Molecular Tools Research Articles

Potential of MALDI-TOF MS biotyping to detect deltamethrin resistance in the dengue vector Aedes aegypti.

Insecticide resistance in mosquitoes is spreading worldwide and represents a growing threat to vector control. Insecticide resistance is caused by different mechanisms including higher metabolic detoxication, target-site modification, reduced penetration and behavioral changes that are not easily detectable with simple diagnostic methods. Indeed, most molecular resistance diagnostic tools are costly and labor intensive and then difficult to use for routine monitoring of insecticide resistance. The present study aims to determine whether mosquito susceptibility status against the pyrethroid insecticides (mostly used for mosquito control) could be established by the protein signatures of legs and/or thoraxes submitted to MALDI-TOF Mass Spectrometry (MS). The quality of MS spectra for both body parts was controlled to avoid any bias due to unconformity protein profiling. The comparison of MS profiles from three inbreeds Ae. aegypti lines from French Guiana (IRF, IR03, IR13), with distinct deltamethrin resistance genotype / phenotype and the susceptible reference laboratory line BORA (French Polynesia), showed different protein signatures. On both body parts, the analysis of whole protein profiles revealed a singularity of BORA line compared to the three inbreeding lines from French Guiana origin, suggesting that the first criteria of differentiation is the geographical origin and/or the breeding history rather than the insecticide susceptibility profile. However, a deeper analysis of the protein profiles allowed to identify 10 and 11 discriminating peaks from leg and thorax spectra, respectively. Among them, a specific peak around 4870 Da was detected in legs and thoraxes of pyrethroid resistant lines compared to the susceptible counterparts hence suggesting that MS profiling may be promising to rapidly distinguish resistant and susceptible phenotypes. Further work is needed to confirm the nature of this peak as a deltamethrin resistant marker and to validate the routine use of MS profiling to track insecticide resistance in Ae. aegypti field populations.

Molecular characteristics and phylogenetic analysis of pigeon paramyxovirus type 1 isolates from pigeon meat farms in Shanghai (2009–2012)

The majority of pigeon paramyxovirus type 1 (PPMV-1) strains are generally non-pathogenic to chickens; however, they can induce severe illness and high mortality rates in pigeons, leading to substantial economic repercussions. The genomes of 11 PPMV-1 isolates from deceased pigeons on meat pigeon farms during passive monitoring from 2009 to 2012 were sequenced and analyzed using polymerase chain reaction and phylogenetic analysis. The complete genome lengths of 11 isolates were approximately 15,192 nucleotides, displaying a consistent gene order of 3′-NP-P-M-F-HN-L-5′. ALL isolates exhibited the characteristic motif of 112RRQKRF117 at the fusion protein cleavage site, which is characteristic of velogenic Newcastle disease virus. Moreover, multiple mutations have been identified within the functional domains of the F and HN proteins, encompassing the fusion peptide, heptad repeat region, transmembrane domains, and neutralizing epitopes. Phylogenetic analysis based on sequences of the F gene unveiled that all isolates clustered within genotype VI in class II. Further classification identified at least two distinct sub-genotypes, with seven isolates classified as sub-genotype VI.2.1.1.2.2, whereas the others were classified as sub-genotype VI.2.1.1.2.1. This study suggests that both sub-genotypes were implicated in severe disease manifestation among meat pigeons, with sub-genotype VI.2.1.1.2.2 displaying an increasing prevalence among Shanghai’s meat pigeon population since 2011. These results emphasize the value of developing pigeon-specific vaccines and molecular diagnostic tools for monitoring and proactively managing potential PPMV-1 outbreaks.

Programming Spatial Cell Sorting by Engineering Cadherin Intracellular Activity.

The spatial sorting of cells into appropriate tissue compartments is essential for embryogenesis and tissue development. Spatial cell sorting is controlled by the interplay between cell surface affinity and intracellular mechanical properties. However, intracellular signaling that can sufficiently sort cell populations remains unexplored. In this study, we engineered chimeric cadherins by replacing the cadherin intracellular domain with cytoskeletal regulators to test their ability to induce spatial cell sorting. Using a fibroblast-based reconstitution system, we observed that Rac1 and RhoA activity in the cadherin tail induced outward and inward sorting, respectively. In particular, RhoA activity embedded cells toward the inside of E-cadherin-expressing spheroids and tumor spheroids, leading to tissue invagination. Despite the simplicity of chimeric cadherin design, our results indicate that differences in cadherin intracellular activities can determine the direction of spatial cell sorting, even when cell surface affinity is not different, and provide new molecular tools to engineer tissue architectures.

Reprogramming tendon healing: a guide to novel molecular tools

Tendons are a frequent site of injury, which greatly impairs the movement and locomotion of patients. Regrettably, injuries at the tendon frequently require surgical intervention, which leads to a long path to recovery. Moreover, the healing of tendons often involves the formation of scar tissue at the site of injury with poor mechanical properties and prone to re-injury. Tissue engineering carries the promise of better and more effective solutions to the improper healing of tendons. Lately, the field of regenerative medicine has seen a significant increase in the focus on the potential use of non-coding RNAs (e.g., siRNAs, miRNAs, and lncRNAs) as molecular tools for tendon tissue engineering. This class of molecules is being investigated due to their ability to act as epigenetic regulators of gene expression and protein production. Thus, providing a molecular instrument to fine-tune, reprogram, and modulate the processes of tendon differentiation, healing, and regeneration. This review focuses particularly on the latest advances involving the use of siRNAs, miRNAs, and lncRNAs in tendon tissue engineering applications.

Development and validation of a TaqMan-based qPCR assay for reliable detection of invasive sailfin catfishes (Pterygoplichthys spp.) through measurements of environmental DNA

Sailfin catfishes (Pterygoplichthys spp.) are a group of fishes introduced to several freshwater habitats across the globe. Sailfin catfishes are a hard-armoured fish with no economic importance as a food species. These catfishes have been reported to be affecting the growth of several economically valuable species that are native to different freshwater ecosystems. In this manuscript, we developed a TaqMan-based assay targeting the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene, to specifically detect the presence of catfishes by environmental DNA (eDNA) measurements. The developed assay was screened against 16 species of fish and mussel that co-occur in the habitats along with sailfin catfishes, and the qPCR reaction did not have any non-target amplifications. The assay was further validated under laboratory conditions by measuring eDNA from tanks having different biomass of catfishes (1 and 0.5 g L−1), and the copy number of the amplified product was directly proportional to the biomass maintained in the tanks. Overall, the study provides a robust molecular tool based on eDNA measurements to facilitate detection of invasive sailfin catfishes and, in turn, contributes towards the management of the invaded habitats.

On the Identity of a rare Morphologically Abnormal Butterfly ray from the east Coast of India Resolved Through Molecular tool

On the Identity of a rare Morphologically Abnormal Butterfly ray from the east Coast of India Resolved Through Molecular tool

Characterizing the Diversity of Oomycetes Associated with Diseased Cotton Seedlings in Alabama.

Many oomycete species are associated with the seedlings of crops, including upland cotton (Gossypium hirsutum L.), which leads to annual threats. The diversity of oomycete species in Alabama needs to be better understood since the last survey of oomycetes associated with cotton in Alabama was 20 years ago-before significant updates to taxonomy and improvements in identification of oomycetes using molecular tools. Our current study aimed to identify oomycetes associated with Alabama cotton seedlings, correlate diversity with soil edaphic factors, and assess virulence toward cotton seed. Thirty symptomatic cotton seedlings were collected independently from 25 fields in 2021 and 2022 2 to 4 weeks after planting. Oomycetes were isolated by plating root sections onto a semiselective medium. The internal transcribed spacer (ITS) region was sequenced to identify the resulting isolates. A seed virulence assay was conducted in vitro to verify pathogenicity, and 347 oomycete isolates were obtained representing 36 species. Northern Alabama soils had the richest oomycete communities and a greater silt and clay concentration than sandier soils in the central and southern coastal plains. Globisporangium irregulare and Phytophthora nicotianae were consistently recovered from cotton roots in both years. Globisporangium irregulare was pathogenic and recovered from all Alabama regions, whereas P. nicotianae was pathogenic but recovered primarily in areas with lower sand content in northern Alabama. Many oomycete species have not been previously reported in Alabama or the southeastern United States. Altogether, this knowledge will help facilitate effective management strategies for cotton seedling diseases caused by oomycetes in Alabama and the United States.

Genome-wide identification of WRKY transcription factors in Casuarina equisetifolia and the function analysis of CeqWRKY11 in response to NaCl/NaHCO3 stresses

BackgroundCasuarina equisetifolia (C. equisetifolia) is a woody species with many excellent features. It has natural resistance against drought, salt and saline-alkali stresses. WRKY transcription factors (TFs) play significant roles in plant response to abiotic stresses, therefore, molecular characterization of WRKY gene family under abiotic stresses holds great significance for improvement of forest trees through molecular biological tools. At present, WRKY TFs from C. equisetifolia have not been thoroughly studied with respect to their role in salt and saline-alkali stresses response. The current study was conducted to bridge the same knowledge gap.ResultsA total of 64 WRKYs were identified in C. equisetifolia and divided into three major groups i.e. group I, II and III, consisting of 10, 42 and 12 WRKY members, respectively. The WRKY members in group II were further divided into 5 subgroups according to their homology with Arabidopsis counterparts. WRKYs belonging to the same group exhibited higher similarities in gene structure and the presence of conserved motifs. Promoter analysis data showed the presence of various response elements, especially those related to hormone signaling and abiotic stresses, such as ABRE (ABA), TGACG (MeJA), W-box ((C/T) TGAC (T/C)) and TC-rich motif. Tissue specific expression data showed that CeqWRKYs were mainly expressed in root under normal growth conditions. Furthermore, most of the CeqWRKYs were up-regulated by NaCl and NaHCO3 stresses with few of WRKYs showing early responsiveness to both stresses while few others exhibiting late response. Although the expressions of CeqWRKYs were also induced by cold stress, the response was delayed compared with other stresses. Transgenic C. equisetifolia plants overexpressing CeqWRKY11 displayed lower electrolyte leakage, higher chlorophyll content, and enhanced tolerance to both stresses. The higher expression of abiotic stress related genes, especially CeqHKT1 and CeqPOD7, in overexpression lines points to the maintenance of optimum Na+/K+ ratio, and ROS scavenging as possible key molecular mechanisms underlying salt stress tolerance.ConclusionsOur results show that CeqWRKYs might be key regulators of NaCl and NaHCO3 stresses response in C. equisetifolia. In addition, positive correlation of CeqWRKY11 expression with increased stress tolerance in C. equisetifolia encourages further research on other WRKY family members through functional genomic tools. The best candidates could be incorporated in other woody plant species for improving stress tolerance.

The Role of Bio-fortification in Enhancing the Nutritional Quality of Vegetables: A Review

Bio-fortification is a process that enhances the nutritional quality of crops, including vegetables, by increasing their micronutrient content. It can be achieved through different approaches such as agronomic, conventional breeding, and transgenic/biotechnological methods. Vegetables are known to be rich in micronutrients, vitamins, antioxidants, and other health-benefiting compounds, making them essential for a balanced diet. However, malnutrition and hidden hunger continue to be global challenges, particularly in developing countries. Micronutrient deficiencies, such as iron, zinc, iodine, and vitamin A, are prevalent in these populations. Conventional breeding focuses on selecting genotypes with desirable nutritional traits without compromising agricultural productivity. Transgenic/biotechnological approaches involve the synthesis of transgenes to enhance the bioavailability of micronutrients in plants. Bio-fortification of vegetables is particularly important as they are rich sources of micronutrients, vitamins, and other health-benefiting compounds. However, improving the nutritional quality of vegetables through conventional breeding has had limited success, and modern molecular tools and techniques offer potential for handling complex traits and developing nutrient-dense varieties. Bio-fortification offers a sustainable solution to address these deficiencies by increasing the nutrient content of crops, particularly plant-based foods. These techniques have shown promising results in increasing the concentration of nutrients, such as iron, in vegetables, thereby improving their nutritional quality.

Modular Low-Copy-Number Plasmid Vectors for Rhodobacterales with Extended Host Range in Alphaproteobacteria.

Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for marine biotechnology, due to their ecological adaptability, wealth of versatile low-copy-number plasmids, and their ability to produce secondary metabolites. However, molecular tools for engineering strains of this bacterial lineage are limited. Here, we expand the genetic toolbox by establishing standardized, modular repABC-based plasmid vectors of four well-characterized compatibility groups from the Roseobacter group applicable in the Rhodobacterales, and likely in further alphaproteobacterial orders (Hyphomicrobiales, Rhodospirillales, Caulobacterales). We confirmed replication of these newly constructed pABC vectors in two members of Rhodobacterales, namely, Dinoroseobacter shibae DFL 12 and Rhodobacter capsulatus B10S, as well as in two members of the alphaproteobacterial order Hyphomicrobiales (synonym: Rhizobiales; Ensifer meliloti 2011 and "Agrobacterium fabrum" C58). Maintenance of the pABC vectors in the biotechnologically valuable orders Rhodobacterales and Hyphomicrobiales facilitates the shuttling of genetic constructs between alphaproteobacterial genera and orders. Additionally, plasmid replication was verified in one member of Rhodospirillales (Rhodospirillum rubrum S1) as well as in one member of Caulobacterales (Caulobacter vibrioides CB15N). The modular construction of pABC vectors and the usage of four compatible replication systems, which allows their coexistence in a host cell, are advantageous features for future implementations of newly designed synthetic pathways. The vector applicability was demonstrated by functional complementation of a nitrogenase mutant phenotype by two complementary pABC-based plasmids in R. capsulatus.

Widespread establishment of adventive populations of Leptopilina japonica (Hymenoptera, Figitidae) in North America and development of a multiplex PCR assay to identify key parasitoids of Drosophila suzukii (Diptera, Drosophilidae)

In recent years, there has been an increase in the adventive establishment and spread of parasitoid wasps outside of their native range. However, lack of taxonomic tools can hinder the efficient screening of field-collected samples to document the establishment and range expansion of parasitoids on continent-wide geographic scales. Here we report that Leptopilina japonica (Hymenoptera, Figitidae), a parasitoid of the globally invasive fruit pest Drosophila suzukii (Diptera, Drosophilidae), is now widespread in much of North America despite not having been intentionally introduced. Surveys in 2022 using a variety of methods detected L. japonica in 10 of 11 surveyed USA States and one Canadian Province where it was not previously known to occur. In most surveys, L. japonica was the most common species of D. suzukii parasitoid found. The surveys also resulted in the detection of Ganaspis cf. brasiliensis (Hymenoptera, Figitidae), the recently-released biological control agent of D. suzukii, in six USA States where it had not previously been found. These new detections are likely a result of intentional biological control introductions rather than spread of adventive populations. A species-specific multiplex PCR assay was developed as a rapid, accurate and cost-effective method to distinguish L. japonica, G. cf. brasiliensis, the closely-related cosmopolitan parasitoid Leptopilina heterotoma (Hymenoptera, Figitidae) and other native parasitoid species. This dataset and the associated molecular tools will facilitate future studies of the spread and ecological impacts of these introduced parasitoids on multiple continents.

Simplified molecular diagnosis of visceral leishmaniasis: Laboratory evaluation of miniature direct-on-blood PCR nucleic acid lateral flow immunoassay.

Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy.

Whole Exome Sequencing as an Effective Molecular Diagnosis Tool for Craniofacial Fibrous Dysplasia with Ocular Complications

Purpose To summarize the clinical manifestations of craniofacial fibrous dysplasia (CFD) patients with ocular complications, and find effective methods to diagnose early. Methods Nine CFD patients with ocular complications, and their parents were recruited in this study. All patients underwent ocular and systemic examinations. Bone lesions from all patients and peripheral blood from patients and their parents were collected for whole exome sequencing (WES). According to the screening for low-frequency deleterious variants, and bioinformatics variants prediction software, possible disease-causing variants were found in multiple CFD patients. The variants were validated by Sanger sequencing. Trio analysis was performed to verify the genetic patterns of CFD. Results All patients were diagnosed with CFD, according to the clinical manifestations, classic radiographic appearance, and pathological biopsy. The main symptoms of the 9 CFD patients, included visual decline (9/9), craniofacial deformity (3/9) and strabismus (2/9), with few extraocular manifestations. The family backgrounds of all the CFD patients indicated that only the patient was affected, and their immediate family members were normal. GNAS variants were identified in all bone lesions from CFD patients, including two variant types: c.601C > T:p.R201C(6/9) and c.602G > A:p.R201H (3/9) in exon 8. The detection rate reached 100% by WES, but only 77.8% by Sanger sequencing. Interestingly, we found GNAS variants could not be detected in peripheral blood samples from CFD patients or their parents, and other potentially disease-causing gene variants related to CFD were not found. Conclusions For CFD patients with bone lesions involving the optic canal or sphenoid sinus regions, ocular symptoms should also be considered. Furthermore, we confirmed that CFD is not inherited, somatic variants in the GNAS gene are the main pathogenic gene causing CFD. Compared to the traditional methods in molecular genetic diagnosis of CFD, WES is more feasible and effective but limited in the type of samples.

Environmental DNA as a tool to reconstruct catch composition for longline fisheries vessels

Global wild-capture fisheries are a large and diverse sector requiring various tools for fisheries-dependant data collection and effective Monitoring, Control and Surveillance (MCS). Here we present a novel protocol to collect eDNA from brine tanks onboard commercial longline vessels to reconstruct catch composition. We collected samples from nine vessels operating out of the Eastern Tuna Billfish Fishery, Australia, validating eDNA results with reliable catch data consisting of seven target and bycatch species. Environmental DNA was highly effective for detecting species retained on vessels without contamination or false positives. For four vessels, logbook data and eDNA were consistent with detections of all species. The remaining vessels detected all species except for rare catches of short-billed spearfish (Tetrapturus angustirostris). Similarities between rank abundance distributions of catch and eDNA reads were observed with logbook data mirrored when eDNA sequences were organised into rank order abundance. The method was effective at identifying highly abundant taxa retained in brine tanks- tuna (Thunnus spp.), swordfish (Xiphias gladius), marlin (Kajijia audax), and Atlantic Pomfret (Brama brama). Further research is required to validate how eDNA and other molecular monitoring tools can be scaled and applied to provide solutions for monitoring challenges in the fisheries sector.

A two-photon fluorescent probe for highly selective detection of Cys over GSH and Hcy based on the Michael addition and transcyclization mechanism and its application in bioimaging and protein straining in SDS-PAGE

BackgroundCysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging. ResultsHere, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 μM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 μg/mL) than classical Coomassie brilliant blue (14.11 μg/mL). SignificanceThe outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method.

Polyfluoroalkyl Tag Decoration Enables Significantly Enhanced Tumor Penetration Ability of a PTK7 Targeting Aptamer.

Aptamers are widely used molecular recognition tools in targeted therapy, but their ability to effectively penetrate deep into solid tumors remains a significant challenge, leading to suboptimal treatment efficacy. Here, we developed a polyfluoroalkyl (PFA) decoration strategy to enhance aptamer recognition, cell internalization, and solid tumor penetration. Our results indicate that PFA with around 11 fluorine atoms significantly improves aptamer internalization both in vitro and in vivo settings. However, we also observed that the use of PFA tags containing 19 and 23 fluorine atoms on aptamers resulted in nonspecific cell anchoring in control cell lines, affecting the specificity of aptamers. Overall, we found that using a chemical modification strategy could enhance the deep tumor penetration ability of aptamers and validate their effectiveness in vivo. This approach has significant practical applications in targeted drug delivery for cancer treatment.

Integrating molecular and radiological screening tools during community-based active case-finding for tuberculosis and COVID-19 in southern Africa

ObjectivesTo evaluate diagnostic yield and feasibility of integrating testing for TB and COVID-19 using molecular and radiological screening tools during community-based active case-finding (ACF). MethodsCommunity-based participants with presumed TB and/or COVID-19 were recruited using a mobile clinic. Participants underwent simultaneous point-of-care (POC) testing for TB (sputum; Xpert-Ultra) and COVID-19 (nasopharyngeal swabs; Xpert-SARS-CoV-2). Sputum culture and SARS-CoV-2 RT-PCR served as reference standards. Participants underwent ultra-portable POC chest-radiography with computer-aided detection (CAD). TB infectiousness was evaluated using smear microscopy, cough aerosol sampling studies (CASS), and chest radiographic cavity detection. Feasibility of POC testing was evaluated via user-appraisals. Results601 participants were enrolled, with 144/601 (24.0%) reporting symptoms suggestive of TB and/or COVID-19. 16/144 (11.1%) participants tested positive for TB, while 10/144 (6.9%) tested positive for COVID-19 (2/144 [1.4%] had concurrent TB/COVID-19). 7/16 (43.8%) individuals with TB were probably infectious. Test-specific sensitivity and specificity (95% CI) were: Xpert-Ultra 75.0% (42.8-94.5) and 96.9% (92.4-99.2); Xpert-SARS-CoV-2 66.7% (22.3-95.7) and 97.1% (92.7-99.2). Area-under-the-curve (AUC) for CAD4TB was 0.90 (0.82-0.97). User appraisals indicated POC Xpert to have ‘good’ user-friendliness. ConclusionsIntegrating TB/COVID-19 screening during community-based ACF using POC molecular and radiological tools is feasible, has a high diagnostic yield, and can identity probably infectious persons.

Redesigning Humans in the Nano-Age

The blurring boundaries between therapy and human enhancement raise a lot of ethical questions that need to be addressed. Therapy is healing and restoring the sick to health; enhancement is improving the normal functioning of the body. With nanomedicine, the boundary becomes even hazier. Nanomedicine refers to the application of nanotechnology to the prevention and treatment of diseases. The European Science Foundation defines it as the science and technology of diagnosing, treating, and preventing disease and traumatic injury, relieving pain, and preserving and improving human health using molecular tools and knowledge of the human body (ESF, 2005). While nanomedicine delivers numerous and unprecedented benefits to the health and general well-being of individuals, nano- enabled human enhancement technologies will be the gateway to the transhumanist vision of immortality. Transhumanism contends that through the advancements in science and technology, we can overcome our physical and cognitive limitations, extend human lifespan, and attain perpetual existence. The blurring boundaries between enhancement will have remarkable consequences on what it means to be a human being. The study will make use of New and Emerging Science and Technology (NEST) Ethics as a framework for addressing dichotomous approaches and polarizing perspectives on human enhancement.

Single primer site-specific nested PCR for accurate and rapid genome-walking

Genome-walking is a molecular tool used to unveil uncharacterized DNA regions flanking a known DNA, which has been widely used in bioscience and related areas. This study developed a reliable and efficient PCR-based genome-walking approach, named as single primer site-specific nested PCR (SPN-PCR). A SPN-PCR set sequentially consists of three single-primer nested PCR amplifications. The primary relaxed thermal cycle promotes outmost nested site-specific primer (NSSP) to partially combine with numerous places on DNA template, synthesizing many single-stranded DNAs (ssDNA). Among them, the target ssDNA is exponentially amplified in the subsequent stringent cycles, as its 3′ part possesses the outmost NSSP complement; but a non-target ssDNA cannot be amplified, because it does not possess such a complement. Stringent secondary and tertiary PCRs also exclusively enrich this target DNA. Finally, the target DNA product becomes predominant. The feasibility of SPN-PCR was validated by genome-walking several selected genes from two divergent species.

Rapidly engineering an osmotic-pressure-tolerant gut bacterium for efficient non-sterile production of bulk chemicals

The concept of next-generation industrial biotechnology (NGIB) has been presented as a green biomanufacturing approach based on the use of low-cost substrates in an open and continuous non-sterilization fermentation process. However, the realization of NGIB, partially through the utilization of extremophiles, has been impeded by the challenges in engineering these extremophiles. A previously unexplored solution for advancing NGIB is to obtain readily engineerable microorganisms with osmotic-pressure tolerance. Here, Jejubacter sp. L23 strain, a Gram-negative, rod-shaped, osmotic-pressure-tolerant bacterium, was isolated from the gut of superworm Zophobas atratus and identified as a new member of the family Enterobacteriaceae, with high 16S rDNA similarity to Escherichia coli (95.2 %). The growth of L23 strain was observed at 10 to 45 °C and pH 4.0 to 10.0 in the presence of 0 to 12 % (w/v) NaCl. We systematically investigated the genetic parts, molecular tools, and manipulation approaches applicable to the L23 strain, and successfully conducted chromosomal genetic engineering, e.g. base-editing and deletion, by using high-efficiency CRISPR systems under low-salt conditions. Furthermore, the metabolic network of L23 strain was rewired to obtain the engineered L23R7 strain, which could display stable expression of exogenous genes and produce bulk chemicals at high yields using inulin, abundant non-food biomass, and salt water without sterilization. For example, 41 g/L isobutanol was produced by utilizing inulin in a 3-L bioreactor through a non-sterile fermentation process. This potent industrial strain holds the promise of promoting the development of NGIB as a sustainable biomanufacturing solution.