Optimized protoplast isolation and transfection with a breakpoint: accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot.

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic loci. Software-predicted guide RNAs (gRNAs) often fail to induce efficient cleavage at target loci. Many target loci are inaccessible due to complex chromatin structure. Currently, there is no suitable tool available to predict the architecture of genomic target sites and their accessibility. Hence, significant time and resources are spent on performing editing experiments with inefficient guides. Although in vitro-cleavage assay could provide a rough assessment of gRNA efficiency, it largely excludes the interference of native genomic context. Transient in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic context. Here, we developed a modified protocol that offers highly efficient protoplast isolation from rice, Arabidopsis, and chickpea, using a sucrose gradient, transfection using PEG (polyethylene glycol), and validation of single guide RNAs (sgRNAs) cleavage efficiency of CRISPR-Cas9. We have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and dicots. We introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice, Arabidopsis, and chickpea protoplasts. Using dual sgRNAs, our CRISPR-deletion strategy offers straightforward detection of genome editing success by simple agarose gel electrophoresis. Sanger sequencing of PCR products confirmed the editing efficiency of specific sgRNAs. Notably, we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability, allowing a pause between isolation and transfection. This high-efficiency protocol for protoplast isolation and transfection enables rapid (less than 7 days) validation of sgRNA cleavage efficiency before proceeding with stable transformation. The isolation and transfection method can also be utilized for rapid validation of editing strategies, evaluating diverse editing reagents, regenerating plants from transfected protoplasts, gene expression studies, protein localization and functional analysis, and other applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00139-7.

Metabolite profiling of Piper longum L. fruit volatiles by two-dimensional gas chromatography and time-of-flight mass spectrometry: Insights into the chemical complexity.

Piper longum L. (long pepper) is an economically and industrially important medicinal plant. However, the characterization of its volatiles has only been analyzed by gas chromatography-mass spectrometry (GC-MS). In the present study, precise characterization of P. longum fruit volatiles has been performed for the first time through advanced two-dimensional gas chromatography-time-of-flight spectrometry (GC×GC-TOFMS). A total of 146 constituents accounting for 93.79% were identified, of which 30 were reported for the first time. All these constituents were classified into alcohols (4.5%), alkanes (8.9%), alkenes (6.71%), esters (6.15%), ketones (0.58%), monoterpene hydrocarbons (1.64%), oxygenated monoterpenes (2.24%), sesquiterpene hydrocarbons (49.61%), oxygenated sesquiterpenes (13.03%), phenylpropanoid (0.23%), and diterpenes (0.2%). Among all the classes, sesquiterpene hydrocarbons were abundant, with germacrene-D (2.87% ± 0.01%) as the major one, followed by 8-heptadecene (2.69% ± 0.03%), ß-caryophyllene (2.43% ± 0.03%), n-heptadecane (2.4% ± 0.04%), n-pentadecane (2.11% ± 0.05%), and so forth. Further, 20 constituents were observed to be coeluted and separated precisely in the two-dimensional column. The investigation provides an extensive metabolite profiling of P. longum fruit volatiles, which could be helpful to improve its therapeutic potential.

Recent advances in spirocyclization of maleimides via transition-metal catalyzed C-H activation.

In recent years, the maleimide scaffold has received a great deal of attention in C-H activation. Several types of products can be constructed using maleimides as a coupling partner. Alkylation, alkenylation, annulation, dehydrogenative annulation and spirocyclization are various reactions shown by maleimides in C-H activation. Thus, the maleimide scaffold has been extensively studied in the last few years in C-H activation owing to its unique reactivity. Among the diverse class of reactions of maleimides, spirocyclization is a less explored reaction. The spirocycles, in particular the spirosuccinimides are interesting candidates in drug discovery and materials chemistry. Therefore the method of spirocyclization of maleimides via C-H activation becomes an important strategy for the synthesis of a diverse array of spirosuccinimides. This review summarizes the reports available in this field from 2015-2023 and also highlights the scopes and prospects of this method.

Overexpression of Setaria italica phosphoenolpyruvate carboxylase gene in rice positively impacts photosynthesis and agronomic traits.

C4 plants have the inherent capacity to concentrate atmospheric CO2 in the vicinity of RuBisCo, thereby increasing carboxylation, and inhibiting photorespiration. Carbonic anhydrase (CA), the first enzyme of C4 photosynthesis, converts atmospheric CO2 to HCO3-, which is utilized by PEPC to produce C4 acids. Bioengineering of C4 traits into C3 crops is an attractive strategy to increase photosynthesis and water use efficiency. In the present study, we isolated the PEPC gene from the C4 plant Setaria italica and transferred it to C3 rice. Overexpression of SiPEPC resulted in a 2-6-fold increment in PEPC enzyme activity in transgenic lines with respect to non-transformed control. Photosynthetic efficiency was enhanced in transformed plants, which was associated with increased ФPSII, ETR, lower NPQ, and higher chlorophyll accumulation. Water use efficiency was increased by 16-22% in PEPC transgenic rice lines. Increased PEPC activity enhanced quantum yield and carboxylation efficiency of PEPC transgenic lines. Transgenic plants exhibited higher light saturation photosynthesis rate and lower CO2 compensation point, as compared to non-transformed control. An increase in net photosynthesis increased the yield by (23-28.9%) and biomass by (24.1-29%) in transgenic PEPC lines. Altogether, our findings indicate that overexpression of C4-specific SiPEPC enzyme is able to enhance photosynthesis and related parameters in transgenic rice.

Derivatives of Cinnamic Acid Esters and Terpenic Diversity in Volatiles of Thirty-Six Sand Ginger (Kaempferia galanga L.) Accessions of Eastern India Revealing Quality Chemovars.

The essential oil of Kaempferia galanga L. commonly known as sand ginger has increased its demand in national and international market for decades. Cinnamic acid esters like ethyl-p-methoxy cinnamate (EPMC) and ethyl cinnamate (EC) are major constituents in its essential oil. In spite of the high demand for the plant as raw material, identification of quality chemovars having high essential oil (EO) yield and constituents is still at an infant stage. With this in mind, we have evaluated the EO yield of 36 accessions from three provinces of Eastern India, which varied within a range of 0.41 ± 0.01 to 2.63 ± 0.03 v/w. Further, a total of 65 compounds were detected by gas chromatography and mass spectrometry (GC-MS) with area percentages varying from 76.16 to 97.3%. EPMC was found to be the major component in 14 accessions with area percentages varying from 10.7% to 41.1%, whereas other 22 accessions showed EC as the major constituent, varying from 16% to 29.1%. Further, a diversity study among accessions was performed by agglomerative hierarchical clustering (AHC) and principal component analysis (PCA) analysis based on the abundance of identified constituents, which categorized all 36 accessions into three clusters. Thus, the present study helps to identify quality chemovar K.g16 and K.g14 with respect to oil yield and constituents, respectively, which could be used to guide commercial cultivation and further improvement of the taxa.

Potential role of Indian long pepper (Piper longum L.) volatiles against free radicals and multidrug resistant isolates.

In the present study, the extracted volatiles from dried leaf and fruit of Piper longum were analysed by gas chromatography-mass spectrometry (GC-MS) and each detected 53 constituents having 92.41% and 96.31% of the total volatiles respectively. E-nerolidol (19.56%), ß-pinene (17.07%) and α-pinene (6.8%) were main constituents in leaf volatiles whereas the fruit volatiles dominated by germacrene-D (23.38%), 8-heptadecene (8.95%) and ß-caryophyllene (8.20%). Antioxidant potential of the volatiles were assessed by DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assays. The fruit volatiles revealed higher radical scavenging activities as compared to leaf. The samples were also evaluated against multidrug resistant (MDR) isolates including one non MDR fungal strain. The fruit volatiles showed a very strong activity against Acinetobacter baumannii than others whereas leaf volatiles possessed strong activity against Klebsiella pneumoniae as compared to other strains. Thus, the dried fruits can be exploited for drug development towards therapeutic purpose.

Morphophysiological alterations in transgenic rice lines expressing PPDK and ME genes from the C4 model Setaria italica.

C4 plants are superior to C3 plants in terms of productivity and limited photorespiration. PPDK (pyruvate orthophosphate dikinase) and NADP-ME (NADP-dependent malic enzyme) are two important photosynthetic C4-specific enzymes present in the mesophyll cells of C4 plants. To evaluate the effect of C4 enzymes in rice, we developed transgenic rice lines by separately introducing Setaria italica PPDK [SiPPDK] and S. italica ME [SiME] gene constructs under the control of the green tissue-specific maize PPDK promoter. Rice plant lines for both constructs were screened using the polymerase chain reaction (PCR), Southern hybridization, and expression analysis. The best transgenic plant lines for each case were selected for physiological and biochemical characterization. The results from qRT-PCR and enzyme activity analysis revealed higher expression and activity of both PPDK and NADP-ME genes compared with the nontransformed and empty-vector-transformed plants. The average photosynthetic efficiency of transgenic plant lines carrying the PPDK and NADP-ME genes increased by 18% and 12%, respectively, and was positively correlated with the increased accumulation of photosynthetic pigment. The decrease in Fv/Fm, increased electron transport rate (ETR), and increased photochemical quenching (qP) compared with nontransformed control plants suggest that transgenic rice plants transferred more absorbed light energy to photochemical reactions than wild-type plants. SiME-transgenic plants displayed reduced leaf malate content and superior performance under water deficit conditions. Interestingly, the transgenic plants showed yield enhancement by exhibiting increased plant height, panicle length, panicle weight and thousand grain weight. Overall, the exogenous foxtail millet C4 gene PPDK enhanced photosynthesis and yield to a greater extent than NADP-ME.

Role of sedoheptulose-1,7 bisphosphatase in low light tolerance of rice (Oryza sativa L.).

Rice grain yield is drastically reduced under low light especially in kharif (wet) season due to cloudy weather during most part of crop growth. Therefore, 50-60% of yield penalty was observed. To overcome this problem, identification of low light tolerant rice genotypes with a high buffering capacity trait such as photosynthetic rate has to be developed. Sedoheptulose-1,7 bisphosphatase, a light-regulated enzyme, plays pivotal role in the Calvin cycle by regenerating the substrate (RuBP) for RuBisCo and therefore, indirectly regulates the influx of CO2 for this crucial process. We found a potential role of SBPase expression and activity in low light tolerant and susceptible rice genotypes by analyzing its influence on net photosynthetic rate and biomass. We observed a significant relationship of yield with photosynthesis, SBPase expression and activity especially under low light conditions. Two tolerant and two susceptible rice genotypes were used for the present study. Tolerant genotypes exhibited significant but least reduction compared to susceptible genotypes in the expression and activity of SBPase, which was also manifested in its photosynthetic rate and finally in the grain yield under low light. However, susceptible genotypes showed significant reduction in SBPase activity along with photosynthesis and grain yield suggesting that tracking the expression and activity of SBPase could form a simple and reliable method to identify the low light tolerant rice cultivars. The data were analyzed using the Indostat 7.5, Tukey-Kramer method through Microsoft Excel 2019 and PAST4.0 software. The significant association of SBPase activity with the grain yield, net assimilation rate, electron transfer rate, biomass and grain weight were observed under low light stress. These traits should be considered while selecting and breeding for low light tolerant cultivars. Thus, SBPase plays a major role in the low light tolerance mechanism in rice.

DNA as a digital information storage device: hope or hype?

The total digital information today amounts to 3.52 × 1022 bits globally, and at its consistent exponential rate of growth is expected to reach 3 × 1024 bits by 2040. Data storage density of silicon chips is limited, and magnetic tapes used to maintain large-scale permanent archives begin to deteriorate within 20 years. Since silicon has limited data storage ability and serious limitations, such as human health hazards and environmental pollution, researchers across the world are intently searching for an appropriate alternative. Deoxyribonucleic acid (DNA) is an appealing option for such a purpose due to its endurance, a higher degree of compaction, and similarity to the sequential code of 0's and 1's as found in a computer. This emerging field of DNA as means of data storage has the potential to transform science fiction into reality, wherein a device that can fit in our palms can accommodate the information of the entire world, as latest research has revealed that just four grams of DNA could store the annual global digital information. DNA has all the properties to supersede the conventional hard disk, as it is capable of retaining ten times more data, has a thousandfold storage density, and consumes 108 times less power to store a similar amount of data. Although DNA has an enormous potential as a data storage device of the future, multiple bottlenecks such as exorbitant costs, excruciatingly slow writing and reading mechanisms, and vulnerability to mutations or errors need to be resolved. In this review, we have critically analyzed the emergence of DNA as a molecular storage device for the future, its ability to address the future digital data crunch, potential challenges in achieving this objective, various current industrial initiatives, and major breakthroughs.

A Clinical Study of Opioid Substitution Therapy in a Tertiary Care Center of Eastern India.

BACKGROUND: Opioid substitution therapy (OST) is an evidence-based intervention for opiate-dependent persons that replaces illicit drug use with medically prescribed, orally administered opiates such as buprenorphine and methadone. OST reduces HIV risk behaviors and harms associated with injecting opioid. Most of the evidence for OST effectiveness has been generated in middle- and high-income countries where programs are mostly located in dedicated health-care settings; evidence regarding the outcomes of OST programs in low-income countries where OST is often provided in grassroots settings such as drop-in centers is limited. AIMS AND OBJECTIVES: To study the sociodemographic variables, HIV ELISA status, HIV risk behavior, comorbid substance use pattern, and required dose of buprenorphine used for treatment of injection drug users (IDUs) attending oral substitution therapy (OST) center at a tertiary health care center. METHODOLOGY: A total of IDUs aged 18-60 years who attended the OST center during 1-year period at a government medical college are included in the study. RESULTS: Majority of the IDUs are male with mean age of 32.8 years. The mean dose of buprenorphine used for the substitution was 4.6 mg/day at the start of therapy. Most of the IDUs are of lower educational status and educated up to primary or middle school. 32.50% of the participants who are unemployed are totally dependent on the family. Most common substance abuse among IDU users were tobacco (74.17%), followed by heroine (57.5%). High-risk behavior found among OST clients was unprotected sexual intercourse (19.17%), sharing needle (11.67%), and sexual intercourse with multiple partners (6.67%). HIV ELISA testing showed positive among 2 (1.67%). CONCLUSION: These findings have relevance to other parts of India and Asia, where injecting drug use is common and is a first step toward filling the gap in knowledge regarding the effectiveness of community-based OST programs delivered in resource-constrained settings.