Synthetic Nucleotide Research Articles

Formulation and Evaluation of Acyclovir Loaded Transferosomal Gel for Transdermal Drug Delivery

Background: A carrying structure for targeted transdermal drug delivery is a transferosome. These unique liposomes are made of an edge activator and phosphatidylcholine. The most common antiviral drug is acyclovir, a synthetic nucleotide nucleoside analog derived from guanine. It works well to cure the varicella-zoster virus and the virus that causes herpes simplex (HSV), primarily HSV-1 and HSV-2. But its skin permeability is minimal. Therefore, this work aimed to use transferosomes to prepare acyclovir so that it could pass through the skin's barrier function. Objective: This study uses a 32-factorial factorial design to develop a transferosomal gel containing acyclovir through thin-film hydration method for painless acyclovir delivery services for skin disease treatment. Material and Methods: The independent variables are the amount of phospholipid (X1) and tween 80 (X2), while the dependent variables are particle size (Y1) and percentage entrapment efficiency (Y2). To create an ideal formulation, the produced transferosomes were assessed for particle size, in vitro drug release amount, and entrapment efficiency (EE%). A Carbopol 934 gel basis was prepared using the optimized acyclovir transferosome formulation, and its drug concentration, pH, spreadability, viscosity, and stability were assessed. Results: With small particles ranging from 176.6 to 324.4 nm, the produced acyclovir transferosomes had a high EE% range from 66.34 to 76.42 %. According to the in vitro release study, there is a negative correlation between in vitro release and EE%. The formulation TF5, including 1%w/w of carbopol 940, provides a superior profile of drug absorption in vitro. Consequently, acyclovir can enter the skin as transferosomes and cross the stratum corneum barrier. Conclusion: Acyclovir can be transformed into a transfersomal gel, which will improve antiviral efficacy, get over the skin's protective layer, prevent adverse oral reactions, and eventually improve patient adherence. Keywords: Transferosome, Transdermal drug delivery system, Acyclovir, 32 Fractional factorial design, Carbopol 934

The United States Food and Drug Administration's Platform Technology Designation to Expedite the Development of Drugs.

Drug development costs can be significantly reduced if proven "platform" technologies are allowed to be used without having to validate their use. The most recent US Food and Drug Administration (FDA) guideline brings more clarity, as well as a greater focus on the most complex technologies that can now be used for faster drug development. The FDA has highlights the use of lipid nanoparticles (LNPs) to package and deliver mRNA vaccines, gene therapy, and short (2-20 length) synthetic nucleotides (siRNA). Additionally, monoclonal antibody cell development is targeted. The FDA provides a systematic process of requesting platform status to benefit from its advantages. It brings advanced science and rationality into regulatory steps for the FDA's approval of drugs and biologicals.

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes.

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

A folding motif formed with an expanded genetic alphabet

Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly stable unimolecular structure (that is, the folded Z-motif, or fZ-motif) that melts at 66.5 °C at pH 8.5. Spectroscopic, gel and two-dimensional NMR analyses show that the folded Z-motif is held together by six reverse skinny dZ−:dZ base pairs, analogous to the crystal structure of the free heterocycle. Fluorescence tagging shows that the dZ−:dZ pairs join parallel strands in a four-stranded compact down–up–down–up fold. These have two possible structures: one with intercalated dZ−:dZ base pairs, the second without intercalation. The intercalated structure would resemble the i-motif formed by dC:dC+-reversed pairing at pH ≤ 6.5. This fZ-motif may therefore help DNA form compact structures needed for binding and catalysis.

PDNA-tachyplesin treatment stimulates the immune system and increases the probability of apoptosis in MC4-L2 tumor cells.

Breast cancer is the most common cancer among women worldwide, and marine creatures are the most abundant reservoir of anticancer medicines. Tachyplesin peptides have shown antibacterial capabilities, but their potential to inhibit cancer growth and trigger cancer cell death has not been investigated. A synthetic tachyplesin nucleotide sequence was generated and inserted into the pcDNA3.1( +) Mammalian Expression Vector. PCR analysis and enzyme digesting procedures were used to evaluate the vectors' accuracy. The transfection efficiency of MCF-7 and MCF10-A cells was 57% and 65%, respectively. The proliferation of MCF-7 cancer cells was markedly suppressed. Administration of plasmid DNA (pDNA) combined with tachyplesin to mice with tumors did not cause any discernible morbidity or mortality throughout treatment. The final body weight curves revealed a significant reduction in weight among mice treated with pDNA/tachyplesin and tachyplesin at a dose of 100 µg/ml (18.4 ± 0.24 gr, P < 0.05; 11.4 ± 0.24 gr P < 0.01) compared to the control group treated with PBS (22 ± 0.31 gr). Animals treated with pDNA/tachyplesin and tachyplesin exhibited a higher percentage of CD4 + Foxp3 + Tregs, CD8 + Foxp3 + Tregs, and CD4 + and CD8 + T cell populations expressing CTLA-4 in their lymph nodes and spleen compared to the PBS group. The groups that received pDNA/tachyplesin exhibited a substantial upregulation in the expression levels of caspase-3, caspase-8, BAX, PI3K, STAT3, and JAK genes. The results offer new possibilities for treating cancer by targeting malignancies using pDNA/tachyplesin and activating the mTOR and NFκB signaling pathways.

An overview of aptamer: Design strategy, prominent applications, and potential challenge in plants

Aptamers, serving as highly efficient molecular recognition and biotechnology tools, have garnered increasing interest in the realm of plant science in recent years. Aptamers are synthetic single-stranded short nucleotides or peptides, that bind targets with high specificity and affinity, triggering precise biological responses. As an alternative to antibodies, aptamers present promising avenues for advancement in biological researches. Aptamers function in a range of fields, encompassing cell signaling, drug development, biosensor technology, as well as botany, agricultural and forestry sciences. In this review, we introduce classifications and screening methods of aptamers, as well as aptamer-based technologies, highlighting their significant contributions to recent advancements. With their powerful functionality and ability to bind targets with high specificity and affinity, aptamers offer promising opportunities for breakthroughs in plant research.

Analysis of the properties of Komagataella phaffii CF-st401, a genetically modified producer of the sweet protein brazzein by using in silico methods

Currently, in order to reduce the consumption of mono- and disaccharides in diets, sweeteners are widely used. At the same time, none of the sweeteners approved for food industry usage matches the organoleptic properties of natural sugars. This circumstance was the basis for the development of technology for producing a new type of food ingredient with a sweet taste - brazzein using the producer strain Komagataella phaffii CF-st401. The purpose of the study was the application of in silico methods to assess the safety of K. phaffii CF-st401 genetically modified (GM) microorganism, which is the producer of the "Sweet protein Brazzein". Material and methods. The research object was the map of K. phaffii CF-st401 plasmid obtained by Biryuch LLC as a result of sequencing the plasmid of GM strain K. phaffii CF-st401, including: a synthetic nucleotide sequence similar to the sequence encoding the brazzein protein in the plant (Pentadiplandra brazzeana) and optimized for expression in the DNA of the recipient strain; the nucleotide sequence of plasmid M4794 from Saccharomyces cerevisiae and a linear fragment of the flanking DNA regions from K. phaffii used for integration into the genome of the recipient strain K. phaffii YIB Δleu2 VKPM Y-476, as well as amino acid sequence data of recombinant brazzein. By using bioinformatics methods (in silico), we investigated the DNA structure of the vector sequence of K. phaffii CF-st401, including the presence of operons responsible for toxin production, antibiotic resistance, and allergenicity. Results. As a result of the studies of K. phaffii CF-st401 vector plasmid, introduced into K. phaffii YIB Δleu2 VKPM Y-4761, it was shown that its regions responsible for the structure of the "Sweet protein Brazzein" coincide by more than 70% with elements of the brazzein P56552 reference protein from the plant P. brazzeana. The absence of selective markers and allergenicity in the vector plasmid was confirmed. Conclusion. The analysis of the structure of the DNA vector sequence of the K. phaffii CF-st401 GM strain confirmed the feasibility of using bioinformatics methods to predict the properties of technological microorganisms when assessing their safety for consumers.

Preference of Bacterial Rhamnosyltransferases for 6-Deoxysugars Reveals a Strategy To Deplete O-Antigens.

Bacteria synthesize hundreds of bacteria-specific or "rare" sugars that are absent in mammalian cells and enriched in 6-deoxy monosaccharides such as l-rhamnose (l-Rha). Across bacteria, l-Rha is incorporated into glycans by rhamnosyltransferases (RTs) that couple nucleotide sugar substrates (donors) to target biomolecules (acceptors). Since l-Rha is required for the biosynthesis of bacterial glycans involved in survival or host infection, RTs represent potential antibiotic or antivirulence targets. However, purified RTs and their unique bacterial sugar substrates have been difficult to obtain. Here, we use synthetic nucleotide rare sugar and glycolipid analogs to examine substrate recognition by three RTs that produce cell envelope components in diverse species, including a known pathogen. We find that bacterial RTs prefer pyrimidine nucleotide-linked 6-deoxysugars, not those containing a C6-hydroxyl, as donors. While glycolipid acceptors must contain a lipid, isoprenoid chain length, and stereochemistry can vary. Based on these observations, we demonstrate that a 6-deoxysugar transition state analog inhibits an RT in vitro and reduces levels of RT-dependent O-antigen polysaccharides in Gram-negative cells. As O-antigens are virulence factors, bacteria-specific sugar transferase inhibition represents a novel strategy to prevent bacterial infections.

Single-Droplet Microsensor for Ultra-Short Circulating EFGR Mutation Detection in Lung Cancer Based on Multiplex EFIRM Liquid Biopsy.

Liquid biopsy is a rapidly emerging field that involves the minimal/non-invasive assessment of signature somatic mutations through the analysis of circulating tumor DNA (ctDNA) shed by tumor cells in bodily fluids. Broadly speaking, the unmet need in liquid biopsy lung cancer detection is the lack of a multiplex platform that can detect a mutation panel of lung cancer genes using a minimum amount of sample, especially for ultra-short ctDNA (usctDNA). Here, we developed a non-PCR and non-NGS-based single-droplet-based multiplexing microsensor technology, "Electric-Field-Induced Released and Measurement (EFIRM) Liquid Biopsy" (m-eLB), for lung cancer-associated usctDNA. The m-eLB provides a multiplexable assessment of usctDNA within a single droplet of biofluid in only one well of micro-electrodes, as each electrode is coated with different probes for the ctDNA. This m-eLB prototype demonstrates accuracy for three tyrosine-kinase-inhibitor-related EGFR target sequences in synthetic nucleotides. The accuracy of the multiplexing assay has an area under the curve (AUC) of 0.98 for L858R, 0.94 for Ex19 deletion, and 0.93 for T790M. In combination, the 3 EGFR assay has an AUC of 0.97 for the multiplexing assay.

Discovery, implications and initial use of semi-synthetic organisms with an expanded genetic alphabet/code.

Much recent interest has focused on developing proteins for human use, such as in medicine. However, natural proteins are made up of only a limited number of canonical amino acids with limited functionalities, and this makes the discovery of variants with some functions difficult. The ability to recombinantly express proteins containing non-canonical amino acids (ncAAs) with properties selected to impart the protein with desired properties is expected to dramatically improve the discovery of proteins with different functions. Perhaps the most straightforward approach to such an expansion of the genetic code is through expansion of the genetic alphabet, so that new codon/anticodon pairs can be created to assign to ncAAs. In this review, I briefly summarize more than 20 years of effort leading ultimately to the discovery of synthetic nucleotides that pair to form an unnatural base pair, which when incorporated into DNA, is stably maintained, transcribed and used to translate proteins in Escherichia coli. In addition to discussing wide ranging conceptual implications, I also describe ongoing efforts at the pharmaceutical company Sanofi to employ the resulting 'semi-synthetic organisms' or SSOs, for the production of next-generation protein therapeutics. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Oligonucleotide drugs attract investment

Oligonucleotides—short, synthetic nucleotide strands that can be used as drugs to modulate gene expression—were at the heart of two deals inked in December. Vertex Pharmaceuticals will pay Entrada Therapeutics $250 million and up to $485 million later to advance treatments for myotonic dystrophy type 1, including a preclinical oligonucleotide therapy from Entrada that potentially restores muscle function. And GSK will pay Wave Life Sciences $170 million up front plus future milestones and royalties as part of a pact to advance up to 11 oligonucleotide programs.

Exploring the Distribution and Orientation of Cell Proliferation as Drivers of Mouse Facial Development

During the development of the face, tissues move, change shape, and fuse in tightly orchestrated patterns to create all the parts of a normal face. These shape changes are driven by factors such as cell signaling, migration, proliferation, and apoptosis. However, the contributions of each of these drivers to morphogenesis are poorly studied. Here, we explore differential cell proliferation as a driver of mouse facial morphogenesis. We quantify patterns in both the spatial distribution and orientation of proliferation in the developing face in 3D over a critical period of murine facial development (E9.5‐E11.5). We use immunostaining with light sheet microscopy (LSM) to capture total and proliferating nuclei. To compare proliferative density in facial tissues, we segment these images using a novel convolutional neural network. We then generate atlases of average proliferation at each half‐day age point within our range and use these to identify relationships between morphology and cell proliferation. We show that regions with more dense proliferation tend to undergo more intensive shape changes. We then simulate outgrowth of the maxillary process using a cell simulation engine, PhysiCell, to demonstrate that differential proliferation is necessary to maintain expected morphology in growing tissues. In addition to differential proliferation, localized orientation of cell division could also affect morphology. In plants and some animal tissues, including murine limb buds, preferentially oriented cell proliferation drives shape change by causing tissue elongation in specific directions. We explore the orientation and distribution of cell proliferation using LSM: we inject pregnant dams with a synthetic nucleotide, EdU, 5 minutes before harvest to mark the daughter cells of proliferative events occurring in the interim. We then compare the angles of the proliferative axes for each pair of daughters relative to the primary direction of tissue growth. Preliminary results suggest that cell proliferation in the maxillary and nasal processes is oriented preferentially towards the axis of tissue growth. These results suggest that both the distribution and orientation of cell proliferation play a role in murine facial morphogenesis. Understanding the mechanisms underlying morphogenesis is important to guide future research that could lead to earlier and more robust diagnosis and treatment of syndromes and facial abnormalities.

Advancement of Prodrug Approaches for Nucleotide Antiviral Agents.

Synthetic nucleoside or nucleotide analogues played a key role to the development of antiviral agents in past decades. However, low membrane permeability and insufficient cellular phosphorylation impaired the biological activity of polar nucleoside drugs because they have to penetrate the cell membrane and be phosphorylated to active metabolite stepwise by intracellular enzymes. To overcome these limitations, diverse lipophilic prodrug modifications based on nucleoside mono-, di-, and triphosphate were designed and put into practice to efficiently deliver nucleoside into the target site, and bypass the rate-limited phosphorylation step. As the most successful prodrug strategy, ProTide technology has led to the discovery of three FDA-approved antiviral agents, including sofosbuvir, tenofovir alafenadmide, and remdesivir, which has been authorized for emergency use in patients of COVID-19 in the US. In recent years, nucleoside di- and triphosphate prodrugs have also made the significant progress. This review will focus on the summary of design approach and metabolic activation path of different nucleotide prodrug strategies. The potential application of nucleotide prodrugs for the treatment of COVID-19 was also described due to the pandemic of SARS-CoV-2.

SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF ABACAVIR AND LAMIVUDINE IN COMBINED DOSAGE FORM

Antiretroviral therapy [ART] has been developed remarkably within last three decades, since the first NRTI (nucleoside reverse transcriptase inhibitor) developed. For the mostly patients the challenge of complete and sustain suppression of viral has been clarify, since the triple therapy arrived. Convenient pill burden and tolerability are the main limiting factor for enhancing the long term benefit of ART. ABACAVIR (ABA) and LAMIVUDINE (LAM) are synthetic nucleotide analog (SNA) that showing a synergistic and potent inhibitory effect on human immunodeficiency virus-I (HIV-I), causative agent of AIDS. ABA and LAM belongs to nucleoside reverse transcriptase inhibitor (NRTI) class. As per new therapeutic strategy these two antiretroviral (ARV) drugs are required for the treatment of AIDS. When ARV regimens changed due to lack to virologic response ABA should be avoided. ABA is getting converted in to the active metabolite (carbovir triphosphate) which is an deoxyguanosine-5’ triphosphate analog by intracellular enzymes. LAM gets converted in its active 5’-triphosphate metabolite through phosphorylation. Chemically, ABA is (1S, cis)-4-[2-amino-6-(cyclopropyl amino)-9H-purin-9-yl]-2-cyclopentene-1-methanol sulphate and LAM is (2R, cis)-4-amino-1-(2-hydroxymethyl-1, 3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one. Fig. 1 and 2 show ABA and LAM’s structure respectively.

Transcriptional processing of an unnatural base pair by eukaryotic RNA polymerase II.

The development of unnatural base pairs (UBPs) has greatly increased the information storage capacity of DNA, allowing for transcription of unnatural RNA by the heterologously expressed T7 RNA polymerase (RNAP) in Escherichia coli. However, little is known about how UBPs are transcribed by cellular RNA polymerases. Here, we investigate how synthetic unnatural nucleotides, NaM and TPT3, are recognized by eukaryotic RNA polymerase II (Pol II) and found that Pol II is able to selectively recognize UBPs with high fidelity when dTPT3 is in the template strand and rNaMTP acts as the nucleotide substrate. Our structural analysis and molecular dynamics simulation provide structural insights into transcriptional processing of UBPs in a stepwise manner. Intriguingly, we identified a novel 3’-RNA binding site after rNaM addition, termed the swing state. These results may pave the way for future studies to design transcription and translation strategies in higher organisms with expanded genetic codes.

Clinical Pharmacology Studies Supporting Oligonucleotide Therapy Development: An Assessment of Therapies Approved and in Development Between 2012 and 2018.

Synthetic nucleotides that utilize RNA‐centric pharmacology can target diseases at the RNA level, thus altering protein expression in ways previously inaccessible to small molecules and therapeutic biologics. Recognizing that the unique pharmacology of oligonucleotides may require specific considerations in pre‐approval assessment, clinical and nonclinical pharmacology studies being conducted for a selected set of oligonucleotide therapies in a 6‐year period were assessed. This investigation focused primarily on the four following areas: (i) drug‐drug interaction (DDI) potential, (ii) organ impairment (i.e., renal and hepatic impairment), (iii) immunogenicity, and (iv) cardiac safety. Data were summarized and assessed from 14 Investigational New Drug programs and 7 New Drug Applications submitted to the US Food and Drug Administration (FDA) from the period of January 2012 to August 2018, encompassing 152 unique studies. The assessment of DDI potential was largely consistent with the recommendations of current DDI‐relevant guidances. Limited data were available to provide recommendations across organ impairment categories. Limited data on immunogenicity indicate impact on pharmacokinetic, the impact on safety and efficacy, although not extensively evaluated, appeared negligible. Cardiac safety evaluation indicated a potential for discordant translation of risk from nonclinical studies to clinical findings. Continued experience with synthetic oligonucleotide therapies will help inform the development of best practices to support their development and regulatory approval.

From Whole Blood to Isolated Pro-Metastasis Immune Cells: An Ex Vivo Approach to Isolate and Manipulate Immune Cells Contributing to Tumor Metastasis.

Immune evasion hallmark has grabbed wide attention in cancer progression on the clinical level. Accordingly, innate and adaptive immune cells isolation and manipulation is essential in order to assess their activity and role in the tumor microenvironment (TME). This could open a gate toward a personalized therapy by a simple aspiration of blood sample from patients. Here, we describe the isolation of peripheral blood mononuclear cells (PBMCs) using Ficoll plus media in order to achieve the highest yield of immune cells that can be further processed and used in isolation of specific immune cells such as macrophages and cytotoxic T cells (CD8+ cells). Among the highly metastatic macrophages are the M2. This protocol describes the optimized techniques to isolate monocytes from whole blood, differentiate them into M2. This is followed by genetic and epigenetic (using synthetic nucleotides of noncoding RNAs) manipulation of these isolated immune cells in a tumor culture media, in addition to measurement of released cytokines using specific ELISA kit. In the last decade, new groups of noncoding RNAs have been emerged which are microRNAs and long noncoding RNAs. First, they were known as "junk DNA" with unknown regulatory functions. Despite the limited knowledge of these molecules, basic expression profiling is proving to be clinically relevant to cancer diagnosis, metastasis, and prognosis. Here, we describe methods used in molecular biology to assess the epigenetic expression of ncRNAs and their impact on other messenger RNA transcripts in M2 macrophages that could serve as future biomarkers in the context of tumor biology and metastasis or could open a gate in the treatment of cancer.

Expression of the Xylanase Gene from Pyromyces finnis in Pichia pastoris and Characterization of the Recombinant Protein

The heterologous expression and characteristics of a new xylanase from Pyromyces finnis are described. The endo-1,4-β-xylanase XylP (EC 3.2.1.8) consists of 223 amino acids and 19 residues of a putative signal peptide in the N-terminal region. The amino-acid sequence of the mature protein has the greatest homology (84%) with the sequence of the native catalytic N-terminal domain of Neocallimastix patriciarum endo-1,4-β-xylanase. A synthetic nucleotide sequence encoding the mature XylP protein was expressed in Pichia pastoris. The purified recombinant enzyme was active with birch xylan and arabinoxylan as substrates. The optimal pH and temperature for enzyme activity were established as 5.0 and 50°C, respectively, with the use of birch xylan. The specific activity of xylanase was 4700 U/mg protein, and KM and Vmax were equal to 0.51 mg/mL and 7395.3 μmol/(min mg), respectively. The recombinant XylP protein showed moderate thermal and high pH stability, as well as resistance to digestive enzymes and protein xylanase inhibitors from cereals. It was also shown that Mg2+, Co2+ and Li+ ions have a positive effect on enzyme activity.

Development of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Method for Simultaneous Detection of Spotted Fever Group Rickettsiae and Malaria Parasites by Dipstick DNA Chromatography.

Spotted fever group (SFG) rickettsiae causes febrile illness in humans worldwide. Since SFG rickettsiosis’s clinical presentation is nonspecific, it is frequently misdiagnosed as other febrile diseases, especially malaria, and complicates proper treatment. Aiming at rapid, simple, and simultaneous detection of SFG Rickettsia spp. and Plasmodium spp., we developed a novel multiple pathogen detection system by combining a loop-mediated isothermal amplification (LAMP) method and dipstick DNA chromatography technology. Two primer sets detecting SFG Rickettsia spp. and Plasmodium spp. were mixed, and amplified products were visualized by hybridizing to dipstick DNA chromatography. The multiplex LAMP with dipstick DNA chromatography distinguished amplified Rickettsia and Plasmodium targeted genes simultaneously. The determined sensitivity using synthetic nucleotides was 1000 copies per reaction for mixed Rickettsia and Plasmodium genes. When genomic DNA from in vitro cultured organisms was used, the sensitivity was 100 and 10 genome equivalents per reaction for Rickettsia monacensis and Plasmodium falciparum, respectively. Although further improvement will be required for more sensitive detection, our developed simultaneous diagnosis technique will contribute to the differential diagnosis of undifferentiated febrile illness caused by either SFG Rickettsia spp. or Plasmodium spp. in resource-limited endemic areas. Importantly, this scheme is potentially versatile for the simultaneous detection of diverse infectious diseases.

Regionalized cell proliferation in the symbiont-bearing gill of the hydrothermal vent mussel Bathymodiolus azoricus

Deep-sea mussels Bathymodiolus spp. harbor high densities of chemosynthetic bacterial symbionts located within their gill epithelial cells. Compared to non-symbiotic coastal mussel relatives of similar size, Bathymodiolus gills are considerably larger, a feature often considered an adaptation to symbiosis because it is related to the presence of intracellular bacteria in epithelial cells located in the lateral zone. In order to document the mechanisms underlying these sizes differences, this study compares gill cell proliferation patterns in Bathymodiolus azoricus and Mytilus edulis using microscopy-based approaches. We used incubation experiments with a synthetic nucleotide (5-ethynyl 2′-deoxyuridine, EdU), detectable throughout novel cell divisions, and phosphohistone H3 immunolabeling, a marker of mitosis. The results revealed proliferation areas in the ciliated zone and in the bacteria-loaded bacteriocytes located close to the frontal zone of gill filaments, swept by the incurrent sea-waterflow, and also in the dorsal region of gills in B. azoricus. Cell proliferation seems far less intensive in M. edulis. This study overall suggests high cell turnover and fast tissue dynamics in symbiont-bearing mussels.