Prospects of chloroplast metabolic engineering for developing nutrient-dense food crops.

Addressing nutritional deficiencies in food crops through biofortification is a sustainable approach to tackling malnutrition. Biofortification is continuously being attempted through conventional breeding as well as through various plant biotechnological interventions, ranging from molecular breeding to genetic engineering and genome editing for enriching crops with various health-promoting metabolites. Genetic engineering is used for the rational incorporation of desired nutritional traits in food crops and predominantly operates through nuclear and chloroplast genome engineering. In the recent past, chloroplast engineering has been deployed as a strategic tool to develop model plants with enhanced nutritional traits due to the various advantages it offers over nuclear genome engineering. However, this approach needs to be extended for the nutritional enhancement of major food crops. Further, this platform could be combined with strategies, such as synthetic biology, chloroplast editing, nanoparticle-mediated rapid chloroplast transformation, and horizontal gene transfer through grafting for targeting endogenous metabolic pathways for overproducing native nutraceuticals, production of biopharmaceuticals, and biosynthesis of designer nutritional compounds. This review focuses on exploring various features of chloroplast genome engineering for nutritional enhancement of food crops by enhancing the levels of existing metabolites, restoring the metabolites lost during crop domestication, and introducing novel metabolites and phytonutrients needed for a healthy daily diet.

Clinical, Serological, and Virological Analysis of 572 Chikungunya Patients From 2010 to 2013 in India.

Background: Chikungunya fever (CHIK) is a major public health concern in India. Characterized by acute fever with joint pain and swelling, most patients recover from this self-limiting illness in 7-10 days, with cessation of joint pain post-acute episode. However, in some patients, joint pain persists, lasting for months or even years. The precise correlates to the chronic phase of this debilitating illness and/or this remarkable heterogeneity in disease manifestation are poorly understood. Methods: We evaluated 572 chikungunya patients from India who were recruited on the basis of positive real-time polymerase chain reaction and/or CHIK virus immunoglobulin (IgM) after receiving consent. Arthralgic conditions were monitored using visual analog score (VAS) 12 weeks after onset of fever in 130 patients. Initial viral load, IgG, and initial neutralization response were assayed and correlated with clinical and VAS information in 40 patients. Results: Our extensive screening revealed that patients with higher initial viral loads during the acute phase of illness had poor prognosis at the post-acute phase with more restricted joint movement and higher VAS. Additionally, patients who showed early seroconversion to neutralizing IgG responses had better prognosis, as many of these patients did not manifest restricted joint movements at the post-acute phase. Conclusions: Our study sheds light on chikungunya disease with respect to disease progression and assesses clinical, virological, and serological parameters of chikungunya disease severity. Importantly, it reveals that initial high viral load and neutralizing IgG response may function in a seemingly contrasting manner to negatively or positively dictate disease outcome.

Chloroplast transformation in new cultivars of tomato through particle bombardment.

A protocol has been established for genetic transformation of the chloroplasts in two new cultivars of tomato (Solanum lycopersicum L.) grown in India and Australia: Pusa Ruby and Yellow Currant. Tomato cv. Green Pineapple was also used as a control that has previously been used for establishing chloroplast transformation by other researchers. Selected tomato cultivars were finalized from ten other tested cultivars (Green Pineapple excluded) due to their high regeneration potential and better response to chloroplast transformation. This protocol was set up using a chloroplast transformation vector (pRB94) for tomatoes that is made up of a synthetic gene operon. The vector has a chimeric aadA selectable marker gene that is controlled by the rRNA operon promoter (Prrn). This makes the plant or chloroplasts resistant to spectinomycin and streptomycin. After plasmid-coated particle bombardment, leaf explants were cultured in 50 mg/L selection media. Positive explant selection from among all the dead-appearing (yellow to brown) explants was found to be the major hurdle in the study. Even though this study was able to find plastid transformants in heteroplasmic conditions, it also found important parameters and changes that could speed up the process of chloroplast transformation in tomatoes, resulting in homoplasmic plastid-transformed plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03954-3.

Awareness data on cervical cancer among females of rural and urban areas of Haryana, India.

A cross-sectional study was done to assess the degree of current awareness and behaviors about cervical cancer among females in urban and rural areas of North India. This survey was conducted on one thousand females (500 rural and 500 urban). A well-structured questionnaire was designed to collect information about participants' knowledge on cancer of cervix uteri such as age, height and weight measurements, marital status, menstrual status, personal hygiene, age at menarche, sexual history, pregnancy and abortion history, use of contraceptive pills for birth-control, smoking, alcohol consumption, and other relevant information. The data was collected by conducting face-to-face interviews after obtaining the verbal consent of the participants. The data has the potential to reduce disease burden by spreading awareness about symptoms and risk factors of cervical cancer as well as implementation of effective early screening strategies.

Carotenoid Pathway Engineering in Tobacco Chloroplast Using a Synthetic Operon.

The carotenoid pathway in plants has been altered through metabolic engineering to enhance their nutritional value and generate keto-carotenoids, which are widely sought after in the food, feed, and human health industries. In this study, the aim was to produce keto-carotenoids by manipulating the native carotenoid pathway in tobacco plants through chloroplast engineering. Transplastomic tobacco plants were generated that express a synthetic multigene operon composed of three heterologous genes, with Intercistronic Expression Elements (IEEs) for effective mRNA splicing. The metabolic changes observed in the transplastomic plants showed a significant shift towards the xanthophyll cycle, with only a minor production of keto-lutein. The use of a ketolase gene in combination with the lycopene cyclase and hydroxylase genes was a novel approach and demonstrated a successful redirection of the carotenoid pathway towards the xanthophyll cycle and the production of keto-lutein. This study presents a scalable molecular genetic platform for the development of novel keto-carotenoids in tobacco using the Design-Build-Test-Learn (DBTL) approach. This study corroborates chloroplast metabolic engineering using a synthetic biology approach for producing novel metabolites belonging to carotenoid class in industrially important tobacco plant. The synthetic multigene construct resulted in producing a novel metabolite, keto-lutein with high accumulation of xanthophyll metabolites. This figure was drawn using BioRender ( https://www.biorender.com ).

Breakthrough Infection With Severe Acute Respiratory Syndrome Coronavirus 2 Among Healthcare Workers in Delhi: A Single-Institution Study.

Introduction This study aimed to determine the breakthrough infection rate of coronavirus disease 2019 (COVID-19) (severe acute respiratory syndrome coronavirus 2 {SARS-CoV-2}) infection in healthcare workers (HCWs) vaccinated with either BBV152 or AZD1222 (ChAdOx1-S) vaccine. Methods A cross-sectional analysis was conducted at a medical college and hospital complex in Delhi, India, through telephonic interviews among HCWs who had received at least one dose of a COVID-19 vaccine during January-March 2021. Breakthrough infections were operationally defined as the occurrence of COVID-19 infection ≥14 days after administration of two doses of either COVID-19 vaccine. Data were entered in Epidata 3.1 (Odense, Denmark: EpiData Association) (single entered) and analyzed with IBM SPSS version 25 (Armonk, NY: IBM Corp.). A p-value < 0.05 was considered statistically significant. Results We enrolled 325 HCWs with a mean (SD) age of 29.1 (9.9) years including 211 (64.9%) males and 114 (35.1%) females. A total of 37 (13.3%, 95% CI 9.8, 17.7) COVID-19 breakthrough infections were observed in the HCWs. Additionally, 20 (6.1%) non-breakthrough infections were reported in the HCWs who were vaccinated with at least a single dose of a COVID-19 vaccine, or both doses, but prior to 14 days since the administration of the second dose. Most breakthrough infections were mild without needing supplemental oxygen for recovery. Conclusion Nearly one in seven HCWs experienced a COVID-19 breakthrough infection in the present study. A history of SARS-CoV-2 natural infection followed by at least one dose of COVID-19 vaccination was associated with significant protection against breakthrough infections.

Seroprevalence of SARS-CoV-2 and Risk Assessment Among Healthcare Workers at a Dedicated Tertiary Care COVID-19 Hospital in Delhi, India: A Cohort Study.

Background Healthcare workers (HCWs) have a substantially higher risk of Covid-19 infection but there is a paucity of information on the risk factors of disease transmission in high-burden real-world settings. The study objective was to determine the seroprevalence of SARS-CoV-2 among healthcare workers in a high-burden Covid-19 setting and to estimate the incidence and identify the risk factors of infection. Methods This was a prospective observational cohort study amongst doctors and nurses working at a dedicated Covid-19 tertiary care government hospital in Delhi, India. A baseline blood sample (2-3ml) was collected from all the participants to test for the presence of total SARS-CoV-2 antibodies. The HCWs that were seronegative (non-reactive) at baseline were followed-up for ≥21≤28 days with the collection of a second blood sample to assess for the incidence of SARS-CoV-2 infection. Results A total of 321 (51.3%, 95% C.I 47.4, 55.3) HCWs were detected with SARS-CoV-2 antibodies on baseline examination. The seroprevalence, when adjusted for assay characteristics, was 54.5% (95% C.I 50.3, 58.6). On bivariate analysis, SARS-CoV-2 antibody positivity lacked statistically significant association with either age, sex, occupation, cumulative duty duration, and smoking status. The incidence of seroconversion in the baseline seronegative cohort on follow-up after 21-28 days was observed in 35 (14.9%) HCWs (n=245). Furthermore, the self-reported adherence to infection prevention and control measures did not show a statistically significant association with antibody positivity in the HCWs, neither at baseline nor on follow-up. Conclusions  The high risk of SARS-CoV-2 transmission in HCWs may be substantially reduced by adherence to Infection Prevention Control (IPC) and protective measures.