein expression. The method is simple and can be used for any eukaryotic gene expression.

A bottleneck in recent gene synthesis technologies is the high cost of oligonucleotide synthesis and post-synthesis sequencing. In this article, a simple and rapid method for low-cost gene synthesis technology was developed based on DNAWorks program and an improved single-step overlap extension PCR (OE-PCR). This method enables any DNA sequence to be synthesized with few errors, then any mutated sites could be corrected by site-specific mutagenesis technology or PCR amplification-assembly method, which can amplify different DNA fragments of target gene followed by assembly into an entire gene through their overlapped region. Eventually, full-length DNA sequence without error was obtained via this novel method. Our method is simple, rapid and low-cost, and also easily amenable to automation based on a DNAWorks design program and defined set of OE-PCR reaction conditions suitable for different genes. Using this method, several genes including Manganese peroxidase gene (Mnp) of Phanerochaete chrysosporium (P. chrysosporium), Laccase gene (Lac) of Trametes versicolor (T. versicolor) and Cip1 peroxidase gene (cip 1) of Coprinus cinereus (C. cinereus) with sizes ranging from 1.0 kb to 1.5 kb have been synthesized successfully.

The production of foreign proteins using selected host with the necessary posttranslational modifications is one of the key successes in modern biotechnology. This methodology allows the industrial production of proteins that otherwise are produced in small quantities. However, the separation and purification of these proteins from the fermentation media constitutes a major bottleneck for the widespread commercialization of recombinant proteins. The major production costs (50-90%) for typical biological product resides in the purification strategy. There is a need for efficient, effective, and economic large-scale bioseparation techniques, to achieve high purity and high recovery, while maintaining the biological activity of the molecule. Aqueous two-phase systems (ATPS) allow process integration as simultaneously separation and concentration of the target protein is achieved, with posterior removal and recycle of the polymer. The ease of scale-up combined with the high partition coefficients obtained allow its potential application in large-scale downstream processing of proteins produced by fermentation. The equipment and the methodology for aqueous two-phase extraction of proteins on a large scale using mixer-settlerand column contractors are described. The operation of the columns, either stagewise or differential, are summarized. A brief description of the methods used to account for mass transfer coefficients, hydrodynamics parameters of hold-up, drop size, and velocity, back mixing in the phases, and flooding performance, required for column design, is also provided.