Reinventing shake flask fermentation: The breathable flask.

Cultivating cells in shake flasks is a routine operation that is largely unchanged since its inception. A glass or plastic Erlenmeyer vessel with the primary gas exchange taking place across various porous plugs is used with media volumes typically ranging from 100 mL to 2 L. Oxygen limitation and carbon dioxide accumulation in the vessel is a major concern for studies involving shake flask cultures. In this study, we enhance mass transfer in a conventional shake flask by replacing the body wall with a permeable membrane. Naturally occurring concentration gradient across the permeable membrane walls facilitates the movement of oxygen and carbon dioxide between the flask and the external environment. The modified flask called the breathable flask, has shown a 40% improvement in mass transfer coefficient (kLa) determined using the static diffusion method. The prokaryotic cell culture studies performed with Escherichia coli showed an improvement of 28%-66% in biomass and 41%-56% in recombinant product yield. The eukaryotic cell culture study performed with Pichia pastoris expressing proinsulin exhibited a 40% improvement in biomass and 115% improvement in protein yield. The study demonstrates a novel approach to addressing the mass transfer limitations in conventional shake flask cultures. The proposed flask amplifies its value by providing a membrane-diffusion-based sensing platform for the integration of low-cost, noninvasive sensing capabilities for real-time monitoring of critical cell culture parameters like dissolved oxygen and dissolved carbon dioxide.

Detection and molecular characterization of 'Candidatus Phytoplasma australasiaticum' in Aegle marmelos: a fruit of high medicinal values in India.

In surveys conducted from 2020 to 2022, five leaf samples each from symptomatic Agele marmelos trees and seedlings, along with five samples from asymptomatic trees and seedlings, were collected in Ayodhya, Uttar Pradesh, India. The DNA extraction from all the samples was subjected to nested PCR assays, using the universal phytoplasma-specific primers set (P1/P7 followed by R16F2n/R16R2). The resulting 1.2 kb amplified products were observed in all the symptomatic samples but not in the asymptomatic samples. Bael phytoplasma strain sequences from the trees and seedlings were found 100% identical within themselves and only two representative sequences (one each from tree and seedling) were deposited in GenBank (NCBI) as PP415872 (AmA-1) and PP415873 (AmA-2). BLASTn searches revealed the maximum (100%) sequence identity with a phytoplasma strain from murraya little leaf strain of Faizabad (GenBank Acc.no. OP984129) and lowest (99.84%) with arecanut crown choking of Shimoga (GenBank Acc. no. OM417502) from Karnataka. Phylogenetic analysis clustered the bael phytoplasma isolates with peanut witches' broom group phytoplasma strains. Virtual RFLP analysis confirmed their identity as 'Ca. P. australasiaticum', a 16SrII-D subgroup strain. This study presents the first identification of a phytoplasma strain in A. marmelos, emphasizing its potential threat to fruit crops and the need for vigilance in nursery practices to prevent further dissemination.

An automatic glucose monitoring system based on periplasmic binding proteins for online bioprocess monitoring.

Glucose is one of the most vital nutrients in all living organisms, so its monitoring is critical in healthcare and bioprocessing. Enzymatic sensors are more popular as a technology solution to meet the requirement. However, periplasmic binding proteins have been investigated extensively for their high sensitivity, enabling microdialysis sampling to replace existing complex and expensive glucose monitoring solutions based on enzymatic sensors. The binding proteins are used as optical biosensors by introducing an environment-sensitive fluorophore to the protein. The biosensor's construction, characterization, and potential application are well studied, but a complete glucose monitoring system based on it is yet to be reported. This work documents the development of the first glucose sensor prototype based on glucose binding protein (GBP) for automatic and continuous glucose measurements. The development includes immobilizing the protein into reusable chips and a low-cost solution for non-invasive glucose sampling in bioprocesses using microdialysis sampling technique. A program was written in LabVIEW to accompany the prototype for the complete automation of measurement. The sampling technique allowed glucose measurements of a few micromolar to 260 mM glucose levels. A thorough analysis of the sampling mode and the device's performance was conducted. The reported measurement accuracy was 81.78%, with an RSD of 1.83%. The prototype was also used in online glucose monitoring of E. coli cell culture. The mode of glucose sensing can be expanded to the measurement of other analytes by switching the binding proteins.

Bioburden detection on surface and water samples in a rapid, ultra-sensitive and high-throughput manner.

Bioburden detection is crucial for food, water, and biopharmaceutical applications as it can directly impact public health. The objective of this study is to develop and validate an assay and protocol for detecting bioburden on solid surfaces, as well as in water, with high sensitivity and accuracy in a rapid manner. Henceforth, a resazurin-based assay optimized for detecting bioburden has been integrated with a previously developed portable multichannel fluorometer. The microbes were isolated from solid surfaces in different laboratory settings by swabbing technique, and stream water was collected for contamination analysis. Based on the results, the assay and protocol can successfully detect bioburden as low as 20 CFU/cm2 and 10 CFU/mL present in both surface and water samples, respectively.

First Report of 'Candidatus Phytoplasma citri' related strain (16SrII-C subgroup) associated with phyllody disease of cumin in India.

Cumin (Cuminum cyminum L.), is an important export-oriented seed spice crop for India. Cumin is popularly used for flavouring food, including soups, pickles and vegetables, and for herbal medicine. India is the largest producer, consumer and exporter of cumin seed with an annual production of 0.795 million tones over an area of 1.09 million hectares. During 2020-21, India exported about 0.299 million tons of cumin worth of Rs 33280 million (Anonymous, 2021). Recently, phytoplasma suspected symptoms were observed in cumin at Agricultural Research Station, Mandor, Jodhpur, Rajasthan, India from 2019. The symptoms related to phytoplasma infection were first recorded after 70-75 days of sowing in the month of January of the years 2019 to 2022. The major symptoms recorded were yellowing, phyllody, witches-broom, yellowing and deformed elongated seeds. Disease incidence was recorded as 0.25-1.0%, 0.5-1.5%, 0.5-2.5 % and 0.5-10.6% during the years 2019, 2020, 2021 and 2022, respectively using quadrate method. In 2022, among different genotypes assessed GC 4, MCU 73, MCU 105, and MCU 32 exhibited lower disease incidences ranging from 0.5% to 1.5%, while MCU 78 recorded the highest disease incidence at 10.6%. To detect the association of phytoplasma with symptomatic cumin samples, genomic DNA was extracted from four representative cumin genotypes (CuPP-MND-01 to CuPP-MND-04) and one asymptomatic cumin plant using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) (Schneider et al., 1995; Gundersen and Lee, 1996) and secA gene specific primers (SecAfor1/SecArev3 followed by nested PCR primers SecAfor5/ SecArev2) (Hodgetts et al. 2008; Bekele et al. 2011). The amplicons of ∼1.25 kb with 16S rRNA gene and ∼600 bp with secA gene specific primers were amplified in all symptomatic cumin plant samples and positive control of brinjal little leaf. PCR amplified products from the four selected positive samples (CuPP-MND-01 to CuPP-MND-04) of 16S rRNA gene and secA gene, were sequenced from both ends. The 1,245 bp sequences were deposited in GenBank (OQ299007-10), which showed 100% sequence identity with each other and 99.4% identity with 'Candidatus Phytoplasma citri' reference strain (GenBank accession: U15442) (Rodrigues Jardim et al. 2023). The phylogenetic analysis and virtual RFLP analysis using 17 restriction enzymes of 16S rRNA gene sequences through iPhyclassifier allowed affiliating the cumin phytoplasma strains with 16SrII-C subgroup strain with a similarity coefficient of 1 to the reference pattern of 16Sr group II, subgroup C (GenBank accession: AJ293216) (Zhao et al. 2009). In addition, the phylogenetic analysis of the secA gene-based sequences (OQ305073-76) further confirmed the close association of 16SrII-C group phytoplasmas with phyllody and witches' broom disease of cumin. Earlier 16SrII-C subgroup phytoplasma has been reported from various crops and weeds in India (Rao et al. 2021). However, no phytoplasma association has been reported earlier with cumin in India and abroad. To the best of our knowledge, this is the first report on the association of 16SrII-C group phytoplasma causing phyllody, witches' broom in cumin genotypes. This report has economic and epidemiological implications and needs immediate attention to reduce export losses due to phytoplasma disease in cumin and to prevent the potential spread to other crops.

A novel, low-cost microfluidic device with an integrated filter for rapid, ultrasensitive, and high-throughput bioburden detection.

Rapid and accurate bioburden detection has become increasingly necessary for food, health, pharmaceutical and environmental applications. To detect bioburden accurately, and in a highly sensitive manner, we have fabricated a novel microfluidic device with an integrated filter to trap the cells. Bioburden is detected on the filter paper in situ using the redox reaction of fluorescent label resorufin and a portable multichannel fluorometer is used for fluorescence measurement. The microfluidic device was fabricated in a facile, low-cost, and rapid way with microwave-induced thermally assisted bonding. To characterize the bonding quality of the microfluidic cassettes, different tests were performed, and the filter paper material and size were optimized. Primary Bacillus subtilis culture bacterial samples were filtered through the device to validate and investigate the performance parameters. Our results show that a limit of detection (LOD) of 0.037 CFU/mL can be achieved through this microfluidic device whereas the LOD in a normal microfluidic cassette in the fluorometer and the golden standard spectrophotometer are 0.378 and 0.128 CFU/mL respectively. The results depict that three to ten times LOD improvement is possible through this microfluidic cassette and more sensitive detection is possible depending on the volume filtered within a rapid 3 min. This novel microfluidic device along with the fluorometer can be used as a rapid portable tool for highly sensitive, accurate and high-throughput bacterial detection for different applications.

First Report of Candidatus Phytoplasma asteris (16SrI-B subgroup) associated with phyllody disease of fenugreek in the world.

Fenugreek (Trigonella foenum-graecum) is a leafy vegetable and spice crop, native to Indian subcontinent and Eastern Mediterranean region. Phytoplasma infection symptoms were observed in fenugreek at ICAR-National Bureau of Plant Genetic Resources Regional Station, Jodhpur and Agricultural Research Station Mandore Jodhpur, Rajasthan, India. The first appearance of phytoplasma suspected symptoms of little leaf was recorded after 50 days of sowing in the months of January 2022. The major symptoms recorded were virescence, phyllody, shoot proliferation, witches-broom, little leaf, yellowing and overall stunted growth in 146 germplasm accessions at NBPGR research farm, Jodhpur and one major commercially cultivated variety RMT 305 at Mandore Jodhpur. Ten samples from symptomatic and five samples from asymptomatic fenugreek plants were collected and processed for total DNA extraction using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) and secA gene specific primers (SecAfor1/SecArev3 and SecAfor2/SecArev3) (Schneider et al. 1995; Gundersen and Lee 1996; Hodgetts et al. 2008). The amplicons of ∼1.25 kb with 16S rRNA and ∼480 bp with secA gene specific primers were amplified in all symptomatic fenugreek samples. In negative control (asymptomatic plants) no amplification was observed with either of gene specific primers in gel electrophoresis. PCR amplified products from the six selected positive samples (FPP-NBPGR-J-01 to FPP-NBPGR-J-04 and FPP-MND-01 to FPP-MND-02) of 16S rRNA and secA gene, were sequenced from both ends. Sequences were deposited in the NCBI GenBank with accession numbers ON756108-ON756113 for 16S rRNA gene sequences and ON745809 to ON745814 for secA gene sequences. BLAST analysis of 16S rRNA and secA sequences revealed 100% sequence identity among themselves and 99.95 to 100% sequence identity with the earlier reported phytoplasma strains of aster yellows group related phytoplasma strains (GenBank Acc. No. MN239504, MN080270) belonging to Ca. P. asteris (16SrI group). Further analyses of the 16S rRNA and secA gene-based phylogenetic tree and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene study demonstrated that the phytoplasma associated with fenugreek phyllody belonged to 16Sr group I ('Ca. P. asteris') and subgroup B (GenBank accession AP006628), with similarity coefficient of 1.0. Earlier association of 16Sr-II-D subgroup (Ca. P. australasiae) with fenugreek as host was reported from Pakistan (Malik et al., 2020). To the best of our knowledge, this is the first report of a 'Ca. P. asteris', 16SrI-B subgroup related phytoplasma strain associated with fenugreek phyllody in the world. The 16SrI-B phytoplasma strain is a widely distributed strain associated with several agricultural and horticultural crops of India (Rao 2021). This is not only the first instance of fenugreek phyllody disease found in India, but also the first instance of fenugreek phyllody caused by 16SrI-B subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as fenugreek is one of the most common seed spice crop being grown all over India.

An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics.

This study describes the cell-free biomanufacturing of a broad-spectrum antiviral protein, griffithsin (GRFT) such that it can be produced in microgram quantities with consistent purity and potency in less than 24 h. We demonstrate GRFT production using two independent cell-free systems, one plant and one microbial. Griffithsin purity and quality were verified using standard regulatory metrics. Efficacy was demonstrated in vitro against SARS-CoV-2 and HIV-1 and was nearly identical to that of GRFT expressed in vivo. The proposed production process is efficient and can be readily scaled up and deployed wherever a viral pathogen might emerge. The current emergence of viral variants of SARS-CoV-2 has resulted in frequent updating of existing vaccines and loss of efficacy for front-line monoclonal antibody therapies. Proteins such as GRFT with its efficacious and broad virus neutralizing capability provide a compelling pandemic mitigation strategy to promptly suppress viral emergence at the source of an outbreak.

Air-permeable redox mediated transcutaneous CO2 sensor.

Standard clinical care of neonates and the ventilation status of human patients affected with coronavirus disease involves continuous CO2 monitoring. However, existing noninvasive methods are inadequate owing to the rigidity of hard-wired devices, insubstantial gas permeability and high operating temperature. Here, we report a cost-effective transcutaneous CO2 sensing device comprising elastomeric sponges impregnated with oxidized single-walled carbon nanotubes (oxSWCNTs)-based composites. The proposed device features a highly selective CO2 sensing response (detection limit 155 ± 15 ppb), excellent permeability and reliability under a large deformation. A follow-up prospective study not only offers measurement equivalency to existing clinical standards of CO2 monitoring but also provides important additional features. This new modality allowed for skin-to-skin care in neonates and room-temperature CO2 monitoring as compared with clinical standard monitoring system operating at high temperature to substantially enhance the quality for futuristic applications.

First report of association of 'Candidatus Phytoplasma asteris' with Moringa oleifera leaf yellowing and stunting disease in India.

Moringa oleifera (family Moringaceae) also known as the 'drumstick tree' is a significant nutritious and medicinal plant that is commonly grown in India and contains a variety of vital phytochemicals. M. oleifera is used in several Indian herbal medicine formulations to treat a variety of illnesses (Kumar and Rao 2021). Typical phytoplasma symptoms of leaf yellowing and stunting were observed in M. oleifera trees up to 10% incidence at Acharya Narendra Dev University of Agriculture & Technology, Ayodhya, Uttar Pradesh, India in November 2021 and stunting with less fruit bearings symptoms with 8% incidence in October 2021 at Jonnalakothapalle village of Mudigubba mandal of Ananthapuramu district in Andhra Pradesh, India (Fig.1a, b). To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from the leaf samples collected from two diseased and two healthy plants from both the locations using CTAB method. The DNAs isolated were analysed by nested polymerase chain reaction (PCR) with universal phytoplasma primer pairs P1/P7 and R16F2n/R16R2 for the 16S rRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/sArev3 and SecAfor2/ SecArev3 for secA gene (Hodgetts et al. 2008). Amplicons of the expected size (~1.25kb from 16S rRNA gene and ~480bp from secA gene) were obtained from symptomatic plants only. The nested PCR products were cloned (pGEM-T Easy Vector, Promega), sequenced (ABA Biotech, India) and the sequences were deposited in GenBank with accession numbers OP358449, OP358450, OP358451, OP358452 for the 16SrRNA gene (~1.25 kb) and OP358443, OP358444, OP358445, OP358446 for the secA gene (~480 bp). BLASTn analysis revealed that the partial 16S rRNA gene sequences of M. oleifera phytoplasma isolate shared up to 99.9% sequence identity with the strain 'Candidatus Phytoplasma asteris' (Accession numbers MN909051, MN909047) and secA gene sequences shared up to 100% sequence identity with 'Ca. Phytoplasma asteris' (Accession numbers KJ434315, KJ462009) belonging to 16SrI group. The 16S rRNA and secA genes sequence-based phylogenetic analysis (Figure 1d,e) showed that the phytoplasma strain associated with M. oleifera leaf yellowing and stunting clustered within the 16SrI phytoplasma group closest to 16SrI-B ('Ca. P. asteris') subgroup strains. Furthermore, the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). To the best of our knowledge, this is the first report of the 16SrI-B subgroup of the phytoplasma strains with M. oleifera in the world. 'Candidatus Phytoplasma asteris' (16SrI-B subgroup) strains have been reported from several other commercial crops and weed hosts in India and efficient leafhopper vectors have been identified (Rao 2021; Reddy 2021). This indicates that the 'Ca. P. asteris'-related strains (16SrI-B) are widespread and infecting several plant species in India. The increasing incidence of the 16SrI-B strain and its wide host range in India strongly suggests further research into the epidemiology involved in the dynamic spread of the disease in order to recommend a suitable management approach.

Identification and multilocus gene characterization of phytoplasmas associated with sweet cherry in India.

Symptoms of leaf roll, swollen nodes, flat branch and witches' broom were observed in five cultivars of sweet cherry from Srinagar, Jammu and Kashmir province, India, during 2019-2021. Phytoplasmas association were confirmed by amplifying 16S rRNA, secA, rp, tuf and secY genes with phytoplasma-specific primers in all symptomatic sweet cherry cultivars in nested PCR assays. Pairwise sequence comparison, phylogeny and virtual RFLP (16S rRNA gene) analyses confirmed the presence of 'Candidatus Phytoplasma asteris' and 'Ca. P. trifolii' strains in the sweet cherry samples. The incidence of flat branch and witches' broom symptoms associated with 'Ca. P. trifolii' varied from 5.8 to 25% in cultivars Bigarreau Nepoleon (Double), Bigarreau Noir Grossa and CITH-Cherry-9. However, incidence of leaf rolling, swollen nodes and bud proliferation associated with 'Ca. P. asteris' was recorded 7.5% in cultivar Stella and 10% in Sunburst, respectively, in the surveyed area. The multigene characterization of sweet cherry phytoplasma strains confirmed the validity of these molecular markers for identification of phytoplasmas enclosed in 16SrI and 16SrVI groups. The presence of phytoplasmas in sweet cherry is the first report from India.

A novel approach to noninvasive monitoring of dissolved carbon dioxide in small-scale cell culture processes.

Disposable small-scale vessels are commonly used in cell culture studies in academia as well as early stages of bioprocess development. These types of research are crucial for our understanding about cells and bioprocesses as they provide important information regarding different parameters affecting cells. Dissolved carbon dioxide (DCO2) is one main parameter affecting cell metabolism. It is also an indicator of cell culture well-being. Despite CO2 being a critical process parameter, there is a lack of appropriate monitoring system for CO2 in small-scale vessels. Here, we present a membrane-based noninvasive method for measuring DCO2 in cell culture medium. The idea was achieved by modifying a T-flask and replacing a small area of it with CO2 permeable silicone membrane. In the proposed method, the concentration of CO2 dissolved in the cell culture medium is determined by measuring the initial diffusion rate of CO2 through a silicone membrane attached to the bottom wall of the T-flask. The measurement method was validated previously, and the efficacy of the noninvasive method was evaluated by growing E.coli, Pichia pastoris, and CHO cells in the proposed prototype. The results obtained from this method were verified with other quantitative data obtained from the process such as optical density (OD), cell density, dissolved oxygen (DO) and pH. The results show that the proposed membrane-based method is an effective way for completely noninvasive monitoring of DCO2 in small-scale cell culture processes. Additional diffusing species such as oxygen could also be measured using the same approach.

Microwave induced thermally assisted solvent-based bonding of biodegradable thermoplastics: an eco-friendly rapid approach for fabrication of microfluidic devices and analyte detection.

There is an increasing interest in low-cost, facile and versatile thermoplastic bonding for microfluidic applications that can be easily transitioned from laboratory prototyping to industrial manufacturing. In addition, owing to the surge in the usage of thermoplastic microfluidics and its adverse effect on the environment, it is prudent to source alternative materials that are biodegradable, providing a sustainable, green approach. To address the problems, here we introduce an environment friendly, low-cost and safe welding technology used in the fabrication of microcassettes from biodegradable cellulose acetate (CA) thermoplastics. The thermally assisted solvent based bonding of the thermoplastics was accomplished in a domestic microwave oven with the aid of a polyether ether ketone (PEEK) vise. To characterize the quality of the bonding, our in-house technique was compared with a conventional thermally assisted solvent bonding configuration using a heat press machine and tested under different conditions. Our microwave induced bonding of CA presents three times reduced bonding time with higher bonding strength, good reliability and does not necessitate the use of cumbersome instrumentation. Finally, we demonstrate an electrophoresis application and vitamin C detection accomplished using this biodegradable microcassette presenting comparable results with traditional techniques, illustrating the potential of this fabrication technique in different microfluidic applications.

Rapid Ultrasensitive and High-Throughput Bioburden Detection: Microfluidics and Instrumentation.

Contamination detection often requires lengthy culturing steps to detect low-level bioburden. To increase the rate of detection and decrease the limit of detection (LOD), a system featuring microfluidics and a multichannel fluorometer has been developed. The eight-channel fluorometer enables parallel testing of multiple samples with the LOD as low as <1 cfu/mL. This low-cost system utilizes the slope of fluorescence intensity that serves as the criterion for bioburden detection. The redox indicator dye resazurin is used to monitor the presence of viable cells in this study and is reduced to resorufin with a high quantum yield at 585 nm. The sample under investigation is spiked with resazurin and loaded in a special-design microfluidic cassette, and the rate of change is observed via the fluorometer. The method was validated using primary Escherichia coli culture in comparison with a spectrophotometer which served as the gold standard. An optimized assay based on Luria-Bertani medium was developed. The impact on the assay sensitivity based on incubation and filtration steps was also explored. The assay is shown to pick up inadvertent contamination from test tubes and pipette tips showing its applicability in real-world settings. The data analysis demonstrated a comparable performance of the multichannel fluorometer vis-a-vis the conventional plate reader. The multichannel system is shown to detect bioburden presence in as low as 20 s for bacterial concentrations ≥5 cfu/mL after 6 h of incubation. Considering its portability, low cost, simplicity of operation, and relevant assay sensitivity, the system is well positioned to detect low-level bioburden in the laboratory, pharmaceutical, and field settings.

Current Research Trends and Prospects for Yield and Quality Improvement in Sesame, an Important Oilseed Crop.

Climate change is shifting agricultural production, which could impact the economic and cultural contexts of the oilseed industry, including sesame. Environmental threats (biotic and abiotic stresses) affect sesame production and thus yield (especially oil content). However, few studies have investigated the genetic enhancement, quality improvement, or the underlying mechanisms of stress tolerance in sesame. This study reveals the challenges faced by farmers/researchers growing sesame crops and the potential genetic and genomic resources for addressing the threats, including: (1) developing sesame varieties that tolerate phyllody, root rot disease, and waterlogging; (2) investigating beneficial agro-morphological traits, such as determinate growth, prostrate habit, and delayed response to seed shattering; (3) using wild relatives of sesame for wide hybridization; and (4) advancing existing strategies to maintain sesame production under changing climatic conditions. Future research programs need to add technologies and develop the best research strategies for economic and sustainable development.

High-Throughput RNA Sequencing of Mosaic Infected and Non-Infected Apple (Malus × domestica Borkh.) Cultivars: From Detection to the Reconstruction of Whole Genome of Viruses and Viroid.

Many viruses have been found associated with apple mosaic disease in different parts of the world. In order to reveal and characterize the viruses and viroids in symptomatic apple plants, next-generation sequencing (RNA seq.) of rRNA-depleted total RNA using Illumina Hiseq2500 was applied to two cultivars, Oregon Spur and Golden Delicious, with symptoms of mosaic and necrosis and one cultivar, Red Fuji, which was asymptomatic. The RNA sequencing detected five viruses, viz., apple necrotic mosaic virus (ApNMV), apple mosaic virus (ApMV), apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), and one viroid i.e., apple hammerhead viroid (AHVd). RT-PCR amplification and sequencing also confirmed the presence of all these five viruses and viroids detected in HTS of total RNA. The complete genomes of five viruses and AHVd were reconstructed. The phylogenetic analysis of these viruses and AHVd revealed genetic diversity by forming subclusters with isolates from other countries. Recombination events were observed in all five viruses while single-nucleotide variants were detected only in ApMV and ApNMV. The absence of ApMV and ApNMV in asymptomatic samples from the same cultivars in an RT-PCR assay indicated that these two viruses are associated with mosaic disease of apples in India. This is the first viral genome analysis of symptomatic and asymptomatic apple plants and the first report of genome characterization of viruses associated with apple mosaic disease from India. High-throughput RNA sequencing is a powerful tool to characterize the genome of viruses and viroids in plants previously undetected by conventional methods. This would also help in the indexing and certification of large-scale germplasm.

Duplex PCR assay for detection of chickpea chlorotic dwarf virus and peanut witches' broom phytoplasma in chickpea.

A duplex PCR assay was standardized by optimizing PCR reaction constituents and cycles for the simultaneous detection of chickpea chlorotic dwarf virus (CpCDV) and a peanut witches' broom (PnWB) phytoplasma associated with the chickpea stunt disease. Coat protein gene and tuf gene specific primers for CpCDV and phytoplasmas were used. Different concentrations of the PCR components such as Taq polymerase, primers and PCR annealing temperature were standardized for the identification of the two agents by a duplex PCR assay. Expected amplicons of 590 bp for CpCDV and 1090 bp for phytoplasmas were consistently amplified from the symptomatic chickpea tissues. That resulted in equally efficient and sensitive in detecting single or mixed infection of CpCDV and PnWB phytoplasma in 148 symptomatic chickpea stunt samples collected in two states of India. The results indicate the robustness in the detection of pathogens present in chickpea showing stunt disease and for theoretical use in epidemiological studies that would help the appropriate disease management strategies.

An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics.

This study describes the cell-free biomanufacturing of a broad-spectrum antiviral protein, griffithsin (GRFT) such that it can be produced with consistent purity and potency in less than 24 hours. We demonstrate GRFT production using two independent cell-free systems, one plant and one microbial. Griffithsin purity and quality were verified using standard regulatory metrics. Efficacy was demonstrated in vitro against SARS-CoV-2 and HIV-1 and was nearly identical to that of GRFT expressed in vivo . The proposed production process is efficient and can be readily scaled up and deployed anywhere in the world where a viral pathogen might emerge. The current emergence of viral variants has resulted in frequent updating of existing vaccines and loss of efficacy for front-line monoclonal antibody therapies. Proteins such as GRFT with its efficacious and broad virus neutralizing capability provide a compelling pandemic mitigation strategy to promptly suppress viral emergence at the source of an outbreak.

What do masks mask? A study on transdermal CO2 monitoring.

Medical professionals have complained of extreme discomfort and fatigue from continuous wearing of N95 respirators (N95) overlaid with surgical masks (SM) and face shields (FS) during COVID-19 pandemic. However, there are no reports on the effect of face coverings on transdermal CO2 (TrCO2) levels (a measure of blood CO2) during moderate activity. In this study, real-time monitoring of TrCO2, heart rate and skin surface temperature was conducted for six subjects aged 20-59 years with and without wearing personal protective equipment (PPE). We initially studied the effect of wearing PPE (N95+SM+FS) at rest. Then, the effect of moderate stepping/walking activity (120 steps per minute for 60 min) while wearing PPE was evaluated. In addition, we investigated the effect of exercising intensity with different masks. We observed a significant difference (p < 0.0001) in TrCO2 levels between without and with PPE during moderate exercise, but not while resting. TrCO2 levels were correlated to exercise intensity independently with masking condition and breathability of masks. For the first time, we present data showing that a properly fitting N95 worn along with SM and FS consistently leads to elevated TrCO2 under moderate exertion, which could contribute to fatigue over long-term use.