Metabolic engineering of hairy root cultures in Beta vulgaris for enhanced production of vanillin, 4-hydroxybenzoic acid, and vanillyl alcohol.

The flavor of vanilla is a complex blend of compounds, with vanillin as the most prominent, along with vanillyl alcohol and 4-hydroxybenzoic acid. Natural vanillin extracted from vanilla beans is expensive, so researchers use heterologous synthesis to produce nature-identical vanillin in plant hosts. Consequently, alternative traditional farming and gathering methods are required to bridge the significant disparity between supply and demand. The current research successfully developed a method to induce hairy root formation from leaves. It integrated the Vanillin synthase (VpVAN) gene into transgenic hairy root lines of Beta vulgaris, synthesizing vanillin-related compounds. The presence of the VpVAN gene in transgenic roots was confirmed using PCR analysis. Additionally, RT-qPCR analysis demonstrated the expression of the VpVAN gene in the transgenic root lines. The transgenic hairy root clones H1, H2, and H5 showed enhanced vanillin production, vanillyl alcohol, and 4-hydroxybenzoic acid. Elicitation with methyl jasmonate (MJ) and salicylic acid (SA) further improved the production of these compounds in B. vulgaris hairy roots. The maximum hairy root biomass was observed after 60 days, with the maximum synthesis of vanillin and 4-hydroxybenzoic acid obtained from hairy root clones H5 and HR2, respectively. Vanillyl alcohol HR2 was obtained on the 45th day of cultivation. Elicitation with wound-associated hormone methyl jasmonate and salicylic acid enhanced the yield of vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid, with a 215-fold increase in vanillin, a 13-fold increase in vanillyl alcohol, and a 21 fold increase in 4-hydroxybenzoic acid. The study results indicate that establishing transgenic hairy root cultures with the VpVAN gene is a promising alternative method for enhancing the production of vanilla flavor compounds such as vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid. A cost-effective protocol has been developed to mass-produce phenolic compounds using a hairy root culture of B. vulgaris. This approach addresses the increasing demand for these substances while reducing the cost of natural vanillin production, making it suitable for industrial-scale applications.

Bridging fungal resistance and plant growth through constitutive overexpression of Thchit42 gene in Pelargonium graveolens.

KEY MESSAGE: Thchit42 constitutive expression for fungal resistance showed synchronisation with leaf augmentation and transcriptome analysis revealed the Longifolia and Zinc finger RICESLEEPER gene is responsible for plant growth and development. Pelargonium graveolens essential oil possesses significant attributes, known for perfumery and aromatherapy. However, optimal yield and propagation are predominantly hindered by biotic stress. All biotechnological approaches have yet to prove effective in addressing fungal resistance. The current study developed transgenic geranium bridging molecular mechanism of fungal resistance and plant growth by introducing cassette 35S::Thchit42. Furthermore, 120 independently putative transformed explants were regenerated on kanamycin fortified medium. Primarily transgenic lines were demonstrated peak pathogenicity and antifungal activity against formidable Colletotrichum gloeosporioides and Fusarium oxysporum. Additionally, phenotypic analysis revealed ~ 2fold increase in leaf size and ~ 2.1fold enhanced oil content. To elucidate the molecular mechanisms for genotypic cause, de novo transcriptional profiles were analyzed to indicate that the auxin-regulated longifolia gene is accountable for augmentation in leaf size, and zinc finger (ZF) RICESLEEPER attributes growth upregulation. Collectively, data provides valuable insights into unravelling the mechanism of Thchit42-mediated crosstalk between morphological and chemical alteration in transgenic plants. This knowledge might create novel opportunities to cultivate fungal-resistant geranium throughout all seasons to fulfil demand.

Molecular cloning and heterologous expression analysis of 1-Deoxy-D-Xylulose-5-Phosphate Synthase gene in Centella asiatica L.

Many genes involved in triterpenoid saponins in plants control isoprenoid flux and constitute the precursor pool, which is channeled into various downstream pathways leading to the synthesis of triterpenoid saponins in C. asiatica. Full-length 1-Deoxy-D-Xylulose-5-Phosphate-Synthase (CaDXS) gene was isolated for the study from the previously annotated Centella asiatica leaves transcriptomic data. The CaDXS gene sequence was submitted to the NCBI databases with GenBank accession number MZ997832. The full-length CaDXS gene contained a 2244 base pair open reading frame that encoded a 747 amino acid polypeptide. The predicted molecular weight (MW) and theoretical pI of DXS are 76.28 kDa and 6.86, respectively. Multiple amino acid sequence alignment of amino acids and phylogenetic studies suggest that CaDXS shares high similarities with DXS from other plants DXS belonging to different families. A phylogenetic tree was constructed using Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6. Structural analysis provided fundamental information about the three-dimensional features and physicochemical parameters of the CaDXS protein. Quantitative expression analysis showed that CaDXS transcripts were maximally expressed in leaf, followed by petiole, roots, and node tissues. CaDXS was cloned into the expression vector pET28a, expressed heterologously in DH5α bacteria, confirmed by sequencing, and subsequently characterized by protein expression and functional complementation. The study focused on understanding the protein structure, biological significance, regulatory mechanism, functional analysis, and gene characterization of the centellosides biosynthetic pathway gene DXS for the first time in the plant. It would provide new information about the metabolic pathway and its relative contribution to isoprenoid biosynthesis.

Impact of economic shocks on mental health during COVID-19: A longitudinal study of adult residents of Indian metropolitan cities.

Background: The causal impact of income shocks on mental well-being in developing countries is an under-researched area. The COVID-19 pandemic, along with the economic recession caused by the lockdowns to reduce transmission, provides a natural experimental setting to examine the causal impact of a decline in monthly per capita expenditure (MPCE) on mental health of the general population in India during the pandemic. Aim: To evaluate the impact of income shocks on mental health of adults in metro cities during the COVID-19 pandemic. Materials and Methods: The data were collected using the abridged version of the Depression Anxiety Stress Schedule administered through a telephonic survey on adult residents of six metropolitan cities in September-August 2020 and July-August 2021. Results: In the present study, 994 adults participated from six metropolitan cities. Average treatment effects were estimated using Propensity Score Matching. The mean normalized scores are significantly higher for respondents whose MPCE had fallen (treated) vis-à-vis respondents whose MPCE had remained same or increased (control): anxiety (0.21 for treated vs -0.19 for control), stress (0.16 vs -0.14), and depression (0.04 vs -0.19). Propensity score matching reveals that the normalized scores for anxiety, stress, and depression were 33 (95% confidence intervals, CI: 20.0-46.7), 25 (95% CI: 12.9-36.9), and 36 (95% CI: 18.6-53.1) higher among the treated group vis-a-vis control group. The ATET was 34 (95% CI: 18.9-48.9), 26 (95% CI: 10.1-42.9), and 32 (955 CI: 12.3-50.7) for these three outcomes, respectively. The post-estimation tests indicated that the results are valid. Conclusions: The study advocates that policies to ensure income security should be made an integral part of the response packages to tackle pandemics like COVID-19.

Co-Creation of Breast Cancer Risk Communication Tools and an Assessment of Risk Factor Awareness: A Qualitative Study of Patients and the Public in India.

BACKGROUND: Low awareness of BC and its associated risk factors causes delays in diagnosis and impacts survival. It is critical to communicate BC risk to patients in a format that they are easily able to understand. Our study aim was to develop easy-to-follow transmedia prototypes to communicate BC risk and evaluate user preferences, alongside exploring awareness of BC and its risk factors. METHODS: Prototypes of transmedia tools for risk communication were developed with multidisciplinary input. A qualitative in-depth online interview study was undertaken using a pre-defined topic guide of BC patients (7), their relatives (6), the general public (6), and health professionals (6). Interviews were analyzed using a thematic approach. FINDINGS: Most participants preferred pictographic representations (frequency format) of lifetime risk and risk factors and storytelling using short animations and comic strips (infographics) for communicating genetic risk and testing: "In a short time, they explained it very well, and I liked it". Suggestions included minimizing technical terminology, decreasing the delivery speed, "two-way dialogue", and using local "language for different locations". There was low awareness of BC, with some understanding of age and hereditary risk factors but limited knowledge of reproductive factors. INTERPRETATION: Our findings support use of multiple context-specific multimedia tools in communicating cancer risk in an easy-to-understand way. The preference for storytelling using animations and infographics is a novel finding and should be more widely explored.

Change in mental health during the COVID-19 pandemic: a longitudinal study of residents of Indian metropolitan cities.

BACKGROUND: Initial studies have reported an increase in the incidence of mental health problems during the early months of COVID-19. Longitudinal studies of changes in mental health undertaken in Low and Middle Income countries during the pandemic remains an under-researched area. AIMS: The current study examines changes in mental health among adult residents of metropolitan cities of India, a middle-income country reporting the second-highest COVID cases and third-highest fatalities, during the pandemic. METHOD: Data was collected, based on a telephonic survey using the internationally accepted abridged Depression Anxiety Stress schedule (DASS-21), in August and September 2020 and July-August 2021. The sample size is 994. The data was analysed using an ordered logit model. RESULTS: At the onset of the pandemic, high levels of anxiety, stress and depression prevailed; their levels reduced after one year. Respondents who have experienced a decline in economic fortunes, have family members with pre-existing co-morbidity or had COVID in the family are significantly less likely to report improvement in mental health; less-educated respondents are also vulnerable. CONCLUSIONS: Specific sub-groups, identified as at risk, need monitoring and continued provisioning of tailor-made mental health services addressing their specific needs. Relief measures targeting economically affected households are also required.

Understanding the Geography of Victimization: A Spatial Analysis of Intimate Partner Violence in India.

Most studies on intimate partner violence (IPV) and its drivers have focused on individual-and household-level characteristics of the victim. Recent studies have acknowledged that it is a community-level phenomenon, using spatial analytical methods to analyze community-level determinants of IPV and its geographic dimensions. Such studies provide mixed evidence on the impact of different factors and need to be supplemented by similar studies-particularly in South Asian countries where IPV is common. The present study examines district-level variations in the incidence of various forms of IPV and identifies its determinants in India, a fast-growing South Asian country with poor gender indicators. The study combines data from the National Family Health Survey, District Level Household Survey, and the decadal Census. It applies spatial analytical methods such as the Global Moran's I, Getis-ord statistic, and Multivariate Local Geary to determine the nature of the spatial distribution of different categories of IPV. Spatial regression models are used to identify the community-level predictors of each category of IPV. The study finds non-random overlapping spatial clusters in the eastern part of India. The study also finds that neighborhoods characterized by low empowerment levels, and with a high child sex ratio, road connectivity, and proportion of socially marginalized groups are more likely to exhibit high levels of all types of IPV-although the impact of these determinants varies across districts. Furthermore, spill-overs in the incidence of IPV between neighboring districts are also observed. The study concludes by recommending the use of localized policies, rather than broad national or state policies, in reducing IPV.

Changes in dietary practices of mother and child during the COVID-19 lockdown: Results from a household survey in Bihar, India.

The outbreak of COVID-19, and the national-level lockdown to contain it, were expected to disrupt supply chains, lead to livelihood loss, and reduce household income. Studies anticipated a decline in food security in India, leading to a near famine-like situation. In this study, we examine the change in Dietary Score (number of food groups consumed out of a possible eight) and proportion of respondents complying with Minimum Dietary Diversity norms (consuming at least four food groups) among women aged 15-49 years and their youngest child (aged between 7 and 36 months) during the lockdown. The present study also analyses whether ownership of ration cards and contacts with the party in power locally helped the household to tide over the crisis. The data was collected through a two-phase primary survey undertaken in January-March 2020 (pre-lockdown period) and October-November 2020 (post-lockdown period). It was undertaken in six districts of Bihar, a state with a history of poor maternal and child health outcomes and dysfunctional delivery of health services. We find that dietary practices of women deteriorated, while that of children remained the same. The deterioration is less among households owning ration cards or having political contacts. The analysis suggests that, during pandemics or similar crisis periods, the need to supplement the supply of staple items through the Public Distribution System with a direct transfer of cash will allow households to maintain diversity in the consumption basket.

Information-sharing experiences of professionals looking after children with cancer: a qualitative exploration from a specialist paediatric oncology unit in India.

Background: Childhood cancer often involves a long-term engagement of children and their parents with health services. During this journey, communications between professionals, parents and young people can be stressful for all the stakeholders. This study explores the communication preferences in paediatric oncology. Objectives: The objective of the present exploratory qualitative study was to understand the views of professionals regarding information exchange during cancer treatment of children and complement these findings with clinic-based ethnographic observation of real-life consultations. Methods: Using qualitative methods, in-depth interviews were conducted with paediatric oncology professionals. The interviews had been audio-recorded and transcribed verbatim. Alongside in-depth interviews, real-life interactions between parents, professionals and children were observed. Data were analysed using a thematic analysis framework as suggested by Braun and Clark. Results: Paediatric oncology professionals (n = 14) were interviewed from diverse professional backgrounds that included consultant paediatric oncologists, junior specialist trainees in paediatric oncology, paediatric oncology nurses, social workers, survivor counsellor and psychologists looking after children with cancer. Additionally, clinic-based ethnographic observations (n = 10) of interactions between professionals, parents and young people were also conducted. The following themes emerged from the interviews: a) Information needs of children were very different from adolescents. Children were more worried about 'here and now'; b) adolescents were, on the other hand, mostly worried about the 'impact of cancer on their broader life, friendships and academics'; c) parents were curious about the outcome, costs and effectiveness of treatment, and different patterns emerged for mothers and fathers; d) information needs were dynamic and different at the start of the treatment, during treatment, at remission or end of life; e) the journey of the clinicians themselves impacted information-sharing practices; and f) direct observation of consultations highlighted the importance of priming parents before delivery of information, having multiple family members during the conversation and managing intense emotions expressed during the session. Conclusion: Paediatric oncology professionals need to be sensitive about the dynamic nature of information needs while interacting with children and parents of children with cancer. The above findings may help tailor the discussions that professionals ought to have with families with a child with cancer. The results may contribute to the understanding as well as to developing training courses on communications in paediatric oncology for low- and middle-income countries.

Comparative Evaluation of the Effectiveness of Rotary Instrumentation over Manual Instrumentation with Ultrasonic Irrigation on Incidence, Duration, and Intensity of Postendodontic Pain: An In vivo Study.

AIM: The aim of this study was to compare the effectiveness of rotary instrumentation over manual instrumentation with ultrasonic irrigation on incidence, duration, and intensity of postendodontic pain (PEP). SUBJECTS AND METHODS: Eighty patients, with asymptomatic irreversible pulpitis in maxillary anterior teeth, were selected and treated with single-visit endodontic treatment. Patients were randomly divided into 2 groups (40 each), Group A (K files using step-back technique) and Group B (ProTaper Next using crown-down technique) along with passive ultrasonic irrigation. Patients were recalled, examined, and asked to fill up questionnaire after 24 h, 48 h, and 7 days. On the basis of response given in the feedback forms, incidence, duration, and intensity of PEP were evaluated. RESULTS: Statistical analysis of the data was carried out using Chi-square test, and level of significance (P < 0.05) was evaluated. More incidence of pain was noticed in Group A when compared with Group B. Significant difference found between two groups (χ 2 = 22.759; P = 0.001). There was also statistically significant difference between two groups at different time intervals. CONCLUSION: Both instrumentation techniques under investigation cause PEP. The incidence of pain was more in manual technique than rotary technique. The duration of pain was higher in manual group than rotary group at different time intervals.

Economic Evaluation of Population-Based BRCA1/BRCA2 Mutation Testing across Multiple Countries and Health Systems.

Clinical criteria/Family history-based BRCA testing misses a large proportion of BRCA carriers who can benefit from screening/prevention. We estimate the cost-effectiveness of population-based BRCA testing in general population women across different countries/health systems. A Markov model comparing the lifetime costs and effects of BRCA1/BRCA2 testing all general population women ≥30 years compared with clinical criteria/FH-based testing. Separate analyses are undertaken for the UK/USA/Netherlands (high-income countries/HIC), China/Brazil (upper-middle income countries/UMIC) and India (low-middle income countries/LMIC) using both health system/payer and societal perspectives. BRCA carriers undergo appropriate screening/prevention interventions to reduce breast cancer (BC) and ovarian cancer (OC) risk. Outcomes include OC, BC, and additional heart disease deaths and incremental cost-effectiveness ratio (ICER)/quality-adjusted life year (QALY). Probabilistic/one-way sensitivity analyses evaluate model uncertainty. For the base case, from a societal perspective, we found that population-based BRCA testing is cost-saving in HIC (UK-ICER = $-5639/QALY; USA-ICER = $-4018/QALY; Netherlands-ICER = $-11,433/QALY), and it appears cost-effective in UMIC (China-ICER = $18,066/QALY; Brazil-ICER = $13,579/QALY), but it is not cost-effective in LMIC (India-ICER = $23,031/QALY). From a payer perspective, population-based BRCA testing is highly cost-effective in HIC (UK-ICER = $21,191/QALY, USA-ICER = $16,552/QALY, Netherlands-ICER = $25,215/QALY), and it is cost-effective in UMIC (China-ICER = $23,485/QALY, Brazil-ICER = $20,995/QALY), but it is not cost-effective in LMIC (India-ICER = $32,217/QALY). BRCA testing costs below $172/test (ICER = $19,685/QALY), which makes it cost-effective (from a societal perspective) for LMIC/India. Population-based BRCA testing can prevent an additional 2319 to 2666 BC and 327 to 449 OC cases per million women than the current clinical strategy. Findings suggest that population-based BRCA testing for countries evaluated is extremely cost-effective across HIC/UMIC health systems, is cost-saving for HIC health systems from a societal perspective, and can prevent tens of thousands more BC/OC cases.

Prediction of Outcomes with a Computational Biology Model in Newly Diagnosed Glioblastoma Patients Treated with Radiation Therapy and Temozolomide.

PURPOSE: Precision medicine has been most successful in targeting single mutations, but personalized medicine using broader genomic tumor profiles for individual patients is less well developed. We evaluate a genomics-informed computational biology model (CBM) to predict outcomes from standard treatments and to suggest novel therapy recommendations in glioblastoma (GBM). METHODS AND MATERIALS: In this retrospective study, 98 patients with newly diagnosed GBM undergoing surgery followed by radiation therapy and temozolomide at a single institution with available genomic data were identified. Incorporating mutational and copy number aberration data, a CBM was used to simulate the response of GBM tumor cells and generate efficacy predictions for radiation therapy (RTeff) and temozolomide (TMZeff). RTeff and TMZeff were evaluated for association with overall survival and progression-free survival in a Cox regression model. To demonstrate a CBM-based individualized therapy strategy, treatment recommendations were generated for each patient by testing a panel of 45 central nervous system-penetrant US Food and Drug Administration-approved agents. RESULTS: High RTeff scores were associated with longer survival on univariable analysis (P < .001), which persisted after controlling for age, extent of resection, performance status, MGMT, and IDH status (P = .017). High RTeff patients had a longer overall survival compared with low RTeff patients (median, 27.7 vs 14.6 months). High TMZeff was also associated with longer survival on univariable analysis (P = .007) but did not hold on multivariable analysis, suggesting an interplay with MGMT status. Among predictions of the 3 most efficacious combination therapies for each patient, only 2.4% (7 of 294) of 2-drug recommendations produced by the CBM included TMZ. CONCLUSIONS: CBM-based predictions of RT and TMZ effectiveness were associated with survival in patients with newly diagnosed GBM treated with those therapies, suggesting a possible predictive utility. Furthermore, the model was able to suggest novel individualized monotherapies and combinations. Prospective evaluation of such a personalized treatment strategy in clinical trials is needed.

Does health insurance ensure equitable health outcomes? An analysis of hospital services usage in urban India.

In this paper, we examine the relationship between socio-economic status (SES) and the usage of in-patient services, and analyze the impact of introducing health insurance in India - a major developing country with poor health outcomes. In contrast to results of similar works undertaken for developed countries, our results reveal that the positive relation between usage of in-patient services and SES persists even in the presence of health insurance. This implies that health insurance is unable to eliminate the inequities in accessing healthcare services that stem from disparities in SES. In fact, insurance aggravates inequity in the healthcare market. The study is based on unit-level data from the 2005-06 Morbidity and Health Care Survey undertaken by National Sample Survey Organization.

Balancing the present and the future: a study of contraceptive use in Calcutta's slums.

Calcutta, an important metropolitan city in eastern India, has a large slum population. Despite the poor economic status of this population, analysis of DHS data (2004-2005) reveals that contraceptive use levels in Calcutta slums is quite high, with a large proportion preferring terminal methods. Econometric analysis reveals that cultural factors are important determinants of contraceptive use. Results also indicate that respondents attempt to balance current needs (by trying to limit family size through use of contraceptives) but also try to provide for the future (by having at least one son before adopting family planning methods).

Is health status of elderly worsening in India? A comparison of successive rounds of national sample survey data.

The increasing greying of India's population raises concerns about the welfare and health status of the aged. One important source of information of health status of the elderly is the National Sample Survey Rounds on Morbidity and Health Care Expenditure. Using unit-level data for 1995-96 and 2004, this paper examines changes in reported health status of the elderly in India and analyses their relationship with living arrangements and extent of economic dependency. It appears that even after controlling for factors like caste, education, age, economic status and place of residence, there has been a deterioration in self-perceived current health status of the elderly. The paper argues that, although there have been changes in the economic condition and traditional living arrangements--with a decline in co-residential arrangements--this is not enough to explain the decline in reported health status and calls for a closer look at narratives of neglect being voiced in developing countries.