Deciphering the chemical constituents and antimicrobial activity of Prangos pabularia Lindl. using LC-MS/MS in combination with experimental evaluation and computational studies.

This study meticulously explores the antimicrobial potential of Prangos pabularia Lindl.'s aerial parts through a comprehensive blend of in vitro and in silico analysis. Extracts with varying polarities underwent LC-MS/MS identification of active components, followed by in vitro and in silico assessments of antimicrobial efficacy against Escherichia coli, Bacillus cereus, Candida albicans, Candida glabrata, and Candida paropsilosis. The methanolic extract exhibited significant antimicrobial activity with a MIC value of 48 µg/mL against all tested strains. Molecular docking revealed the compound 9-(3-methylbut-2-enoxy)-furo-(3,2-g)-chromen-7-one's highest binding affinity against the penicillin-binding protein (PBP) bacterial drug target molecule. Other compounds also displayed substantial interactions with key antimicrobial drug target proteins. Further, Molecular dynamics simulations affirmed the stability of protein and ligand conformations. Collectively, these results underscore Prangos pabularia Lindl.'s aerial parts as a promising botanical resource in combating diverse microbial infections. This comprehensive approach not only validates it's in vitro antimicrobial properties but also provides molecular insights into interaction mechanisms, advancing our comprehension of the plant's therapeutic potential.

Measuring Coverage of Essential Maternal Postnatal Care Services in the Squatter Settlements of Islamabad Capital Territory in Pakistan.

This study aimed to assess the coverage of essential postnatal maternal care services among women residing in the slums of Islamabad. A community-based, cross-sectional study was conducted to assess the coverage of essential postnatal care (PNC) services. Using random sampling, 416 women living in the squatter settlements of Islamabad Capital Territory were selected as study participants. Data was analyzed by using SPSS version 22. Descriptive statistics were employed to display frequencies for categorical variables, whereas mean, median, and standard deviation were calculated for continuous variables. The analysis of data showed that 93.5 percent of the women utilized postnatal services at least once after delivery. Approximately 9 percent and 4 percent of women received all eight recommended services within 24 h of birth and beyond 24 h of birth, respectively. Effective PNC services were received by only 1 percent of the women. The study revealed that the utilization of effective PNC was very low. The majority of the women delivered in health institutions and received their first PNC checkups, but follow-up for the recommended checkups was very low. These results can help health professionals and policymakers in designing programs and developing efficient strategies that would improve PNC service utilization in Pakistan.

'The wife should do as her husband advises': Understanding factors influencing contraceptive use decision making among married Pakistani couples-Qualitative study.

This paper explores the perceptions and attitudes of married couples which prevent them from using modern contraceptive for purpose of family planning, based on semi-structured interviews with 16 married couples from rural Pakistan. This study, with married couples, not using any modern contraceptives, discussed issues of spousal communication and religious norms using qualitative methods. Despite near universal knowledge of modern contraceptives among married Pakistani women, the use continues to be low, with high unmet need. Understanding the couple context about reproductive decision making, pregnancy and family planning intentions is imperative to helping individuals fulfil their reproductive desires. Married couples may have varying intentions and desires about family size; a lack of alignment between partners may lead to unintended pregnancies and affect uptake and use of contraception. This study specifically explored the factors which prevent married couples from using LARCs for family planning, despite their availability, at affordable prices in the study area of rural Islamabad, Pakistan. Findings show differences between concordant and discordant couples regarding desired family size, contraceptive communication and influence of religious beliefs. Understanding the role that male partners play in family planning and use of contraceptives is important in preventing unintended pregnancies and improving service delivery programmes. This study also helped identify the challenges married couples, particularly men have in understanding family planning and contraceptive use. The results also show that while men's participation in family planning decision making is limited, there is also a lack of programs and interventions for Pakistani men. The study findings can support development of appropriate strategies and implementation plans.

Pharmacovigilance in High-Income Countries: Current Developments and a Review of Literature.

The world bank has classified 80 economies based on their Gross National Income (GNI) per capita as High-Income. European Medicines Agency (EMA), Food and Drug Administration (FDA), and Pharmaceuticals and Medical Devices Agency (PMDA) are the major regulatory stakeholders driving global pharmacovigilance regulations. The purpose of this article is to describe pharmacovigilance systems and processes in high-income countries, particularly those that are also members of the International Conference on Harmonization (ICH). All high-income countries are members of the WHO PIDM. The income level of a country has a direct relationship with medicine safety measures. All ten pioneering members of the Uppsala monitoring centre are from high-income countries and were the first responders after the thalidomide tragedy by making drug evaluation committees, introducing the ADR reporting forms and taking safety measures. Despite access to the VigiBase, some countries have separate databases for managing and analyzing data like Canada Vigilance online database, FDA Adverse Event Reporting System, the French pharmacovigilance database and European Union's system Eudravigilance. All high-income countries have robust pharmacovigilance systems. USFDA and EMA are the world leaders in the field of pharmacovigilance. Most high-income countries follow EMA guidelines. Medicine safety is directly influenced by a country's income level.

Isolation and Characterization of Flavonoid Naringenin and Evaluation of Cytotoxic and Biological Efficacy of Water Lilly (Nymphaea mexicana Zucc.).

Despite its limited exploration, Nymphaea mexicana Zucc. can be beneficial if pharmacology, isolation, and biological evaluation are given attention. It is an aquatic species that belongs to the family Nymphaeaceae. The thrust area of the work was the extraction, isolation, and biological evaluation of different extracts of the N. mexicana Zucc. plant. The primary goal of this research was to assess the antimicrobial, antioxidant, and anticancer activities of the extracts and to isolate the target naringenin compound. Comparative FT IR analysis of different extracts of this plant revealed the presence of functional groups of plant secondary metabolites, including polyphenols, flavonoids, terpenoids, esters, amines, glycosides, alkanes, alkaloids, fatty acids, and alcohols. Moderate free radical scavenging potential has been achieved for the various extracts via reducing power and DPPH assays. While cytotoxic activity was evaluated by colorimetric and lactate dehydrogenase cell viability tests on potent cancer cell lines. Lung adenocarcinoma epithelial cells (A-549), and breast cells (MC-7) were treated with MeOH extract. The antimicrobial activity against bacterial strains was evaluated using Gram-positive and -negative cultures, where maximum and minimum inhibition zones were recorded for different strains, including 1.6-25.6 µg/mL for Streptococcus aureus, using the agar well diffusion method. In addition, the anti-inflammatory activity of different extracts of N. mexicana Zucc. was evaluated in a nitrite radical scavenging assay with high concentrations of secondary metabolites, which are important against human pathogens and other diseases.

Implications of COVID-19 and "super floods" for routine vaccination in Pakistan: The reemergence of vaccine preventable-diseases such as polio and measles.

With multiple waves and variants, the coronavirus disease 2019 (COVID-19) pandemic has affected routine vaccination programs globally. Its impact is also visible in Pakistan as routine health services continue to be disrupted. Consequently, thousands of children have emerged as vulnerable in the face of vaccine-preventable diseases (VPDs), which have already started causing outbreaks in the country. Infections with polio and measles have been significantly reported, especially during the last few years. This reemergence of both diseases is posing great challenges for the country at local, national, and global levels. These impacts are being multiplied by the 2022 flooding - called "super floods" - in the country. Hence, relevant stakeholders, such as the Pakistani government and the World Health Organization (WHO), need to revisit the entire vaccination program to address and resolve issues occurring at the management or local levels. It is highly important to pay attention to the context that provides a fertile ground to negatively affect vaccine uptake.

ROS generated from biotic stress: Effects on plants and alleviation by endophytic microbes.

Aerobic living is thought to generate reactive oxygen species (ROS), which are an inevitable chemical component. They are produced exclusively in cellular compartments in aerobic metabolism involving significant energy transfer and are regarded as by-products. ROS have a significant role in plant response to pathogenic stress, but the pattern varies between necrotrophs and biotrophs. A fine-tuned systemic induction system is involved in ROS-mediated disease development in plants. In regulated concentrations, ROS act as a signaling molecule and activate different pathways to suppress the pathogens. However, an excess of these ROS is deleterious to the plant system. Along with altering cell structure, ROS cause a variety of physiological reactions in plants that lower plant yield. ROS also degrade proteins, enzymes, nucleic acids, and other substances. Plants have their own mechanisms to overcome excess ROS and maintain homeostasis. Microbes, especially endophytes, have been reported to maintain ROS homeostasis in both biotic and abiotic stresses by multiple mechanisms. Endophytes themselves produce antioxidant compounds and also induce host plant machinery to supplement ROS scavenging. The structured reviews on how endophytes play a role in ROS homeostasis under biotic stress were very meager, so an attempt was made to compile the recent developments in ROS homeostasis using endophytes. This review deals with ROS production, mechanisms involved in ROS signaling, host plant mechanisms in alleviating oxidative stress, and the roles of endophytes in maintaining ROS homeostasis under biotic stress.

Assessment of Effective Coverage of Antenatal Care and Associated Factors in Squatter Settlements of Islamabad Capital Territory, Pakistan: An Analytical Cross-Sectional Study.

BACKGROUND: Effective coverage of antenatal care (ANC) goes beyond contact coverage and assesses the quality of service provided. We used World Health Organization's recommended positive pregnancy guidelines to assess effective coverage and factors associated with the utilization of ANC among women in squatter settlements of Islamabad Capital Territory. METHODS: We conducted a household survey in the study area with 416 women who had given birth in the past one year. Face-to-face interviews were conducted after the selection of study subjects was done through a systematic random sampling approach. Statistical analysis was carried out using Statistical Package for the Social Sciences 22 (SPSS 22; IBM corp. Armonk, NY). Effective ANC coverage was defined as four or more ANC visits along with all WHO-recommended interventions received at least once during ANC. Adjusted odds ratios (adjOR) with 95% CI were calculated using binary logistic regression to determine the independent effects of all associated factors on the outcome. RESULTS: Of the 416 women interviewed, 399 (95.6%) had availed ANC services at least once. The coverage of 4+ ANC visits was 92% but effective coverage was only received by 35% women. The proportion of women who received nutritional interventions, maternal and fetal assessment and other preventive measures was 68%, 51% and 80.8% respectively. Maternal education (adjOR, 95% CI = 4.8[2.4-9.3]), family income (2.3[1.1-5.1]), multiparity (1.7[1.1-2.9]), place of first ANC visit (4.2[1.7-10.5]) and distance from a health facility (2.2[1.3-3.6]) were independently associated with the non-utilization of effective ANC. CONCLUSION: Despite a very high crude coverage of ANC services, the study shows a very low proportion of women receiving effective coverage. This stresses the importance of measuring the proportion of the population that receives health services with quality to monitor progress toward achieving universal health coverage.

Implementing High-Reliability Organization Principles at Biological Diagnostic Laboratories: Case Study at National Institute of Health, Islamabad.

Introduction: Healthcare organizations are complex systems where healthcare professionals, patients, biological materials, and equipment constantly interact and provide feedback with highly consequential outcomes. These are the characteristics of a complex adaptive system. Healthcare delivery requires coordination but it necessarily relies on delegation of essential functions. It is thus essential to have an engaged workforce to ensure optimal outcomes for patients. Thus human performance factors play a key role in ensuring both the presence of excellent healthcare provision and the absence of outcomes that must be avoided-"never events." Methods: The commitment of management was a precondition for the implementation of the high-reliability organization (HRO) principles. A team from middle management was engaged and provided with appropriate management tools for identifying, prioritizing, assessing, and applying solutions for the safety concern in their operating systems. Results: This article documents efforts at the National Institute of Health (NIH) to adapt the principles of HROs to diagnostic laboratories and vaccine production facilities at its campus in Islamabad, Pakistan, and seeks to draw some lessons for how this approach can be usefully replicated in such facilities elsewhere. Conclusion: Public health institutes such as NIH deliver vital products and services that are inherently risky to produce, where the consequence of failure can be catastrophic. Adopting the HRO principles is an approach to improving not just safety, but also the overall organizational performance in any setting, including low-resource settings, and can serve as an implementable process for other institutions.

A Qualitative Study of Stakeholders' Views on Pharmacovigilance System, Policy, and Coordination in Pakistan.

Objectives: Due to the absence of necessary rules, poor coordination, and various challenges, the pharmacovigilance system of Pakistan is not optimally functional at all levels of the health system. The objective of the study was to assess the stakeholders' perceptions of the current ADR reporting system and to identify the pharmacovigilance policy issues and problems of effective coordination. Methodology: Stakeholders from a broad range of disciplines, academia, regulatory authorities, the pharmaceutical industry, international health organizations, as well as pharmacovigilance experts, and healthcare professionals were included in the study. A total of 25 stakeholders throughout Pakistan were interviewed during exploratory semi-structured interviews. The interviews were recorded digitally, transcribed, coded, compared, and grouped according to their similarity of themes. Participants provided insights into gaps, limitations, and challenges of Pakistan's current ADR reporting system, issues with proposed pharmacovigilance rules, and coordination difficulties. Results: The majority of the participants considered the ADR reporting system in Pakistan to be improving but in a nascent phase. The identified gaps, challenges, limitations of the system, and barriers to reporting were labeled as reasons for limited functioning. Almost all stakeholders were aware of the existence of draft pharmacovigilance rules; however, participants in the industry were familiar with the contents and context of draft pharmacovigilance rules. Bureaucratic red tape and lack of political will appeared to be the top reasons for delaying the approval of the pharmacovigilance rules. Wider consultation, advocacy, and awareness sessions of policymakers and HCPs were suggested for early approval of rules. Participants unanimously agreed that the approval of rules shall improve the quality of life and reduce the economic burden along with morbidity and mortality rates. The need for greater and collaborative coordination among the stakeholders in promoting medicines' safety was highlighted. All participants suggested the use of media and celebrities to disseminate the safety information. Conclusion: Participants showed partial satisfaction with the way pharmacovigilance in Pakistan is moving forward. However, stakeholders believed that engagement of multi-stakeholders, approval of pharmacovigilance rules, and the establishment of pharmacovigilance centers in provinces, hospitals, and public health programs (PHPs) shall support in achieving the desired results.

Surface sterilization for isolation of endophytes: Ensuring what (not) to grow.

Endophytic microbiota opens a magnificent arena of metabolites that served as a potential source of medicines for treating a variety of ailments and having prospective uses in agriculture, food, cosmetics, and many more. There are umpteen reports of endophytes improving the growth and tolerance of plants. In addition, endophytes from lifesaving drug-producing plants such as Taxus, Nothapodytes, Catharanthus, and so forth have the ability to produce host mimicking compounds. To harness these benefits, it is imperative to isolate the true endophytes, not the surface microflora. The foremost step in endophyte isolation is the removal of epiphytic microbes from plant tissues, called as surface sterilization. The success of surface sterilization decides "what to grow" (the endophytes) and "what not to grow" (the epiphytes). It is very crucial to use an appropriate sterilant solution, concentration, and exposure time to ensure thorough surface disinfection with minimal damage to the endophytic diversity. Commonly used surface sterilants include sodium hypochlorite (2%-10%), ethanol (70%-90%), mercuric chloride (0.1%), formaldehyde (40%), and so forth. In addition, the efficiency could further be improved by pretreatment with surfactants such as Triton X-100, Tween 80, and Tween 20. This review comprehensively deals with the various sterilants and sterilization methods for the isolation of endophytic microbes. In addition, the mechanisms and rationale behind using specific surface sterilants have also been elaborated at length.

Global Agri-Food Sector: Challenges and Opportunities in COVID-19 Pandemic.

COVID-19 pandemic has been catastrophic for almost everything including the global economy. Among many sectors, the food and the agriculture sector was the worst hit following the immediate lockdown and market shutdowns. Though some stability was prevalent from supply side till date, however, the severe restrictions put in place to curb the spread of pandemic have endangered the supply of agricultural and food articles contemporaneously across borders and from field to fork. While the income decline due to price falland supplies chain disruptions due to pandemic have escalated the food shortages in several of developing and developed countries. Nevertheless the global demand for food items has remained more or less unchanged owing to their inelastic demand. Even within the global level, the scenario of food security and supply chain stability has been substantially deplorable for emerging and less developing countries due to their lack of insulation to the global shocks or pandemics. Notably, the technological backwardness, excessive know-how dependence and denied accessibility on several grounds lead to poverty and food hunger in these countries. At the policy level, a holistic approach specifically targeted towards the developing and less developed economies is highly warranted to ensure an appreciable progress towards the minimisation of sensitivity with regard to agriculture and food security. Apart from the measures to insulate them from global shocks, additional steps need to be taken to alleviate their technological backwardness and denied accessibility on certain socio-cultural norms.

Role of social network in decision making for increasing uptake and continuing use of long acting reversible (LARC) methods in Pakistan.

INTRODUCTION: Despite evidence from recent Demographic Health Surveys that show 98% of the adult Pakistani population have an awareness of at least one modern contraceptive method, only 25% of married couples in Pakistan used a modern method of contraception. Of the modern contraceptive methods, LARC usage has increased only from 2.1 to 3%. This low uptake is puzzling in the context of high awareness of LARC methods and its availability through public sector facilities at subsidized costs. This study aimed to understand the social influences in initiating and continuing use of an LARC methods for contraception in a rural setting in Pakistan. METHODS: In-depth interviews were conducted with 27 women who were using a LARC method for contraception. Data was managed using NVivo 12 and themes were identified using a content analysis approach to analyze the transcripts. RESULTS: Four key themes, supported by sub-themes relating to a temporal model, were identified to explain women's experiences with initiating and continuing use of a LARC. The themes were (i) Use of trusted networks for information on LARCs; (ii) Personal motivation and family support in decision to use LARC; (iii) Choice of LARC methods and access to providers; and (iv) Social and professional support instrumental in long term use of LARC. Results highlight the significant role of immediate social network of female family members in supporting the women in initiating LARCs and maintaining the method's use. CONCLUSION: This study contributes to an in depth understanding of the decision-making process of women who adopted LARC and maintained its use. Women who proceeded to use an LARC and who persisted with its use despite the experience of side effects and social pressures, were able to do so with support from other female family members and spouse.

Assessment of the Current State of Pharmacovigilance System in Pakistan Using Indicator-Based Assessment Tool.

Objectives: Pakistan felt the need for an effective and robust pharmacovigilance (PV) system after one of the deadliest drug-related tragedies causing more than 300 deaths in 2012. The country set up its national PV center in 2015 and joined WHO's Program for International Drug Monitoring (PIDM) in 2018 as a full member. The current study was aimed to evaluate the PV system's functionality, identify the gaps, areas of improvement, and a strategy to lead a functional PV system in Pakistan. Methods: The descriptive cross-sectional study was conducted by providing an interviewer-administered questionnaire of the PV system across Pakistan by utilizing the Indicator based Pharmacovigilance assessment tool (IPAT). By a convenience sampling method 36 study participants were selected from the Drug Regulatory Authority of Pakistan (DRAP), drug administration of provincial health departments of 4 provinces and federally affiliated areas, 5 national public health programs, and 23 public and private hospitals. The assessment includes document review, interviews of the key informants by structured open-ended questions, and a review of websites of relevant organizations. Results: Drug Regulatory Authority of Pakistan (DRAP) with a national PV center received a 75% overall performance score on IPAT. To be regarded as "minimally functioning," a country's PV and drug safety system must meet all core indicators. DRAP scored 80.76% on the core indicators so cannot be deemed functional at this time. The only province with a regional PV center, Punjab, had scored 72.13% on relevant parameters. Despite receiving funding from the Global Fund, none of the National Public Health Programs (PHPs) have PV centers or associated activities. All hospitals except two private hospitals could not qualify the minimum requirements for functional PV. The absence of a legal framework for mandatory ADR reporting, lack of drug information center, budgetary constraints, no active surveillance activities, the nonexistence of pharmacovigilance risk assessment expert committee, and insufficient coordination among stakeholders were identified as major gaps. Conclusion: The results of the study reveal that Pakistan's PV system is not fully functional at all levels. A two-phased strategy encompassing the non-financial and financial interventions is proposed to improve the PV systems at the national, provincial, PHPs, and hospitals levels.

Learning Active Implementation Frameworks: the role of implementation teams in a case study from Pakistan.

In Pakistan, although coverage of Maternal, Newborn, and Child Health (MNCH) services has increased, the attributable disease burden remains high, indicating quality of these services remains suboptimal. To address this quality gap, challenges associated with the implementation of MNCH services will need to be addressed and effective use of the various MNCH guidelines will need to be supported, evaluated, and continuously improved. Even though the application of the field of implementation science and practice in the low- and middle-income settings has been limited, it is our belief, based on the experience described in this article that these competencies could enhance health professionals' ability to, not only successfully integrate MNCH guidelines into health systems, but to also support their effective and sustainable use. To address this capacity gap in Pakistan, the Health Services Academy, as a member of the World Health Organization's Human Reproduction Program (HRP) Alliance for Research Capacity Strengthening (RCS), has engaged, over the course of 16 months, in the 'Implementation for the Professional Learner Program' in 2019. This innovative implementation science and practice capacity-building program is developed and conducted by The World Health Organization (WHO) Collaborating Centre for Research Evidence for Sexual and Reproductive Health at the University of North Carolina at Chapel Hill (UNC). The initial cohort of this Program also included Palestine's West Bank, and Egypt. The objectives of this Program were to cultivate implementation science and practice competencies, and to support the development of national, community-based or institution-based implementation teams. The expected outcomes of this program included, further enhancement of the capacity of local health professionals in implementation science, systemic change and the effective use of innovations in practice at sub-national/regional levels.

Novel coronavirus disease (COVID-19): a pandemic (epidemiology, pathogenesis and potential therapeutics).

The coronavirus disease (COVID-19) is highly pathogenic viral infection caused by SARS-CoV-2. Currently, COVID-19 has caused global health concern. It is assumed that COVID-19 has zoonotic origin based on the large number of infected people who were exposed to the wet market in Wuhan City, China. The phylogenetic analysis has revealed that SARS-CoV-2 has significant sequence similarity with severe acute respiratory syndrome-like (SARS-like) bat viruses, thus bats could be primary possible reservoir. The intermediate host and there subsequent transfer is not known yet, although human to human transfer is widely confirmed. The transmission of COVID-19 infection from one person to another resulted in the isolation of patients who were subsequently given a variety of treatments. To monitor the current outbreak, robust steps have been taken around the globe to reduce the transmission of COVID-19 infection particularly banning international and domestic flights, inducting lockdowns in vulnerable areas, social distancing etc. No clinically approved antiviral drug or vaccine against COVID-19 is reported yet. However, in clinical trials, few broad-spectrum antiviral drugs were evaluated against COVID-19 infection which resulted in clinical recovery. In this article emergence and pathogenicity of COVID-19 infection along with potential therapeutic strategies are analyzed to combat the COVID-19 pandemic.

Correction: The Knowledge Translation Status in Selected Eastern-Mediterranean Universities and Research Institutes.

[This corrects the article DOI: 10.1371/journal.pone.0103732.].

Capacity building on health diplomacy: a training experience from Pakistan.

Modern-day globalization means that many health issues cannot be resolved by the affected country alone, and this necessitates political consultations, diplomatic negotiations and cross-border solutions. A few examples that require health diplomacy efforts are: halting resentment towards immunization, addressing the burden of noncommunicable diseases, enabling access to drugs and technology, and liberalizing trade to reduce the cost of drugs. The agenda of Sustainable Development Goals (SDGs) demands a concerted effort to achieve the ambitious targets. This article reports the experience of health diplomacy training imparted to mid-level and senior officials in the public as well as private sector in Pakistan. Training was geared to inculcate an understanding of global health diplomacy and governance, and to develop an appreciation of the relationship of global health with other disciplines such as foreign affairs, economics, trade, climate change and human rights. Participants included health professionals, experts from departments other than health, government officials and diplomats. This training was expected to enhance their knowledge of health systems dynamics that are influenced by foreign policy and diplomatic discourses.

Effect of tobacco use on tooth loss among patients visiting the out-patient dental department of a tertiary care hospital in Pakistan.

OBJECTIVE: To determine the factors influencing tooth loss and to assess the strength of association between the factors and the loss.s. METHODS: The unmatched case-control study was conducted at the Pakistan Institute of Medical Sciences, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, from March to April 2016, and comprised data of patients who visited the outpatient dental department. Face-to-face interviews and clinical examination were done. Data was analysed using SPSS 20. RESULTS: Of the 376 subjects, there were 188(50%) in each of the two groups. The mean age among the cases was 37.01±12.077 years and among the controls it was 28.06±9.591 years. Tobacco consumption and age >35 were significant predictors of tooth loss (p<0.05 each). Tobacco users with high duration and frequency had a significant value for increased chances of tooth loss (p<0.001). Both the smokers and smokeless tobacco users had more chances of tooth loss (p<0.001). CONCLUSIONS: There was negative implications of smoking on oral health which increased the chance of tooth loss.