Abstract

Bacteria extracellular vesicles (BEVs), characterized as the lipid bilayer membrane-surrounded nanoparticles filled with molecular cargo from parent cells, play fundamental roles in the bacteria growth and pathogenesis, as well as facilitating essential interaction between bacteria and host systems. Notably, benefiting from their unique biological functions, BEVs hold great promise as novel nanopharmaceuticals for diverse biomedical potential, attracting significant interest from both industry and academia. Typically, BEVs are evaluated as promising drug delivery platforms, on account of their intrinsic cell-targeting capability, ease of versatile cargo engineering, and capability to penetrate physiological barriers. Moreover, attributing to considerable intrinsic immunogenicity, BEVs are able to interact with the host immune system to boost immunotherapy as the novel nanovaccine against a wide range of diseases. Towards these significant directions, in this review, we elucidate the nature of BEVs and their role in activating host immune response for a better understanding of BEV-based nanopharmaceuticals’ development. Additionally, we also systematically summarize recent advances in BEVs for achieving the target delivery of genetic material, therapeutic agents, and functional materials. Furthermore, vaccination strategies using BEVs are carefully covered, illustrating their flexible therapeutic potential in combating bacterial infections, viral infections, and cancer. Finally, the current hurdles and further outlook of these BEV-based nanopharmaceuticals will also be provided.

Introduction

Vesiculation is a crucial and fundamental process across all kinds of species to produce the extracellular vesicles that serve as essential mediators of basic physiological events [1]. These nanovesicles are filled with molecular patterns originated from parent cells, including metabolites, nucleic acids, proteins, and signaling molecules, to maintain cell growth and homeostasis [2, 3]. Biological regulation by extracellular vesicles is widespread across both prokaryotes and eukaryotes [4]. Notably, bacteria, as one of the major inhabitants in the human body, establish intricate relationships with host health and disease, wherein bacterial extracellular vesicles (BEVs) are indispensably involved in these processes [5, 6]. With the increasing and deep understanding of their biological function, BEVs are found to influence various cellular behaviors, including the transport of genetic information, phage infection, mediation of metabolism, as well as interaction between bacteria-bacteria and bacteria-host [7, 8].

Particularly, BEVs are characterized as nanosized nanoparticles, surrounded by lipid-bilayer membranes, ranging from 20 to 400 nm in diameter [7]. In account of the diversity of bacterial types and biogenesis mechanisms, the BEVs could carry versatile cargos inherited from mother cells, such as lipopolysaccharides (LPS), endotoxins, genetic information, cytosolic and membrane proteins [9]. By thanking the unique structure and intrinsic properties of BEVs, these naturally occurring nanovesicles attract the research interest to be developed as novel nanopharmaceuticals, prompting further exploration of their biomedical applications [10, 11]. Generally, BEVs are widely evaluated as biotherapeutics in different forms. Firstly, these nanovesicles with hollow structures could serve as novel drug delivery platforms, facilitating the transport of diverse bioactive molecules and therapeutic cargo to the recipient cells at the lesion site [12]. Thanks to the stability of naturally-occurring membrane structure, the BEVs-based drug delivery platform could carry the therapeutic genetic tools (e.g. siRNA, CRISPR-Cas9, etc.), protecting them from enzymatic degradation or hydrolysis in the complex physiological environment [13, 14]. Besides, benefiting from the ease of modification, BEVs-based drug delivery platforms could efficiently load the small molecular therapeutics [14], or directly produce the synthetic cargo (e.g. antigens, enzymes, therapeutic proteins, etc.) on the BEVs by editing the desired gene in parent bacteria. The BEVs-based drug carriers are also featured in their capability for targeting delivery toward the disease area to enhance drug accumulation and availability [15]. Moreover, the BEVs drug delivery platform can integrate with functional materials, to facilitate combinational therapy (e.g. photodynamic therapy, photothermal therapy, etc.) and maximize the synergistic therapeutic efficiency [16].

Conclusion and further perspective

In this review, we comprehensively summarize the development of BEV-based nanopharmaceuticals to facilitate disparate biomedical applications in recent years. Amongst the unique advantages and functions of BEVs, we demonstrated the tremendous potential of applying these naturally occurring nanovesicles to establish a myriad of innovative therapeutic strategies for new-generation pharmaceuticals evolution, especially in the versatile bioactive cargo delivery and powerful vaccination approaches.

Taking the unique merits of nanovesicles, the structural stability, ease of cargo loading, promising penetration across physiological barriers, and specific targeting capabilities endow the BEVs application to be beneficial in delivery for a wide range of therapeutics. Particularly, the naturally occurring membrane structure of BEVs enables targeting delivery of therapeutic genetic tools (e.g., siRNA, DNA, CRISPR-Cas9, etc.) in the physiological, preserving stability and bioactivity, thus enhancing gene therapy. Leveraging the ease of genetic modification in parent bacteria, protein cargo, such as enzymes and antigens, can also be directly expressed within BEVs, optimizing loading efficiency compared to conventional delivery platforms. Furthermore, advancements in nanotechnology and material science have witnessed the integration of functional materials with BEV delivery platforms to achieve combinational therapy, thereby further improving the synergistic therapeutic efficacy.

Abstract

Background

    Demonstrating that the data produced in metabolic phenotyping investigations (metabolomics/metabonomics) is of good quality is increasingly seen as a key factor in gaining acceptance for the results of such studies. The use of established quality control (QC) protocols, including appropriate QC samples, is an important and evolving aspect of this process. However, inadequate or incorrect reporting of the QA/QC procedures followed in the study may lead to misinterpretation or overemphasis of the findings and prevent future metanalysis of the body of work.

Objective

      The aim of this guidance is to provide researchers with a framework that encourages them to describe quality assessment and quality control procedures and outcomes in mass spectrometry and nuclear magnetic resonance spectroscopy-based methods in untargeted metabolomics, with a focus on reporting on QC samples in sufficient detail for them to be understood, trusted and replicated. There is no intent to be proscriptive with regard to analytical best practices; rather, guidance for reporting QA/QC procedures is suggested. A template that can be completed as studies progress to ensure that relevant data is collected, and further documents, are provided as on-line resources.

Key reporting practices

     Multiple topics should be considered when reporting QA/QC protocols and outcomes for metabolic phenotyping data. Coverage should include the role(s), sources, types, preparation and uses of the QC materials and samples generally employed in the generation of metabolomic data. Details such as sample matrices and sample preparation, the use of test mixtures and system suitability tests, blanks and technique-specific factors are considered and methods for reporting are discussed, including the importance of reporting the acceptance criteria for the QCs. To this end, the reporting of the QC samples and results are considered at two levels of detail: “minimal” and “best reporting practice” levels.

Introduction

     Progress in science is based on the principle that measurements are repeatable; that is to say, it should be possible for scientists in other laboratories, equipped with the same, or similar expertise, resources and infrastructure, to replicate the results of published work within acceptable levels of confidence. Properly documented studies allow other scientists to critically assess and judge the original experimental design and should also enable researchers in other laboratories to repeat the experiment under the same conditions and support or refute the findings. However, a reproducibility crisis in science has been recently highlighted by Munafò et al. (2017) and the lack of transparency in reporting can lead to ambiguity within experimental design, data collection, as well as data processing and interpretation, a situation which is also true for metabolomics. Moreover, any metabolomic data derived from instrumental analyses must be sufficiently reliable that decisions based on it can be reported with confidence; there should be no “leap of faith” from data to knowledge. If the metabolomic data cannot be trusted, then the answer to the posed question is of little value.

Discussion and conclusions

     The use of both QA and QC procedures is recognized as fundamental for achieving reliable and consistent data in metabolomics. The recommendations presented here are not meant to constrain research but to ensure that, where these systems have been employed, this is stated and what has been done is clearly explained. In this opinion paper there is no official authority to mandate the procedures that must be used when employing QA/QC in untargeted metabolomics. However, the recommendation here is that authors state what they did, and to state this as transparently and as completely as possible. In other words, they should deliberately and purposefully avoid obfuscation. The documentation of the QA and QC of the analytical process of an untargeted metabolomics experiment enables experimenters to demonstrate that the work was conducted appropriately, and that the editors and reviewers, and therefore ultimately readers can have confidence in the resulting conclusions. However, recognizing that untargeted metabolic phenotyping is a discovery methodology any hypotheses emerging from it are inevitably provisional. Nevertheless, armed with knowledge that the data are of good quality provides the motivation to devote the resources required to undertake further targeted analysis, using quantitative and validated methods, to confirm, or refute, these hypotheses.

Abstract

Keywords

1. Introduction

2. DNA damage response

3. DDR and radiotherapy failure

4. DDR inhibitors decrease radio-resistance

5. DDR roles in chemotherapy failure

6. DDR inhibitors decrease chemo-resistance

7. Conclusions

Funding

Availability of data and material

Ethics approval and consent to participate

Consent for publication

Declaration of Competing Interest

Acknowledgements

References

Abstract

Up to now, many improvements have been made in providing more therapeutic strategies for cancer patients. The lack of susceptibility to common therapies like chemo- and radio-therapy is one of the reasons why we need more methods in the field of cancer therapy. DNA damage response (DDR) is a set of mechanisms which identifies DNA lesions and triggers the repair process for restoring DNA after causing an arrest in the cell cycle. The ability of DDR in maintaining the genome stability and integrity can be favorable to cancerous cells which are exposed to radiation therapy or are treated with chemotherapeutic agents. When DDR mechanisms are error-free in cancer cells, they can escape the expected cellular death and display resistance to treatment. In this regard, targeting different components of DDR can help to increase the susceptibility of advanced tumors to chemo- and radio-therapy.

1. Introduction

DNA-damaging agents originating whether from endogenous or exogenous resources are known to threat the viability of every cell of our body [1]. Besides to the cellular issues arising from the great number of mutations and genome instability, the lack of proper responses can establish generations of these defective cells and put our whole body in danger [1]. DNA damage response or DDR is a precise mechanism which contains sensing the lesions, establishing signals, and finally initiating repair processes by the means of these signals [2]. During DDR, cell cycle is also affected by the mediators of DDR and is temporarily stopped in order to inhibit the replication of a defective DNA [3]. In some cases, DNA deteriorations are irreparable and thus, cell death is the only option on the table [3].

In addition to normal cells, cancerous cells are also able to use the same mechanisms for their chemotherapy- and/or radiotherapy-induced damaged DNA. Until DDR is working properly in a cancerous cell, DNA damages would not lead to cell death and thus, treatment would not be considered successful [1]. This means that despite all the advantageous impacts of DDR, it stands in the way of overcoming a great challenge in cancer therapy: resistance to our common methods, chemo- and radio-therapy. Recently, a great number of researchers have worked on targeting distinct components of DDR for enhancing the efficacy of cytotoxic treatments [4]. In this paper, we review the process of DDR briefly and explain the role of its key players in decreasing the susceptibility of cancer cells to common therapeutic procedures. Additionally, the usage of DDR inhibitors on different types of cancer would be discussed for giving an insight for the treatment of advanced stages of this lethal disease.

Highlights

Abstract

Graphical abstract

Keywords

1. Introduction

2. Materials and methods

3. Results

4. Discussion

Credit authorship contribution statement

Appendix A. Supplementary material

References

Background: Eponyms are commonly used in medicine, but there are no specific studies of the use of eponyms in clinical chemistry. Methods: Clinical chemistry eponyms were manually collected from books, review articles and journal articles from 1847 through 2020. Eponym usage was examined by searching titles and abstracts in PubMed. Custom Python scripts were used to first permute eponyms into multiple forms, and then to search PubMed using Biopython. The eponyms identified in PubMed were further focused on 2 clinical chemistry journals Clinica Chimica Acta [CCA] and Clinical Chemistry [CCJ]. Results: The manual collection identified >300 eponyms in clinical chemistry. The Biopython search of PubMed identified a subset of 97 unique eponyms in 33,232 articles. PubMed identified 26 eponyms used in 130 CCA articles; whereas a full-text search identified 1187 articles. In comparison, PubMed identified 36 eponyms used in 158 CCJ articles; whereas a full-text CCJ search identified 708 articles. PubMed shows that the journals CCA and CCJ had a peak number of eponym citations in 1977 followed by a steady decline. Conclusions: Eponyms have been frequently used in clinical chemistry with 97 eponyms in common use in PubMed. Overall, the use of clinical chemistry eponyms appears to be declining.

1. Introduction

Naming chemical reactions, chemical reagents, mathematical equations, units, coefficients, constants, laws, diseases, and clinical signs and symptoms, after people (eponyms), places (toponyms) or mythical beings (mythonyms) is not uncommon, and there are a number of compendia of medical eponyms [1–3]. Likewise, in the field of clinical pathology there are compendia of eponyms used in hematology [4], and microbiology [5], but we are not aware of a compendium of eponyms relating specifically to clinical chemistry. There are some general works published at the end of the 19th century and the beginning of the 20th century that list named “tests (reagents)” [6], reagents and reactions relevant to physiological-pathological chemistry [7], pharmacology/ toxicology [8], “tests and reagents known by their author’s names” of practical utility for “the busy chemist, microscopist, and pharmacist” [9], and reactions and reagents for the doctor, analyst, pharmacist, and chemist [10]. Although these lists contain some clinical chemistry eponyms, by and large, they are general lists with an emphasis on chemistry tests and reagents. Eponyms abound in clinical chemistry, including some derived from general laboratory sciences (e.g., Erlenmeyer flask), or from the intersection of clinical chemistry with physics (e.g., Rayleigh scattering), biochemistry (e.g., Michaelis-Menten kinetics), and statistics (e.g., Kruskall-Wallis test). Recently, we examined the natural history of the Malloy-Evelyn Reaction, and traced the coinage and chronology of the adoption of this eponym [11]. We have now extended our study of eponyms and have compiled a list of eponyms relating specifically to clinical chemistry methods, tests, reagents, apparatus, tables, formulae, and coefficients, and examined their usage in clinical chemistry over time.

Abstract

Graphical abstract

Keywords

1. Introduction

2. Experiment section

3. Results and discussion

4. Conclusions

Declaration of Competing Interest

Acknowledgements

Appendix A. Supplementary data

References

Compared to the synthetic nanomaterials, the ones modified from biology and capable of multimodal imaging and therapeutic functions have received increasingly interest for tumor theranostics due to their intrinsic biocompatibility and biodegradability. In this work, we firstly prepared the hematoporphyrin-melanin nanoconjugates (HMNCs) whose hematoporphyrin part was originated from the endogenous hemoglobin and melanin part was extracted from the cuttlefish ink. In the case of HMNCs, the hematoporphyrin part could be excited by ultrasound to produce cytotoxic singlet oxygen for sonodynamic therapy (SDT), while the melanin part with strong near-infrared absorbance possessed rapid and efficient photothermal conversion for photothermal therapy (PTT). The in vitro cell experiments confirmed the high biocompatibility of HMNCs, and the combined SDT-PTT achieved much high therapeutical efficacy towards cancer cells in comparison to SDT or PTT alone. Furthermore, in vivo administration of HMNCs at 40 mg kg− 1 brings no noticeable side effects for mice blood and major organs, showing their high in vivo biosafety. The HMNCs could accumulate in tumor area after intravenously injection so that they provided high contrast for tumor photoacoustic and thermal imaging, and thereafter the tumor growth was highly inhibited through synergistic SDT-PTT in comparison to SDT or PTT alone. Therefore, the HMNCs modified from biology can be served as multifunctional nanoagents for tumor theranostic, and it would inspire to develop novel agents modified from biology and then utilize them for biology.

1. Introduction

Malignant tumors with rapid and uncontrolled growth brings a heavy burden on physical, mental and economic state of patients. To treat these tumors, apart from the clinically used therapeutical approaches, several emerging treatment modalities have been explored by taking advantage of the advanced nanotechnology, such as photosensitizer-based photodynamic therapy [1,2], nutrition-depletioninduced starvation therapy [3,4], nanocarriers-based chemotherapy [5–7], and sonosensitizer-driven sonodynamic therapy (SDT) [8–11] et al. Among these therapies, SDT stands out because it utilizes unharmful ultrasound (US) as the energy source which possesses higher tissue-penetration depth than photodynamic therapy, so that sonosensitizers accumulated within tumor can be efficiently in-situ excited to produce cytotoxic radical oxygen species (ROS) to destroy cancer cells, while bringing a low side-effect for the normal tissue [9]. Currently, a number of sonosensitizers have been prepared which can be basically categorized into inorganic semiconductors (such as TiO2 nanoparticles [12] and its nanocomposites [13,14]) and organic molecules including hypocrellins [15], protoporphyrin [16], hematoporphyrin [17–19] and et al. However, the therapeutical efficacy of tumor SDT is rather limited by the low oxygen level in solid tumors as well as the short life time (~200 ns) and diffusion distance (<20 nm) of ROS [2]. Therefore, it is urgent to improve the therapeutical efficacy of SDT.

Introduction

Materials and Methods

Results and Discussion

Conclusions

References

ABSTRACT

RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg2þ, neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg2þ in folded conformations of RNA have been studied extensively; however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate, and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/molecular dynamics simulations are used to capture the energetics and atomic-level details of Mg2þ-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg2þ, as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg2þ with two or more nonsequential phosphate groups. Notably, these results indicate a push-pull mechanism in which the Mg2þ-RNA interactions actually lead to destabilization of specific nonsequential phosphate-phosphate interactions (i.e., pushed apart), whereas other interactions are stabilized (i.e., pulled together), a balance that stabilizes unfolded states and facilitates the folding of Twister, including the formation of hydrogen bonds associated with the tertiary structure. This study establishes a better understanding of how Mg2þ-ion interactions contribute to RNA structural properties and stability.

INTRODUCTION

RNA molecules have highly diverse structures ranging from simple helices to highly heterogeneous folded conformations that are essential for their wide range of cellular functions (1–3). Specifically, ribozymes, a distinct class of enzymes, exhibit complex tertiary structures and catalyze self-cleavage or the cleavage of phosphodiester bonds of substrate RNA, with metal ions typically playing a central role in the catalytic activity (4–6). To assume their tertiary structures, RNAs must overcome large unfavorable electrostatic interactions associated with their polyanionic phosphodiester backbone (7). To facilitate this, positively charged ions screen the highly negative potential, allowing the RNA secondary structures to collapse into compact tertiary conformations (8–11). Typically divalent ions, most often Mg2þ, facilitate the formation of tertiary interactions required for the full folding of RNA (12–14). However, the inability to visualize the ions during folding represents a key barrier to understanding the role of divalent ions in folding of RNA (15). Studies have used classical molecular dynamics (MD) or other theoretical approaches to investigate Mg2þ-RNA binding, but they were limited to native conformations because of their inability to overcome the issues associated with the Mg2þ exchange rates (16–22). The exchange rate of water complexed to Mg2þ is on the ms timescale (6.7 105 s 1 ), and the exchange rate of Mg2þ with phosphate is on the ms timescale (0.5–2.5 103 s 1 ) (23), which is beyond the timescale of typical atomistic MD simulations (20,24) such that only limited insights into Mg2þ-RNA interactions are accessible (25). Alternatively, simulations using coarse-grained models of nucleic acids provided insights into how Mg2þ can serve to nucleate the folding of key tertiary interactions with the Mg2þ-RNA interactions being dominated by specific interactions even in unfolded states (19,26).

1- Introduction

2- Material and methods

3- Results

4- Discussion

References

Abstract

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites.

Introduction

Mites and ticks are members of the chelicerates, the largest group of terrestrial animals after insects. The two-spotted spider mite, T. urticae, and other spider mites are important crop pests worldwide. This herbivore species is at the extreme end of the generalist-to specialist spectrum and can feed on a staggering 1,100 plant species. Not surprisingly, it is currently reported as the ‘most resistant’ pest worldwide, as it developed resistance to more than 90 acaricides (Mota-Sanchez and Wise, 2019; Van Leeuwen and Dermauw, 2016; Van Leeuwen et al., 2015). In 2011, a 90 Mb high-quality Sanger-sequenced genome became available for this species (Grbic et al., 2011). This allowed to disentangle some of the molecular mechanisms underlying resistance, whether to man-made pesticides or plant secondary compounds. The extreme adaptation potential of T. urticae was associated with specific gene expansions in known detoxification enzyme families, such as cytochrome P450 monooxygenases, glutathione-S-transferases, carboxyl-choline esterases, an unexpected repertoire of ABC and MFS transporters, and a proliferation of cysteine peptidases (Dermauw et al., 2013a; Dermauw et al., 2013b; Grbic et al., 2011; Santamaría et al., 2012).

1- Introduction

2- Structural elements of mRNA

3- Improving the stability and translation of mRNA

4- Avoiding immunogenicity of mRNA

5- 5 mRNA delivery

6- Preclinical and clinical advances of mRNA therapeutics

7- Concerns over the mRNA medicinal industry

8- Summary and perspective

References

Abstract

Messenger RNA (mRNA)-based therapeutics hold the potential to cause a major revolution in the pharmaceutical industry because they can be used for precise and individualized therapy, and enable patients to produce therapeutic proteins in their own bodies without struggling with the comprehensive manufacturing issues associated with recombinant proteins. Compared with the current therapeutics, the production of mRNA is much cost-effective, faster and more flexible because it can be easily produced by in vitro transcription, and the process is independent of mRNA sequence. Moreover, mRNA vaccines allow people to develop personalized medications based on sequencing results and/or personalized conditions rapidly. Along with the great potential from bench to bedside, technical obstacles facing mRNA pharmaceuticals are also obvious. The stability, immunogenicity, translation efficiency, and delivery are all pivotal issues need to be addressed. In the recently published research results, these issues are gradually being overcome by state-of-the-art development technologies. In this review, we describe the structural properties and modification technologies of mRNA, summarize the latest advances in developing mRNA delivery systems, review the preclinical and clinical applications, and put forward our views on the prospect and challenges of developing mRNA into a new class of drug.

INTRODUCTION

Messenger RNA (mRNA) has become an attractive subject of basic and applied research since it was first discovered in 1960s (Brenner et al., 1961). Accordingly, the understanding of mRNA has shifted from a simple link between DNA and protein to a versatile molecule that regulates the functions of genes in all living organisms. Based on this change, numerous types of mRNA-based therapeutics have emerged. In 1990, Wolff et al. firstly reported that intramuscular injection of mRNA into the skeletal muscle of mice led to the expression of encoding proteins (Wolff et al., 1990). Since then, mRNA- based therapeutics have been exploited in a variety of applications, including cancer immunotherapy, infectious disease vaccines, protein substitution and cellular genetic engineering. In 2001, ex vivo mRNA transfected dendritic cells entered clinical trial for the first time (Heiser et al., 2002), and hundreds of mRNA-based clinical trials have been launched over the past two decades. However, mRNA was not considered a new class of drug in the first decades after its discovery. Obstacles such as instability and immunogenicity have hampered its development, making it less pursued than DNA in gene therapy (Burnett and Rossi, 2012; Crooke et al., 2018; Geall et al., 2013; Hajj and Whitehead, 2017; Kallen and Thess, 2014; Kreiter et al., 2011; Reautschnig et al., 2017; Sahin et al., 2014; Van et al., 2015). In recent years, these key problems have been mainly solved by introducing modified nucleosides into mRNA sequences and developing various RNA packaging and delivery systems. A lot of evidences not only proved that mRNA can mediate superior transfection efficiency and longer protein expression time, but also revealed the main advantages of mRNA over DNA. The advantages of mRNA include: (1) mRNA does not need to enter the nucleus to be functional.

1- Introduction

2- Materials and methods

3- Results

4- Discussion

References

Abstract

Ribonuclease P (RNase P) is an RNA processing enzyme essential for production of functional tRNAs. Bacterial RNase P is a ribozyme, i.e., an RNA-based enzyme, which functions in all bacteria including those growing at high temperatures (≥55 °C). We examined three bacterial RNase P ribozymes, one from a mesophilic bacterium and two from thermophilic bacteria, to understand the factor(s) providing efficient catalytic ability under conditions of high temperature. Thermophilic RNase P ribozymes show structural adaptations to allow correct folding at high temperature. The presence of a molecular crowder significantly enhanced the catalytic efficiency of thermophilic RNase P ribozyme reactions at 55 °C, while it modestly reduced the upper limit of the reaction temperature.

Introduction

Ribonuclease P (RNase P) is a class of RNA processing enzymes, the primary role of which is to remove 50 leader sequences from precursor tRNAs (Fig. 1A) [1]. Bacterial RNase P enzymes are commonly composed of one large RNA (typically ca. 300e400 nucleotides) and one small basic protein (ca. 120 amino acids). Bacterial RNase P is classed as a ribozyme because its catalytic ability originates from its RNA component, which can promote the sitespecific cleavage of pre-tRNAs in vitro without the protein component [1,2]. Bacterial RNase P ribozymes have a modular architecture consisting of two structural domains [1,3,4]: the Cdomain (catalytic domain) containing elements essential for catalysis, and the S-domain (specificity domain) that plays an important role in the specific recognition of pre-tRNA substrates (Fig. 1B) [5,6]. As cleavage of the 50 leader sequence is an essential step in production of functional tRNAs, RNase P ribozymes have to function in all bacterial species growing under various environmental conditions, including various growth temperatures. The RNase P ribozyme is also considered to have emerged at an early stage in the molecular evolution of life and to have functioned in the last universal common ancestor (LUCA) [7], which was proposed to have lived in hydrothermal vents [8]. Bacteria in several phylums, such as Aquificales, Firmicutes, Thermotogae, and DeinococcuseThermus, are known to grow under conditions of high temperature (65 Ce90 C). They also contain RNase P ribozymes [9e12], which need to be active at their growth temperature. The RNA components of these ribozymes may have adaptations for the high temperature growth conditions, and may require the assistance of external factors including their protein components, other intracellular macromolecules that provide molecular crowding conditions, and inorganic/organic cations other than Mg2þ. Thermophilic RNase P ribozymes from thermophilic bacteria are also interesting from the viewpoint of the RNA world hypothesis because it has been proposed that the RNA world may have emerged at hydrothermal vents [13,14].

1. Introduction

2. Materials and methods

3. Results

4. Discussion

Funding

Appendix A. Supplementary data

References

Abstract

We previously identified 7 low/lower-middle income countries (LMICs; Afghanistan, Pakistan, India, Mongolia, Yemen, Nigeria, Tunisia) which have excess burden of vitamin D deficiency and could benefit enormously from food fortification with vitamin D. A key challenge is finding a suitable industrially-manufactured food vehicle that is consumed in sufficient amounts by the population at-risk. We used FAO Food Balance Sheet data (from 2003-2013) to model the potential impact of four different food vehicles (edible plant-based oil, wheat flour, maize flour, and milk), and at different addition levels, on the average per capita vitamin D supply in all 7 LMICs. Daily per capita supply for ˜95 foods was calculated and vitamin D supply determined using dietary analysis software with no addition and following stepwise additions of vitamin D to the four food vehicles. The daily per capita vitamin D supply without fortification ranged from 0.4 to 3.3 μg (≤2 μg/d in six LMICs). We applied a vitamin D intake of 5 μg/d as a benchmark because it maintains serum 25-hydroxyvitamin D ≥ 25 nmol/L in ˜90% of individuals. Modelling showed that fortifying edible oil with vitamin D at the 7.5 μg/100 g (guideline) and 15 μg/100 g levels allowed vitamin D supply in 1 and 3 of the 7 LMICs, respectively, to attain ≥5 μg/d (range: 5.8–11.0 μg/d). Fortifying milk at the 1.0 μg/100 g and 2.0 μg/100 g guideline levels, allowed 2 and 3 LMICs, respectively, to attain ≥5 μg/d (range: 5.2–9.8 μg/d). Fortifying wheat flour at the 1.4 μg/100 g (guideline) and 2.8 μg/100 g allowed 5 and 6 LMICs, respectively, to attain ≥5 μg/d (range: 5.3–18.6 μg/d). Maize flour had low impact due to consumption levels. In conclusion, using these levels of addition, at least one food vehicle was able to increase per capita vitamin D supply to ≥5 μg/d in each of the LMICs.

Introduction

While there has been considerable and on-going emphasis placed on the issue of vitamin D deficiency in the higher income country setting, in recent years there has been growing concern about vitamin D deficiency as a possible public health concern in low- and lower-middle income countries (LMICs). Several systematic literature reviews (SLRs) of global vitamin D status [1,2], as well as our recent LMIC-focussed SLR [3], highlight the general lack of available data on prevalence of vitamin D status for the majority of LMICs. It is of note, however, that where such data does exist, several LMICs have been consistently shown to have extremely high and worrisome estimates of the prevalence of low vitamin D status [1–3], potentially putting the health and wellbeing of significant numbers of individuals within these populations at risk. These reported prevalence estimates in many cases far exceed the suggested guide of where more than 20% of the population (overall, or within identifiable subgroups) has circulating 25-hydroxyvitamin D (25(OH)D) concentrations < 25/30 nmol/L, public health intervention should be considered [4]. Of particular concern, within the LMICs, infants, young children and women of child-bearing age were shown to be at highest risk [2,3], subgroups of the population which the World Health Organisation (WHO) emphasise in terms of ensuring adequate nutrition [5,6].

In terms of public health interventions that could be considered to address the excess prevalence of vitamin D deficiency in these LMICs, the World Health Organisation-Food and Agriculture Organization (WHO-FAO) have indicated that of the four key strategies for the control of micronutrient malnutrition, food fortification is the preferred approach in terms of sustainability, uptake and cost-effectiveness [7]. The Global Fortification Data Exchange has recently suggested that twothirds of all countries mandate food fortification to combat micronutrient malnutrition, yet many are not necessarily translating policy into improved nutrition [8]. They suggest food fortification could be the next global health success story – if countries close the gaps [8]. Thus, food fortification with vitamin D could have enormous impact in the atrisk LMICs, once done effectively. The WHO-FAO stress that a key challenge is finding a suitable, culturally appropriate, industriallymanufactured food vehicle that is consumed in sufficient amounts by the populations at-risk [7]. Black et al. [9] have also suggested that beyond the range of food vehicles used for fortification, careful consideration must also be given to the concentration of vitamin D used in each to optimize the effectiveness and minimize risk of excessive intakes. While this can be achieved by modelling usual food consumption intakes in representative populations [7,9], there is a dearth of intake data in LMICs [3,4]. In the absence of such data, we have recently shown that estimates of average per capita supply of vitamin D in LMICs, as calculated using information from FAO Food Balance Sheets (FBS), may be of use as proxy measures for vitamin D intake within the population [3].

1- Introduction

2- Unfolded protein response signaling pathway

3- ER stress regulates autophagy and mitochondrial and lysosomal dysfunction in cancer

4- ER stress-mediated oxidative stress and inflammatory responses in cancer

5- Conclusion

References

Abstract

The endoplasmic reticulum (ER) acts as a moving organelle with many important cellular functions. As the ER lacks sufficient nutrients under pathological conditions leading to uncontrolled protein synthesis, aggregation of unfolded/misfolded proteins in the ER lumen causes the unfolded protein response (UPR) to be activated. Chronic ER stress produces endogenous or exogenous damage to cells and activates UPR, which leads to impaired intracellular calcium and redox homeostasis. The UPR is capable of recognizing the accumulation of unfolded proteins in the ER. The protein response enhances the ability of the ER to fold proteins and causes apoptosis when the function of the ER fails to return to normal. In different malignancies, ER stress can effectively induce the occurrence of autophagy in cells because malignant tumor cells need to re-use their organelles to maintain growth. Autophagy simultaneously counteracts ER stress-induced ER expansion and has the effect of enhancing cell viability and non-apoptotic death. Oxidative stress also affects mitochondrial function of important proteins through protein overload. Mitochondrial reactive oxygen species (ROS) are produced by calcium-enhanced ER release. The accumulation of toxic substances in ER and mitochondria in mitochondria destroys basic organelle function. It is known that sustained ER stress can also trigger an inflammatory response through the UPR pathway. Inflammatory response is thought to be associated with tumor development. This review discusses the emerging links between UPR responses and autophagy, oxidative stress, and inflammatory response signals in ER stress, as well as the potential development of targeting this multifaceted signaling pathway in various cancers.

Introduction

The endoplasmic reticulum (ER) is an organelle that forms a large membrane-like structure in its cytoplasm. The membranous structure of ER has a series of functions such as folding of newly synthesized proteins, maintenance of calcium homeostasis and phospholipid synthesis, and regulation of intracellular signaling pathways [1–3]. The structure of ER can be divided into a nuclear envelope domain, which is integrated in rough ER, and an ER domain for the synthesis of ribosomes. It also contains membranes, Golgi, vacuoles, mitochondria, peroxisomes, late endosomes and lysosomes, which act to promote lipid transfer to the membrane for calcium signal transmission [4]. ER is primarily used to transport and integrate proteins (secreted and membrane proteins, respectively), helping them to fold and transport (extracellular or cell membranes), lipid biosynthesis and maintenance of calcium homeostasis. In protein translation, ER is also a site that modifies N-linked glycosylation and is closely related to cellular signaling pathways [5–7]. For example, advanced ER stress hepatocytes have the function of reducing drug toxicity; the secreted proteins are synthesized in large amounts by abundant ER in other cells, and detoxification is exerted; sarcoplasmic reticulum as another special form of ER is more intramuscular cells in Exercise contraction and relaxation play a role [8]. Once the homeostasis is destroyed, the protein cannot be properly folded, including lack of molecular chaperone or cellular energy, as well as Ca2+ deficiency, redox environmental damage, protein variation and disulfide bond reduction [9]. Eukaryotic cells respond rapidly to ER dysfunction through a series of adaptive pathways called ER stress. And activate the unfolded protein reaction (UPR) [10,11].

1- Introduction

2- Theory

3- Material and methods

4- Results and discussion

5- Conclusions

References

Abstarct

During gastric digestion, hydrolysis of proteins by pepsin contributes largely to the breakdown of protein-rich food. We hypothesized that the effect of pepsin is limited by its diffusivity, which is co-determined by the food structure and the local pH in the food during digestion. To investigate the principle mechanism of enzyme diffusion in food matrices, we used enhanced green fluorescent protein (EGFP) as probe to study the diffusivity of proteins in whey protein isolate gels, using fluorescence correlation spectroscopy (FCS). Gels made with different ionic strength showed distinctive elastic moduli but did not show differences in diffusivity of EGFP. Some models for diffusion in hydrogels yield good description of the obtained data, and can approximate the enzyme diffusion in diverse food matrices. However, the enzyme pepsin is more complicated than the probe EGFP, to yield more accurate predictions, electrostatic and enzyme-substrate interaction also need to be considered.

Introduction

The digestion kinetics of food are dependent on the structure of the food that is digested. Food structure influences the oral processing, the gastric disintegration rate, and the consequent gastric emptying towards the duodenum (Singh, Ye, & Ferrua, 2015). The gastric disintegration of food invovles physical and chemical processes, including the peristalsis of the stomach, acid hydrolysis and enzymatic reactions (Bornhorst & Singh, 2014). Among these processes, hydrolysis of proteins by pepsin contributes largely to the breakdown of protein-rich food in the stomach. This hydrolysis can limited by the diffusion of pepsin and the local pH in the solid food matrix during digestion. The hydrolysis kinetics of egg white protein gels and whey protein gels differed strongly from that of the same proteins in solution, which is likely due to the diffusion limitation in gels for both the pepsin and the hydrolysates (Luo, Boom, & Janssen, 2015). Compared to acid-induced dairy gels, a rennet-induced casein gel consists of compact protein aggregates in the acidic gastric environment, and the rennet gel had much slower proteolysis kinetics than that of acid-induced gels (Floury et al., 2018). Thus, a quantitative investigation of pepsin diffusion in food structures may contribute to the understanding of food breakdown and digestion kinetics. We previously measured the diffusivity of pepsin in whey protein isolate (WPI) gels by fluorescence correlation spectroscopy (FCS). We found that the pepsin does not penetrate deep into the gel but remains in a thin layer below the surface of the gel. A second finding was that the diffusivity of pepsin depends strongly on the concentration of the protein gels (Luo, Borst, Westphal, Boom, & Janssen, 2017). Fluorescence Correlation Spectroscopy (FCS) was used for its non-invasiveness and suitability to be used within protein gels. In FCS, a confocal laser microscope is coupled with a photon detector to measure fluorescence intensity fluctuations in a small focal volume. If these fluctuations originate primarily from the diffusion of the fluorophores through the focal volume, autocorrelation analysis can quantify the diffusion rate of the fluorophore. Whey protein gels were used before as model for protein-based solid foods (Luo et al., 2017). Whey protein gelation is generally a two-step process. After heat denaturation, protein oligomers form primary aggregates with different shapes and sizes depending on the pH and the salt concentration. These primary aggregates then form large self-similar aggregates that precipitate or gel above a critical concentration (Aymard et al., 1996; Nicolai, Britten, & Schmitt, 2011).

1- Introduction

2- Materials and methods

3- Results

4- Discussion

References

Abstract

Photosynthesis is an important driver of calcium carbonate deposition on tropical coral reefs largely due to the symbiosis of numerous invertebrates with photosynthetic dinoflagellates in the family Symbiodiniaceae. In bioeroding sponges, however, similar symbioses appear to support the decalcification of carbonate substrates. Compared to its role in calcification, the relative importance of photosynthesis to decalcification processes is less known. Here, the daytime and night-time chemical bioerosion rates of the common Indo-Pacific sponge Cliona orientalis were examined under varying levels of photosynthetic activity and/or varying abundances of intracellular dinoflagellates. Photosynthesis was manipulated either by preconditioning the sponges with the photosynthetic inhibitor diuron (DCMU), or by exposing them to short-term heat stress to achieve bleaching (loss of symbionts). DCMU reduced symbiont numbers and diminished their ability to evolve oxygen. Thermal stress caused a significantly greater loss of symbionts, but photosynthesis was less inhibited. In both cases, decreases in photosynthetic activity and symbiont densities led to proportionately lowered daytime chemical bioerosion rates. Moreover, increased rates of night-time bioerosion were linked to greater daytime rates of photosynthesis, rather than to the night-time respiration of the sponge holobiont. Our findings support the conclusion that photosynthetic products (photosynthates) and/or by-products (oxygen) stimulate sponge bioerosion. This work further reveals the importance of symbionts in the ecology of such sponges and in their ability to sustain high bioerosion activity in otherwise nutrient-poor ecosystems.

Introduction

Tropical coral reefs thrive in shallow clear waters where sunlight powers productive and diverse food chains. Symbioses between invertebrates and a variety of photosynthetic microorganisms enable high rates of productivity on reefs despite low environmental concentrations of inorganic nutrients (Muscatine and Porter, 1977; Seckbach, 2004; Yellowlees et al., 2008). The evolutionary success that results from hosting phototrophic microbes such as dinoflagellates, diatoms, chlorophytes, rhodophytes, or cyanobacteria is seen in a wide range of diverse invertebrates belonging to taxa such as Cnidaria, Mollusca, Foraminifera, Platyhelminthes, and Porifera (Trench, 1993; Stat et al., 2006; Lipps and Stanley, 2016). Presently almost 200 sponge species (Porifera) are known to host microbial photosynthetic symbionts (“photosymbiotic” sponges), which may contribute to the nutrition and health of the sponge (e.g. Wilkinson, 1987; Hill et al., 2011; Thacker and Freeman, 2012). Sponge species that receive substantial contributions in this way are often named “phototrophic” after the nutritional mode of their symbionts, or “mixotrophic” given that they also depend on filter-feeding by specialized food-trapping sponge cells. The nutrition of sponges is often further supplemented by inputs from intimate associations formed with heterotrophic bacterial populations of varying density and diversity (e.g. Hentschel et al., 2003; Taylor et al., 2007).