A non-invasive ultrasensitive diagnostic approach for COVID-19 infection using salivary label-free SERS fingerprinting and artificial intelligence.

Clinical diagnostics for SARS-CoV-2 infection usually comprises the sampling of throat or nasopharyngeal swabs that are invasive and create patient discomfort. Hence, saliva is attempted as a sample of choice for the management of COVID-19 outbreaks that cripples the global healthcare system. Although limited by the risk of eliciting false-negative and positive results, tedious test procedures, requirement of specialized laboratories, and expensive reagents, nucleic acid-based tests remain the gold standard for COVID-19 diagnostics. However, genetic diversity of the virus due to rapid mutations limits the efficiency of nucleic acid-based tests. Herein, we have demonstrated the simplest screening modality based on label-free surface enhanced Raman scattering (LF-SERS) for scrutinizing the SARS-CoV-2-mediated molecular-level changes of the saliva samples among healthy, COVID-19 infected and COVID-19 recovered subjects. Moreover, our LF-SERS technique enabled to differentiate the three classes of corona virus spike protein derived from SARS-CoV-2, SARS-CoV and MERS-CoV. Raman spectral data was further decoded, segregated and effectively managed with the aid of machine learning algorithms. The classification models built upon biochemical signature-based discrimination method of the COVID-19 condition from the patient saliva ensured high accuracy, specificity, and sensitivity. The trained support vector machine (SVM) classifier achieved a prediction accuracy of 95% and F1-score of 94.73%, and 95.28% for healthy and COVID-19 infected patients respectively. The current approach not only differentiate SARS-CoV-2 infection with healthy controls but also predicted a distinct fingerprint for different stages of patient recovery. Employing portable hand-held Raman spectrophotometer as the instrument and saliva as the sample of choice will guarantee a rapid and non-invasive diagnostic strategy to warrant or assure patient comfort and large-scale population screening for SARS-CoV-2 infection and monitoring the recovery process.

Clinical characterization and Genomic analysis of COVID-19 breakthrough infections during second wave in different states of India

During March to June 2021 India has experienced a deadly second wave of COVID-19 with an increased number of post-vaccination breakthrough infections reported across the country. To understand the possible reason of these breakthroughs we collected 677 clinical samples (throat swab/ nasal swabs) of individuals who had received two doses (n=592) and one dose (n=85) of vaccines (Covishield and Covaxin,) and tested positive for COVID-19, from 17 states/Union Territories of country. These cases were telephonically interviewed and clinical data was analyzed. A total of 511 SARS-CoV-2 genomes were recovered with genome coverage of higher than 98% from both the cases. Analysis of both the cases determined that 86.69% (n=443) of them belonged to the Delta variant along with Alpha, Kappa, Delta AY.1 and Delta AY.2. The Delta variant clustered into 4 distinct sub-lineages. Sub-lineage-I had mutations: ORF1ab-A1306S, P2046L, P2287S, V2930L, T3255I, T3446A, G5063S, P5401L, A6319V and N-G215C; Sub-lineage -II : ORF1ab- P309L, A3209V, V3718A, G5063S, P5401L and ORF7a-L116F; Sub-lineage -III : ORF1ab- A3209V, V3718A, T3750I, G5063S, P5401L and Spike-A222V; Sub-lineage -IV ORF1ab- P309L, D2980N, F3138S and spike - K77T. This study indicated that majority of the clinical cases in the breakthrough were infected with the Delta variant and only 9.8% cases required hospitalization while fatality was observed in only 0.4% cases. This clearly suggests that the vaccination does provide reduction in hospital admission and mortality.

Anesthetic management and outcomes of patients with Steven-Johnson Syndrome-A retrospective review study.

BACKGROUND AND AIMS: Steven-Johnson Syndrome (SJS) is a rare and severe form of erythema exudative multiforme. Multisystem involvement in SJS and the suspicion of precipitation of the disease with exposure to anesthetic drugs makes anesthesia a challenging task. The concerns during anesthesia are the mucosal lesions and special care that is required to prevent injury to the oropharynx and larynx during airway management and also the drugs used for anesthesia. In the literature, very few isolated case reports or case series are available. Here, we have analyzed the cases of SJS coming for ophthalmic anesthesia, taking into consideration factors like mode of presentation, precipitating factors, associated diseases, types of anesthesia, anesthetic modifications, and various drugs used during anesthesia. MATERIAL AND METHODS: The electronic medical records of 497 cases of SJS who required interventions like ophthalmic examination or surgery (either under local or general anesthesia) over a period of 18 months were analyzed retrospectively. The records were reviewed to obtain the concerned details like anesthesia-inducing agents, muscle relaxants, inhalational agents, and analgesics. The problems concerned with monitoring and intubation were also noted. The data were analyzed and presented as frequency and percentage. RESULTS: Patient age ranged between 9 months and 72 years. Many surgeries were conducted under general anesthesia (441) although a few required local (peribulbar block) anesthesia (56). The drugs administered for general anesthesia were sevoflurane, isoflurane, propofol, thiopentone, vecuronium, and atracurium and those administered for pain management were fentanyl, tramadol, butorphanol, and paracetamol. The patients who were sensitive to nonsteroidal anti-inflammatory drugs (NSAIDs) and paracetamol were not administered the same. None of the patients reacted adversely to the different drugs used for anesthesia. CONCLUSION: Identifying the precipitating factors, understanding the pathophysiology and its implications for anesthesia will help in successfully managing anesthesia in the rare cases of SJS.

On the adhesion-cohesion balance and oxygen consumption characteristics of liver organoids.

Liver organoids (LOs) are of interest in tissue replacement, hepatotoxicity and pathophysiological studies. However, it is still unclear what triggers LO self-assembly and what the optimal environment is for their culture. Hypothesizing that LO formation occurs as a result of a fine balance between cell-substrate adhesion and cell-cell cohesion, we used 3 cell types (hepatocytes, liver sinusoidal endothelial cells and mesenchymal stem cells) to investigate LO self-assembly on different substrates keeping the culture parameters (e.g. culture media, cell types/number) and substrate stiffness constant. As cellular spheroids may suffer from oxygen depletion in the core, we also sought to identify the optimal culture conditions for LOs in order to guarantee an adequate supply of oxygen during proliferation and differentiation. The oxygen consumption characteristics of LOs were measured using an O2 sensor and used to model the O2 concentration gradient in the organoids. We show that no LO formation occurs on highly adhesive hepatic extra-cellular matrix-based substrates, suggesting that cellular aggregation requires an optimal trade-off between the adhesiveness of a substrate and the cohesive forces between cells and that this balance is modulated by substrate mechanics. Thus, in addition to substrate stiffness, physicochemical properties, which are also critical for cell adhesion, play a role in LO self-assembly.

Genetic engineering of AtAOX1a in Saccharomyces cerevisiae prevents oxidative damage and maintains redox homeostasis.

This study aimed to validate the physiological importance of Arabidopsis thaliana alternative oxidase 1a (AtAOX1a) in alleviating oxidative stress using Saccharomyces cerevisiae as a model organism. The AOX1a transformant (pYES2AtAOX1a) showed cyanide resistant and salicylhydroxamic acid (SHAM)-sensitive respiration, indicating functional expression of AtAOX1a in S. cerevisiae. After exposure to oxidative stress, pYES2AtAOX1a showed better survival and a decrease in reactive oxygen species (ROS) when compared to S. cerevisiae with empty vector (pYES2). Furthermore, pYES2AtAOX1a sustained growth by regulating GPX2 and/or TSA2, and cellular NAD (+)/NADH ratio. Thus, the expression of AtAOX1a in S. cerevisiae enhances its respiratory tolerance which, in turn, maintains cellular redox homeostasis and protects from oxidative damage.

Primary Multidrug Resistant Tuberculosis and Utility of Line Probe Assay for Its Detection in Smear-Positive Sputum Samples in a Tertiary Care Hospital in South India.

In a high tuberculosis burdened country like India, rapid, cost-effective, and reliable diagnostic tools for tuberculosis are an urgent need of the hour to prevent inappropriate treatment strategies and further spread of resistance. This study aimed to estimate the proportion of new smear-positive tuberculosis cases with primary resistance to rifampicin and/or isoniazid as well as identify the common mutations associated with it. Sputum of 200 newly diagnosed smear-positive cases of 1+ score and above was directly subjected to Line Probe Assay using the GenoType MTBDRplus assay kit. All samples were inoculated onto solid media and 61 samples were inoculated in automated liquid culture also. The Line Probe Assay gave hundred percent interpretable results with 2.5% of the study population showing resistant pattern. Only 1% of the cases were primary multidrug resistant tuberculosis and 1.5% showed isoniazid monoresistance. S531L and C15T were the most common genetic mutations seen for rifampicin and isoniazid resistance, respectively. 40% had absent rpoB wild type 8 band indicating probable silent mutation after clinical correlation. The average turnaround time for Line Probe Assay was far less (3.8 days) as compared to solid and liquid cultures (35.6 days and 13.5 days, resp.).

Long-term culture and expansion of primary human hepatocytes.

Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low expression of the human papilloma virus (HPV) genes E6 and E7 coupled with inhibition of epithelial-to-mesenchymal transition. We show that E6 and E7 expression upregulates the OSM receptor gp130 and that OSM stimulation induces hepatocytes to expand for up to 40 population doublings, producing 1013 to 1016 cells from a single human hepatocyte isolate. OSM removal induces differentiation into metabolically functional, polarized hepatocytes with functional bile canaliculi. Differentiated hepatocytes show transcriptional and toxicity profiles and cytochrome P450 induction similar to those of primary human hepatocytes. Replication and infectivity of hepatitis C virus (HCV) in differentiated hepatocytes are similar to those of Huh7.5.1 human hepatoma cells. These results offer a means of expanding human hepatocytes of different genetic backgrounds for research, clinical applications and pharmaceutical development.

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana.

BACKGROUND AND AIMS: The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III. METHODS: Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system. KEY RESULTS: In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants. CONCLUSIONS: These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by regulating cellular redox homeostasis and ROS generation when electron transport through the COX pathway is disturbed at complex III.

Morphometry of Suprascapular Notch and its Correlation with that of Scapula.

Suprascapular notch is the common site for the entrapment of the suprascapular nerve. Suprascapular nerve compression usually presents with vague pain across the scapula or dull shoulder ache. It is more common in over head athlets like volleyball players. Hence the study of morphology and morphometry of the notch is important clinically.

A study on the morphology of the suprascapular notch and its distance from the glenoid cavity.

INTRODUCTION: A suprascapular nerve entrapment can occur at the suprascapular notch or at the spinoglenoid notch. So, the size and shape of the suprascapular notch are associated with suprascapular entrapment neuropathy as well as with an injury to the suprascapular nerve in arthroscopic procedures. The knowledge on the variations along the course of the nerve is important in understanding the source of the entrapment syndrome. MATERIAL AND METHODS: The present study was carried out on 104 scapulae which were obtained from the Department of Anatomy, NRI Medical College and from other nearby medical colleges. The suprascapular notches in the scapulae were classified, based on the descriptions of Rengachary et al and Ticker et al. The distance between the suprascapular notch and the supraglenoid tubercle, and the distance between the posterior rim of the glenoid cavity and the medial wall of the spinoglenoid notch at the base of the scapular spine, were determined. The data were analyzed statistically. RESULTS: Based on the Rengachary classification, the type III notch was more common. The suprascapular foramen was observed in 2 scapulae. In 56.73% scapulae, the superior transverse diameter was greater than the maximum depth. The U shaped notch (69.23%) was more common. 2.88% and 8.65% scapulae fell short of the mentioned respective safe zone distances from the margin of the glenoid cavity. CONCLUSION: Such studies may be useful in understanding the role of the notch in causing nerve entrapment and to prevent iatrogenic nerve injuries while posterior approaches are made to the shoulder joint.

Molecular characterization of hepatitis A virus from a large outbreak from Kerala, India.

BACKGROUND & OBJECTIVES: Hepatitis A is highly prevalent in India and mainly presents as a sporadic disease. This study investigated an outbreak of viral hepatitis at Medical College Hospital area, Kottayam, Kerala state, India during January 2005. METHODS: Blood (133), faecal (1), sewage (4), and water samples (13) were collected. Sera were tested for IgG- and IgM-anti-HAV and IgM antibodies against hepatitis E (IgM-anti-HEV). Sewage, faeces and water samples were tested for HAV RNA in nested RT-PCR and HAV RNA positive samples were further processed for RNA quantitation using Real Time PCR. RESULTS: Of the 1180 total cases, 540 were reported from Medical college area. Two deaths were reported among doctors. Patients from the community gave a previous history of visit to medical college hospital area. The sewage treatment plant at the campus was non-functional since 1990 and the untreated sewage was constantly overflowing and getting mixed with a canal. At the time of the study, all the water sources were superchlorinated. HAV RNA was present in the faeces of hepatitis A patient (1.36 x 10(7) copies/ml), sewage tank (2.57 x 10(3) copies/ml and the canal (<100 copies/ml). None of the 13 water samples concentrated 10,000-fold and the soil sample showed presence of HAV RNA. Phylogenetic analysis based on 5'-non-coding and P2 regions showed HAV-genotype IIIA in all samples. INTERPRETATION & CONCLUSION: The aetiological agent of the present outbreak was found to be HAV. Epidemic hepatitis A (genotype-IIIA) is emerging in Indian adults, emphasizing the need for definite policy for control.

tla1, a DNA insertional transformant of the green alga Chlamydomonas reinhardtii with a truncated light-harvesting chlorophyll antenna size.

DNA insertional mutagenesis and screening of the green alga Chlamydomonas reinhardtii was employed to isolate tla1, a stable transformant having a truncated light-harvesting chlorophyll antenna size. Molecular analysis showed a single plasmid insertion into an open reading frame of the nuclear genome corresponding to a novel gene ( Tla1) that encodes a protein of 213 amino acids. Genetic analysis showed co-segregation of plasmid and tla1 phenotype. Biochemical analyses showed the tla1 mutant to be chlorophyll deficient, with a functional chlorophyll antenna size of photosystem I and photosystem II being about 50% and 65% of that of the wild type, respectively. It contained a correspondingly lower amount of light-harvesting proteins than the wild type and had lower steady-state levels of Lhcb mRNA. The tla1 strain required a higher light intensity for the saturation of photosynthesis and showed greater solar conversion efficiencies and a higher photosynthetic productivity than the wild type under mass culture conditions. Results are discussed in terms of the tla1 mutation, its phenotype, and the role played by the Tla1 gene in the regulation of the photosynthetic chlorophyll antenna size in C. reinhardtii.