Thread-powered cell lysis and isotachophoresis: unlocking microbial DNA for diverse molecular applications.

Central to the domain of molecular biology resides the foundational process of DNA extraction and purification, a cornerstone underpinning a myriad of pivotal applications. In this research, we introduce a DNA extraction and purification technique leveraging polypropylene (PP) threads. The process commences with robust cell lysis achieved through the vigorous agitation of interwoven PP threads. The friction between the threads facilitates cell lysis especially those microbes having tough cell wall. For purification of DNA, thread-based isotachophoresis was employed which makes the whole process swift and cost-effective. Lysed cell-laden threads were submerged in a trailing electrolyte which separated DNA from other cellular contents. The process was performed with a tailored ITP device. An electric field directs DNA, cell debris, trailing electrolyte, and leading electrolyte toward the anode. Distinct ion migration resulted in DNA concentrating on the PP thread's anode-proximal region. The SYBR green dye is used to visualize DNA as a prominent green zone under blue light. The purified DNA exhibits high purity levels of 1.82 ± 0.1 (A260/A280), making it suitable for various applications aiming at nucleic acid detection.

Human Norovirus Detection: How Much Are We Prepared?

Norovirus (NoV) is known to be the second nonbacterial enteric pathogen after rotavirus that causes acute gastroenteritis. They can be spread from person to person through fecal-oral routes. Infection can lead to severe diarrhea, causing stomach pain, vomiting, and nausea. Rapid detection of NoV can control huge economic and productive losses. Genotyping various emerging NoV strains is important to compare the severity among different strains. Conventional immunological and molecular methods have evolved and contributed to developing detection techniques. Immunological (enzyme-linked immunosorbent assay) and molecular detection (reverse transcriptase polymerase chain reaction [RT-PCR], RT-quantitative PCR, loop-mediated isothermal amplification, nucleic acid sequence-based alignment, recombinase polymerase amplification) methods have been mainly used. The development of biosensors using aptasensor, affinity peptides, nanoparticles, microfluidics, and so on, are currently the most researched topics. The availability of next-generation sequencing technologies has greatly influenced the diagnosis of NoV. The complementation of advanced technologies is helpful in identification of new variants. In this study, techniques that are useful in detecting NoV are discussed. This review has investigated the availability of recent methods used in the detection, present status, and futuristic plan of action in case of outbreak and pandemic.

Carbon dots and their interactions with recognition molecules for enhanced nucleic acid detection.

Carbon Dots (C-dots) have exceptional fluorescence and incident wavelength alteration capabilities because of their π-π* electron transitions between the surface-trapped charges. They have clear, considerate and cost-effective applications in the domain of bio-sensing, optical imaging, medical diagnostics, fluorescence chemotherapy, forensics, and environmentology. Advances in the production process of C-dots can change their optical and chemical characteristics, allowing them to interact with a variety of chemicals and ions that can be exploited for the DNA detection in point-of-care devices. In the current scenario of pathogenic disease prevention, metagenomics and industrial processes, alternative genetic material identification is critical. This review focuses on the existing carbon dots-based DNA detection technologies and their interactions with other components such as metallic salts, dyes, and biological chemicals based on their surface charge distribution (positive or negative) employed in the DNA diagnostic devices and biosensors with their operating mechanism regarding their target component. These intriguing scientific discoveries and technologies will be extensively examined to translate them into real-world solutions which will have a significant societal and economic impact on overall well-being and innovation.

Isothermal nucleic acid amplification and its uses in modern diagnostic technologies.

Nucleic acids are prominent biomarkers for diagnosing infectious pathogens using nucleic acid amplification techniques (NAATs). PCR, a gold standard technique for amplifying nucleic acids, is widely used in scientific research and diagnosis. Efficient pathogen detection is a key to adequate food safety and hygiene. However, using bulky thermal cyclers and costly laboratory setup limits its uses in developing countries, including India. The isothermal amplification methods are exploited to develop miniaturized sensors against viruses, bacteria, fungi and other pathogenic organisms and have been applied for in situ diagnosis. Isothermal amplification techniques have been found suitable for POC techniques and follow WHO's ASSURED criteria. LAMP, NASBA, SDA, RCA and RPA are some of the isothermal amplification techniques which are preferable for POC diagnostics. Furthermore, methods such as WGA, CPA, HDA, EXPAR, SMART, SPIA and DAMP were introduced for even more accuracy and robustness. Using recombinant polymerases and other nucleic acid-modifying enzymes has dramatically broadened the detection range of target pathogens under the scanner. The coupling of isothermal amplification methods with advanced technologies such as CRISPR/Cas systems, fluorescence-based chemistries, microfluidics and paper-based sensors has significantly influenced the biosensing and diagnosis field. This review comprehensively analyzed isothermal nucleic acid amplification methods, emphasizing their advantages, disadvantages and limitations.

DNA compaction enhances the sensitivity of fluorescence-based nucleic acid assays: a game changer in point of care sensors?

Fluorescence-based nucleic acid assays frequently exhibit a feeble signal at low analyte concentrations, necessitating complex, expensive methods such as the development of sequence-specific oligo tags, molecular beacons, and chemical modifications to maintain high detection sensitivity. Hence, there is growing interest in accomplishing fluorescence enhancement in nucleic acid assays using robust and cost-effective strategies. The study exploits the use of two compaction agents, PEG 8000 and CTAB, to compact the ITS-2 amplicon of the fungus Candida albicans and evaluates the effect of both of these agents on the fluorescence intensity of SYTO-9 labelled nucleic acids. Conventional fluorometric measurements showed that both CTAB and PEG 8000 enhanced the emission intensity by ∼1.2- and 2-fold, respectively. Furthermore, we leveraged paper-based spot tests and distance-based assays to validate the effect of DNA compaction for enhancing sensitivity in the point-of-care context. The spot assay performed on paper with compacted samples showed an increase in the emission intensity of SYTO-9 and this was manifested by an elevated G channel intensity in the order of PEG 8000 compacted > CTAB compacted > amplified. Moreover, in the distance-based assay, the PEG 8000 compacted sample was found to migrate farther compared to CTAB compacted and amplified DNA samples at amplicon concentrations, 15 µg ml-1 and 39.65 µg ml-1. The limit of detection (LOD) for PEG 8000 and CTAB compacted samples on both paper-spot and distance-based assays were found to be 0.4 µg ml-1 and 0.5 µg ml-1, respectively. Hence our work provides an overview of employing DNA compaction as an approach for enhancing the sensitivity of fluorescence-based point-of-care nucleic acid assays without the need for cumbersome sensitivity enhancement methods.

CRISPR/Cas technology: Opportunities for phytopathogenic viruses detection.

Detection and monitoring of viruses are essential for healthy plants and prosperity. Recent development in CRISPR/Cas system in diagnosis has open an avenue well suited for pathogen detection. Variety of CRISPR associated proteins are being discovered, suggesting array of application and detection strategies in diagnosis. Phytopathogenic viruses are diverse with respect to their nucleic acid compositions, which presents a challenge in developing a single device applicable for almost all viruses. The review describes about the efficient use of CRISPR/Cas Technology in diagnosis, such as SHERLOCK, DETECTR and SATORI. These methods are different in their characteristic to identify specific nucleic acids and processing the detectable signals. These technologies are in their infancy and lot of scope is there to develop commercial kits. Plant tissue culture-based industries, climate control green houses, indoor cultivation facilities etc. has been considered as few examples. This review will be beneficial for researchers seeking to develop detection mechanism based on CRISPR/Cas technology. The outcome in the form of cost-effective detection of viruses will be boon for agro-based industries, which are facing challenges through virus contamination.

Paper-based microfluidic devices for food adulterants: Cost-effective technological monitoring systems.

Analytical sciences have witnessed emergent techniques for efficient clinical and industrial food adulterants detection. In this review, the contributions made by the paper-based devices are highlighted for efficient and rapid detection of food adulterants and additives, which is the need of the hour and how different categories of techniques have been developed in the past decade for upgrading the performance for point-of-care testing. A simple strategy with an arrangement for detecting specific adulterants followed by the addition of samples to obtain well-defined qualitative or quantitative signals for confirming the presence of target species. The paper-based microfluidics-based technology advances and prospects for food adulterant detection are discussed given the high-demand from the food sectors and serve as a valued technology for food researchers working in interdisciplinary technological frontiers.

Apoplastic gamma-glutamyl transferase activity encoded by GGT1 and GGT2 is important for vegetative and generative development.

Gamma-glutamyl transferase (GGT; EC 2.3.2.2) is the only enzyme capable of degrading glutathione (GSH) in extra-cytosolic spaces. In plant cells, the GGT1 and GGT2 isoforms are located in the apoplast, bound respectively to the cell wall and the plasma membrane. GGT1 is expressed throughout plants, mainly in the leaves and vascular system, while GGT2 is more specifically expressed in seeds and trichomes, and weakly in roots. Their role in plant physiology remains to be clarified, however. Obtaining the ggt1/ggt2 double mutant can offer more clues than the corresponding single mutants, and to prevent any compensatory expression between the two isoforms. In this work, ggt1/ggt2 RNAi (RNA interference) lines were generated and characterized in the tissues where both isoforms are expressed. The seed yield was lower in the ggt1/ggt2 RNAi plants due to the siliques being fewer in number and shorter in length, with no changes in thiols and sulfur compounds. Proline accumulation and delayed seed germination were seen in one line. There were also fewer trichomes (which contain high levels of GSH) in the RNAi lines than in the wild type, and the root elongation rate was slower. In conclusion, apoplastic GGT silencing induces a decrease in the number of organs with a high GSH demand (seeds and trichomes) as a result of resource reallocation to preserve integrity and composition.

Proteome readjustments in the apoplastic space of Arabidopsis thaliana ggt1 mutant leaves exposed to UV-B radiation.

Ultraviolet-B radiation acts as an environmental stimulus, but in high doses it has detrimental effects on plant metabolism. Plasma membranes represent a major target for Reactive Oxygen Species (ROS) generated by this harmful radiation. Oxidative reactions occurring in the apoplastic space are counteracted by antioxidative systems mainly involving ascorbate and, to some extent, glutathione. The occurrence of the latter and its exact role in the extracellular space are not well documented, however. In Arabidopsis thaliana, the gamma-glutamyl transferase isoform (GGT1) bound to the cell wall takes part in the so-called gamma-glutamyl cycle for extracellular glutathione degradation and recovery, and may be implicated in redox sensing and balance. In this work, oxidative conditions were imposed with Ultraviolet-B radiation (UV-B) and studied in redox altered ggt1 mutants. The response of ggt1 knockout Arabidopsis leaves to UV-B radiation was assessed by investigating changes in extracellular glutathione and ascorbate content and their redox state, and in apoplastic protein composition. Our results show that, on UV-B exposure, soluble antioxidants respond to the oxidative conditions in both genotypes. Rearrangements occur in their apoplastic protein composition, suggesting an involvement of Hydrogen Peroxide (H2O2), which may ultimately act as a signal. Other important changes relating to hormonal effects, cell wall remodeling, and redox activities are discussed. We argue that oxidative stress conditions imposed by UV-B and disruption of the gamma-glutamyl cycle result in similar stress-induced responses, to some degree at least. Data are available via ProteomeXchange with identifier PXD001807.

RNA interference: concept to reality in crop improvement.

The phenomenon of RNA interference (RNAi) is involved in sequence-specific gene regulation driven by the introduction of dsRNA resulting in inhibition of translation or transcriptional repression. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in opening a new vista for crop improvement. RNAi technology is precise, efficient, stable and better than antisense technology. It has been employed successfully to alter the gene expression in plants for better quality traits. The impact of RNAi to improve the crop plants has proved to be a novel approach in combating the biotic and abiotic stresses and the nutritional improvement in terms of bio-fortification and bio-elimination. It has been employed successfully to bring about modifications of several desired traits in different plants. These modifications include nutritional improvements, reduced content of food allergens and toxic compounds, enhanced defence against biotic and abiotic stresses, alteration in morphology, crafting male sterility, enhanced secondary metabolite synthesis and seedless plant varieties. However, crop plants developed by RNAi strategy may create biosafety risks. So, there is a need for risk assessment of GM crops in order to make RNAi a better tool to develop crops with biosafety measures. This article is an attempt to review the RNAi, its biochemistry, and the achievements attributed to the application of RNAi in crop improvement.

Transadmittance Type Universal Current-Mode Biquad Filter Using VDTAs.

A new resistorless single-input-multi-output (SIMO) universal transadmittance (TA) type filter employing two voltage differencing transconductance amplifiers (VDTA) and two grounded capacitors is proposed. The proposed topology realizes simultaneously low pass (LP), high pass (HP), and band pass (BP) filter functions. Band rejects (BR) and all pass (AP) filters are also realizable through appropriate connections of currents. The proposed configuration also offers independent control of natural angular frequency (ω 0) and bandwidth (BW) and low active and passive sensitivities. The workability of proposed configuration has been demonstrated through PSPICE simulations with TSMC CMOS 0.18 µm process parameters.

Scale-up of an alkaline protease from Bacillus pumilus MTCC 7514 utilizing fish meal as a sole source of nutrients.

Fish meal grades SL1 and SL2 from Sardine (Sardinella longiceps) and NJ from Pink Perch (Nemipterus japonicas) were evaluated as a sole source of carbon and nitrogen in the medium for alkaline protease production by Bacillus pumilus MTCC 7514. The analysis of the fish meal suggests that the carbon and nitrogen contents in fish meal are sufficient to justify its choice as replacement for other nutrients. Protease production increased significantly (4,914 U/ml) in medium containing only fish meal, compared with the basal medium (2,646 U/ml). However, the elimination of inorganic salts from media reduced the protease productivity. In addition, all the three grades of fish meal yielded almost the same amounts of protease when employed as the sole source of carbon and nitrogen. Nevertheless, the best results were observed in fish meal SL1 medium. Furthermore, protease production was enhanced to 6,966 U/ml and 7,047 U/ml on scaling up from flask (4,914 U/ml) to 3.7 and 20 L fermenters, respectively, using fish meal (10 g/l). Similarly, the corresponding improvement in productivities over flask (102.38 U/ml/h) was 193.5 and 195.75 U/ml/h in 3.7 and 20 L fermenters, respectively. The crude protease was found to have dehairing ability in leather processing, which is bound to have great environmental benefits.

Compensatory expression and substrate inducibility of gamma-glutamyl transferase GGT2 isoform in Arabidopsis thaliana.

γ-Glutamyl transferases (GGT; EC 2.3.2.2) are glutathione-degrading enzymes that are represented in Arabidopsis thaliana by a small gene family of four members. Two isoforms, GGT1 and GGT2, are apoplastic, sharing broad similarities in their amino acid sequences, but they are differently expressed in the tissues: GGT1 is expressed in roots, leaves, and siliques, while GGT2 was thought to be expressed only in siliques. It is demonstrated here that GGT2 is also expressed in wild-type roots, albeit in very small amounts. GGT2 expression is enhanced in ggt1 knockout mutants, suggesting a compensatory effect to restore GGT activity in the root apoplast. Supplementation with 100 µM glutathione (GSH) resulted in the up-regulation of GGT2 gene expression in wild-type and ggt1 knockout roots, and of GGT1 gene expression in wild-type roots. Glutathione recovery was hampered by the GGT inhibitor serine/borate, suggesting a major role for apoplastic GGTs in this process. These findings can explain the ability of ggt1 knockout mutants to retrieve exogenously added glutathione from the growth medium.

Treatment of Casualties in a Forward Hospital of Indian Army : Nine year Experience.

BACKGROUND: To analyze the outcome of the management of casualties in a level II trauma centre of a forward hospital of Armed Forces over a nine year period. Retrospective analysis of all casualties received in a single forward hospital of Indian Army was carried out. METHOD: During 9 years (1990-1998), a total of 5737 casualties were received in a single level II zonal hospital of the Army in a forward area. Majority of the injuries were caused by bullets, or by fragments of improvised explosive devices. A policy of aggressive resuscitation and early primary repair of injuries was followed. General surgeons routinely performed craniotomies, thoracotomies, laparotomies, stabilization of fractures by fixators and repair of vascular injuries. RESULT: 38% of patients had injuries to several body parts (polytrauma), resulting in a total of 8578 injuries. Region-wise distribution of injuries was as follows : 14.2% head and neck injuries, 13.3% chest wounds, 13.5% abdominal injury and 59% extremity wounds. The overall mortality rate was 3.6%. The complication rate was about 7% with infection as the major complication. The results of primary repair of colonic injuries were similar to those of staged repairs. The results after primary closure of war wounds were better than those treated with delayed primary closure in selected cases. CONCLUSION: Prompt evacuation, speedy resuscitation and early definitive repair of war injuries results in low mortality and morbidity. A motivated and dedicated team and adequate availability of blood and ancillary services adds to the excellent outcome. The policy of primary repair of colonic and selected soft tissue injuries appears justified in selected cases.

Delirium Tremens.

The varied clinical manifestations and management of 14 male patients with delirium tremens (DT) have been studied. Eight patients were initially hospitalised for diseases unrelated to ethanol abuse i.e. 2 each for gun shot wound, myocardial infarction and stroke, and one each for pneumonia and gastroenteritis. One patient was going through withdrawal because of prodrome of viral hepatitis before he was hospitalised for uncontrolled agitation and delirium. Two known cases of mild essential hypertension on dietary therapy reported for agitation, abnormal behaviour, a single episode of tonic clonic seizure and hypertensive encephalopathy as they could not/did not get alcohol for 3 days. Three patients presented denovo with DT without concomitant illness. The other features besides delirium and hallucinations were tremulousness in 10, tachycardia in 12, fever in 3, diaphoresis in 2 and tonic clonic seizures in 4 patients. The symptoms fluctuated markedly at short intervals and 2 patients did not have any features of sympathetic overactivity. Altered hepatic biochemical parameters and ketonuria with normal blood sugar were noted in 4 and one patients respectively. Other biochemical parameters including serum electrolytes were normal. CT scan brain done for 5 patients revealed subdural haematoma in one. Cerebro spinal fluid (CSF) and EEG findings were noncontributory. All made good recovery with heavy doses of intravenous vitamin B complex, glucose and oral benzodiazepine. Short course of haloperidol was used in 2 patients. Two patients developed pancreatitis during follow up. All patients made complete recovery, and 8 patients have been followed for 8 to 12 months without relapse. The reason for hospitalisation in such cases is often unrelated to alcohol abuse; hence a detailed history of alcoholism is mandatory to identify those at risk as well as for prompt treatment and decreasing the mortality.