Advancing biological investigations using portable sensors for detection of sensitive samples.

Portable biosensors are emerged as powerful diagnostic tools for analyzing intricately complex biological samples. These biosensors offer sensitive detection capabilities by utilizing biomolecules such as proteins, nucleic acids, microbes or microbial products, antibodies, and enzymes. Their speed, accuracy, stability, specificity, and low cost make them indispensable in forensic investigations and criminal cases. Notably, portable biosensors have been developed to rapidly detect toxins, poisons, body fluids, and explosives; they have proven invaluable in forensic examinations of suspected samples, generating efficient results that enable effective and fair trials. One of the key advantages of portable biosensors is their ability to provide sensitive and non-destructive detection of forensic samples without requiring extensive sample preparation, thereby reducing the possibility of false results. This comprehensive review provides an overview of the current advancements in portable biosensors for the detection of sensitive materials, highlighting their significance in advancing investigations and enhancing sensitive sample detection capabilities.

Profiling of conserved orthologs and miRNAs for understanding their role in salt tolerance mechanism of Sesuvium portulacastrum L.

BACKGROUND: Sesuvium portulacastrum is a facultative halophyte capable of thriving in a saline environment. Despite molecular studies conducted to unravel its salt adaptation mechanism, there is a paucity of information on the role of salt-responsive orthologs and microRNAs (miRNAs) in this halophyte. Here, we searched the orthology to identify salt-responsive orthologs and miRNA targets of Sesuvium using the Arabidopsis genome. METHODS: The relative fold change of orthologs, conserved miRNAs, and miRNA targets of Sesuvium was analyzed under 100 mM (LS) and 250 mM NaCl (HS) treatment at 24 h using qRT-PCR. The comparison between the expression of Sesuvium orthologs and Arabidopsis orthologs (Arabidopsis eFP browser database) was used to identify differentially expressed genes. RESULTS: Upon salt treatment, we found that SpCIPK3 (1.95-fold in LS and 2.90-fold in HS) in Sesuvium roots, and SpNHX7 (1.61-fold in LS and 6.39-fold in HS) and, SpSTPK2 (2.54-fold in LS and 7.65-fold in HS) in Sesuvium leaves were upregulated in a salt concentration-specific manner. In Arabidopsis, these genes were either downregulated or did not show significant variation, implicating its significance in the halophytic nature of Sesuvium. Furthermore, miRNAs like miR394a, miR396a, and miR397a exhibited a negative correlation with their targets-Frigida interacting protein 1, Cysteine proteinases superfamily protein, and Putative laccase, respectively under different salt treatments. CONCLUSION: The study revealed that the high salt tolerance in Sesuvium is associated with distinct transcriptional reprogramming, hence, to gain holistic mechanistic insights, global-scale profiling is required.

De Novo Mining and Validating Novel Microsatellite Markers to Assess Genetic Diversity in Maruca vitrata (F.), a Legume Pod Borer.

Maruca vitrata (Fabricius) is an invasive insect pest capable of causing enormous economic losses to a broad spectrum of leguminous crops. Microsatellites are valuable molecular markers for population genetic studies; however, an inadequate number of M. vitrata microsatellite loci are available to carry out population association studies. Thus, we utilized this insect's public domain databases for mining expressed sequence tags (EST)-derived microsatellite markers. In total, 234 microsatellite markers were identified from 10053 unigenes. We discovered that trinucleotide repeats were the most predominant microsatellite motifs (61.53%), followed by dinucleotide repeats (23.50%) and tetranucleotide repeats (14.95%). Based on the analysis, twenty-five markers were selected for validation in M. vitrata populations collected from various regions of India. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) ranged from 2 to 5; 0.00 to 0.80; and 0.10 to 0.69, respectively. The polymorphic loci showed polymorphism information content (PIC), ranging from 0.09 to 0.72. Based on the genetic distance matrix, the unrooted neighbor-joining dendrogram differentiated the selected populations into two discrete groups. The SSR markers developed and validated in this study will be helpful in population-level investigations of M. vitrata to understand the gene flow, demography, dispersal patterns, biotype differentiation, and host dynamics.

Comprehensive mechanisms of heavy metal toxicity in plants, detoxification, and remediation.

Natural and anthropogenic causes are continually growing sources of metals in the ecosystem; hence, heavy metal (HM) accumulation has become a primary environmental concern. HM contamination poses a serious threat to plants. A major focus of global research has been to develop cost-effective and proficient phytoremediation technologies to rehabilitate HM-contaminated soil. In this regard, there is a need for insights into the mechanisms associated with the accumulation and tolerance of HMs in plants. It has been recently suggested that plant root architecture has a critical role in the processes that determine sensitivity or tolerance to HMs stress. Several plant species, including those from aquatic habitats, are considered good hyperaccumulators for HM cleanup. Several transporters, such as the ABC transporter family, NRAMP, HMA, and metal tolerance proteins, are involved in the metal acquisition mechanisms. Omics tools have shown that HM stress regulates several genes, stress metabolites or small molecules, microRNAs, and phytohormones to promote tolerance to HM stress and for efficient regulation of metabolic pathways for survival. This review presents a mechanistic view of HM uptake, translocation, and detoxification. Sustainable plant-based solutions may provide essential and economical means of mitigating HM toxicity.

Gene Coexpression Analysis Identifies Genes Associated with Chlorophyll Content and Relative Water Content in Pearl Millet.

Pearl millet is a significant crop that is tolerant to abiotic stresses and is a staple food of arid regions. However, its underlying mechanisms of stress tolerance are not fully understood. Plant survival is regulated by the ability to perceive a stress signal and induce appropriate physiological changes. Here, we screened for genes regulating physiological changes such as chlorophyll content (CC) and relative water content (RWC) in response to abiotic stress by using "weighted gene coexpression network analysis" (WGCNA) and clustering changes in physiological traits, i.e., CC and RWC associated with gene expression. Genes' correlations with traits were defined in the form of modules, and different color names were used to denote a particular module. Modules are groups of genes with similar patterns of expression, which also tend to be functionally related and co-regulated. In WGCNA, the dark green module (7082 genes) showed a significant positive correlation with CC, and the black (1393 genes) module was negatively correlated with CC and RWC. Analysis of the module positively correlated with CC highlighted ribosome synthesis and plant hormone signaling as the most significant pathways. Potassium transporter 8 and monothiol glutaredoxin were reported as the topmost hub genes in the dark green module. In Clust analysis, 2987 genes were found to display a correlation with increasing CC and RWC. Furthermore, the pathway analysis of these clusters identified the ribosome and thermogenesis as positive regulators of RWC and CC, respectively. Our study provides novel insights into the molecular mechanisms regulating CC and RWC in pearl millet.

Aptamer-based CRISPR-Cas powered diagnostics of diverse biomarkers and small molecule targets.

CRISPR-Cas systems have been widely used in genome editing and transcriptional regulation. Recently, CRISPR-Cas effectors are adopted for biosensor construction due to its adjustable properties, such as simplicity of design, easy operation, collateral cleavage activity, and high biocompatibility. Aptamers' excellent sensitivity, specificity, in vitro synthesis, base-pairing, labeling, modification, and programmability has made them an attractive molecular recognition element for inclusion in CRISPR-Cas systems. Here, we review current advances in aptamer-based CRISPR-Cas sensors. We briefly discuss aptamers and the knowledge of Cas effector proteins, crRNA, reporter probes, analytes, and applications of target-specific aptamers. Next, we provide fabrication strategies, molecular binding, and detection using fluorescence, electrochemical, colorimetric, nanomaterials, Rayleigh, and Raman scattering. The application of CRISPR-Cas systems in aptamer-based sensing of a wide range of biomarkers (disease and pathogens) and toxic contaminants is growing. This review provides an update and offers novel insights into developing CRISPR-Cas-based sensors using ssDNA aptamers with high efficiency and specificity for point-of-care setting diagnostics.

Phosphorylation of the auxin signaling transcriptional repressor IAA15 by MPKs is required for the suppression of root development under drought stress in Arabidopsis.

Since plants are sessile organisms, developmental plasticity in response to environmental stresses is essential for their survival. Upon exposure to drought, lateral root development is suppressed to induce drought tolerance. However, the molecular mechanism by which the development of lateral roots is inhibited by drought is largely unknown. In this study, the auxin signaling repressor IAA15 was identified as a novel substrate of mitogen-activated protein kinases (MPKs) and was shown to suppress lateral root development in response to drought through stabilization by phosphorylation. Both MPK3 and MPK6 directly phosphorylated IAA15 at the Ser-2 and Thr-28 residues. Transgenic plants overexpressing a phospho-mimicking mutant of IAA15 (IAA15DD OX) showed reduced lateral root development due to a higher accumulation of IAA15. In addition, MPK-mediated phosphorylation strongly increased the stability of IAA15 through the inhibition of polyubiquitination. Furthermore, IAA15DD OX plants showed the transcriptional downregulation of two key transcription factors LBD16 and LBD29, responsible for lateral root development. Overall, this study provides the molecular mechanism that explains the significance of the MPK-Aux/IAA module in suppressing lateral root development in response to drought.

Engineering drought and salinity tolerance traits in crops through CRISPR-mediated genome editing: Targets, tools, challenges, and perspectives.

Prolonged periods of drought triggered by climate change hamper plant growth and cause substantial agricultural yield losses every year. In addition to drought, salinity is one of the major abiotic stresses that severely affect crop health and agricultural production. Plant responses to drought and salinity involve multiple processes that operate in a spatiotemporal manner, such as stress sensing, perception, epigenetic modifications, transcription, post-transcriptional processing, translation, and post-translational changes. Consequently, drought and salinity stress tolerance are polygenic traits influenced by genome-environment interactions. One of the ideal solutions to these challenges is the development of high-yielding crop varieties with enhanced stress tolerance, together with improved agricultural practices. Recently, genome-editing technologies, especially clustered regularly interspaced short palindromic repeats (CRISPR) tools, have been effectively applied to elucidate how plants deal with drought and saline environments. In this work, we aim to portray that the combined use of CRISPR-based genome engineering tools and modern genomic-assisted breeding approaches are gaining momentum in identifying genetic determinants of complex traits for crop improvement. This review provides a synopsis of plant responses to drought and salinity stresses at the morphological, physiological, and molecular levels. We also highlight recent advances in CRISPR-based tools and their use in understanding the multi-level nature of plant adaptations to drought and salinity stress. Integrating CRISPR tools with modern breeding approaches is ideal for identifying genetic factors that regulate plant stress-response pathways and for the introgression of beneficial traits to develop stress-resilient crops.

Selenoprotein: Potential Player in Redox Regulation in Chlamydomonas reinhardtii.

Selenium (Se) is an essential micro-element for many organisms, including Chlamydomonas reinhardtii, and is required in trace amounts. It is obtained from the 21st amino acid selenocysteine (Sec, U), genetically encoded by the UGA codon. Proteins containing Sec are known as selenoproteins. In eukaryotes, selenoproteins are present in animals and algae, whereas fungi and higher plants lack them. The human genome contains 25 selenoproteins, most of which are involved in antioxidant defense activity, redox regulation, and redox signaling. In algae, 42 selenoprotein families were identified using various bioinformatics approaches, out of which C. reinhardtii is known to have 10 selenoprotein genes. However, the role of selenoproteins in Chlamydomonas is yet to be reported. Chlamydomonas selenoproteins contain conserved domains such as CVNVGC and GCUG, in the case of thioredoxin reductase, and CXXU in other selenoproteins. Interestingly, Sec amino acid residue is present in a catalytically active domain in Chlamydomonas selenoproteins, similar to human selenoproteins. Based on catalytical active sites and conserved domains present in Chlamydomonas selenoproteins, we suggest that Chlamydomonas selenoproteins could have a role in redox regulation and defense by acting as antioxidants in various physiological conditions.

Identification and structural analysis of novel malathion-specific DNA aptameric sensors designed for food testing.

Malathion is an organophosphate chemical (OPC) and a toxic contaminant that adversely impacts food quality, human health, biodiversity, and the environment. Due to its small size and unavailability of sensitive sensors, detection of malathion remains a challenging task. Often chromatographic methods employed to analyze OPCs suffer from several shortcomings, including cost, immobility, laboriousness, and unsuitability for point-of-care settings. Hence, developing a specific and sensitive diagnostic sensor for quick and inexpensive food testing is essential. We discovered four unique malathion-specific ssDNA aptamers; designed two independent sensing strategies using fluorescence labeling and Thioflavin T (ThT) displacement. Selected aptamers formed the G4-quadruplex-like (G4Q) structure, which helped develop a label-free detection approach with a 2.01 ppb limit of detection. Additionally, 3D structures of aptamers were generated and validated using a series of computational modeling programs. Furthermore, we explored structural features using CD spectroscopy and molecular docking, probing ligands' binding mode, and revealed vital intermolecular interactions with aptamers. Subsequently, the novel sensors were optimized to detect malathion from food samples. The novel sensors could be further developed to meet the demands of sensing and quantifying toxic contaminants from real food samples in field conditions.

Assessment of genetic diversity and volatile content of commercially grown banana (Musa spp.) cultivars.

Banana is an important fruit crop in the tropics and subtropics; however, limited information on biomarkers and signature volatiles is available for selecting commercial cultivars. Clonal fidelity is a major contributor to banana yield and aroma; however, there are no useful biomarkers available to validate clonal fidelity. In this study, we performed the molecular profiling of 20 banana cultivars consisting of diploid (AA or AB) and triploid (AAA or AAB or ABB) genomic groups. We screened 200 molecular markers, of which 34 markers (11 RAPD, 11 ISSR, and 12 SSR) yielded unequivocally scorable biomarker profiles. About 75, 69, and 24 allelic loci per marker were detected for RAPD, ISSR, and SSR markers, respectively. The statistical analysis of molecular variance (AMOVA) exhibited a high genetic difference of 77% with a significant FST value of 0.23 (p < 0.001). Interestingly, the UBC-858 and SSR CNMPF-13 markers were unique to Grand Nain and Ardhapuri cultivars, respectively, which could be used for clonal fidelity analysis. Furthermore, the analysis of banana fruit volatilome using headspace solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GCMS) revealed a total of fifty-four volatile compounds in nine banana cultivars with 56% of the total volatile compounds belonging to the ester group as the significant contributor of aroma. The study assumes significance with informative biomarkers and signature volatiles which could be helpful in breeding and for the authentic identification of commercial banana cultivars.

Engineering Novel Aptameric Fluorescent Biosensors for Analysis of the Neurotoxic Environmental Contaminant Insecticide Diazinon from Real Vegetable and Fruit Samples.

BACKGROUND: Diazinon is a widely used organophosphorus neurotoxic insecticide. It is a common environmental contaminant and a hazardous agri-waste. Its detection is critical to control entry into food systems and protect the environment. METHODS: In this study, three single-stranded DNA aptamers specific for diazinon were discovered using the systematic evolution of ligands by the exponential enrichment (SELEX) process. Since aptamer-based sensors are quick and straightforward to analyze, they could potentially replace the time-consuming and labor-intensive traditional methods used for diazinon detection. RESULTS: Here, we show the engineering of novel sensors for diazinon detection with a high affinity (Kd), specificity, and high sensitivity at the ppb level. Moreover, the aptamers were helpful in the simultaneous detection of two other structurally relevant insecticides, fenthion, and fenitrothion. Furthermore, the real vegetable and fruit samples confirmed the specific detection of diazinon using DIAZ-02. CONCLUSIONS: We developed novel biosensors and optimized the assay conditions for the detection of diazinon from food samples, such as vegetables and fruit. The biosensor could be adopted to analyze toxicants and contaminants in food, water, and nature as point-of-care technology.

Development of novel fluorescence-based and label-free noncanonical G4-quadruplex-like DNA biosensor for facile, specific, and ultrasensitive detection of fipronil.

Fipronil is a broad-spectrum insecticide widely used in agriculture and residential areas; its indiscriminate use leads to environmental pollution and poses health hazards. Early detection of fipronil is critical to prevent the deleterious effects. However, current insecticide analysis methods such as HPLC, LC/MS, and GC/MS are incompetent; they are costly, immobile, time-consuming, laborious, and need skilled technicians. Hence, a sensitive, specific, and cheap biosensor are essential to containing the contamination. Here, we designed two novel biosensors-the first design relied on fluorescent labeling/quenching, while the second sensor focused on label-free detection using Thioflavin T displacement. Altogether, we identified four candidate aptamers, predicted secondary structures, and performed 3D molecular modeling to predict the binding pocket of fipronil in FiPA6B aptamer. Furthermore, the aptameric sensors showed high sensitivity to fipronil of sub-ppb level LOD, attributed to stringent experimental design. The biosensors displayed high specificity against other phenylpyrazole insecticides and demonstrated robust sensitivity for fipronil in real samples like cabbage and cucumber. Notably, to the best of our knowledge, this is the first demonstration of noncanonical G4-quadruplex-like aptamer binding to fipronil, verified using CD spectroscopy. Such aptasensors possess considerable potential for real-time measurements of hazardous insecticides as point-of-care technology.

Novel DNA Aptameric Sensors to Detect the Toxic Insecticide Fenitrothion.

Fenitrothion is an insecticide belonging to the organophosphate family of pesticides that is widely used around the world in agriculture and living environments. Today, it is one of the most hazardous chemicals that causes severe environmental pollution. However, detection of fenitrothion residues in the environment is considered a significant challenge due to the small molecule nature of the insecticide and lack of molecular recognition elements that can detect it with high specificity. We performed in vitro selection experiments using the SELEX process to isolate the DNA aptamers that can bind to fenitrothion. We found that newly discovered DNA aptamers have a strong ability to distinguish fenitrothion from other organophosphate insecticides (non-specific targets). Furthermore, we identified a fenitrothion-specific aptamer; FenA2, that can interact with Thioflavin T (ThT) to produce a label-free detection mode with a Kd of 33.57 nM (9.30 ppb) and LOD of 14 nM (3.88 ppb). Additionally, the FenA2 aptamer exhibited very low cross-reactivity with non-specific targets. This is the first report showing an aptamer sensor with a G4-quadruplex-like structure to detect fenitrothion. Moreover, these aptamers have the potential to be further developed into analytical tools for real-time detection of fenitrothion from a wide range of samples.

Concerns regarding 'off-target' activity of genome editing endonucleases.

Genome editing (GE) tools ensure targeted mutagenesis and sequence-specific modification in plants using a wide resource of customized endonucleases; namely, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), and the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) system. Among these, in recent times CRISPR/Cas9 has been widely used in functional genomics and plant genetic modification. A significant concern in the application of GE tools is the occurrence of 'off-target' activity and induced mutations, which may impede functional analysis and gene activity studies. Moreover, the 'off-target' activity results in either not reported or unknown, difficult to detect, produce non-quantifiable cellular signaling and physiological effects. In the past few years, several experimental methods have been developed to identify undesired mutations and to curtail 'off-target' cleavage. Improvement in target specificity and minimizing 'off-target' activity will offer better applications of GE technology in plant biology and crop improvement.