Follow-up of immune response in patients with common variable immunodeficiency following SARS-CoV-2 vaccination.

The COVID-19 pandemic highlighted the importance of effective vaccination strategies in controlling the spread of infectious diseases. SARS-CoV-2 vaccine has demonstrated high efficacy in preventing COVID-19 infection in the general population. However, the efficacy of this vaccine in patients with predominantly antibody deficiencies, such as common variable immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA), should be closely monitored. CVID and XLA are rare genetic disorders that impair the immune system's ability to produce antibodies, which are crucial for fighting infections. Patients with these disorders have a higher risk of severe disease and mortality from COVID-19 due to their compromised immune systems. In this study, we evaluated the humoral and cellular immune responses after four doses of mRNA-1273 and one BNT162b2 bivalent vaccine in a cohort of patients with CVID and XLA. The response in this population was lower than in the control group. However, the administration of the third dose improved the number of patients with seroconversion and the intensity of the humoral response, as well as the number of patients with a positive cellular response. Finally, the administration of the fourth and fifth doses improves the antibody titer and neutralization against wild type variant, but not against the prevalent XBB1.5 variant.

Analysis of HLA Alleles in Different Cohorts of Patients Infected by L. infantum from Southern Spain.

Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania, which is endemic in certain areas of Europe, such as southern Spain. The disease manifests in various clinical phenotypes, including visceral, cutaneous, mucosal, or asymptomatic leishmaniasis. This diversity in clinical outcomes may be influenced by the host immune response, with human leukocyte antigen (HLA) molecules playing a crucial role in determining susceptibility and progression of the infection. This study explores the association between specific HLA variants and Leishmania infantum infection. We recruited four cohorts: a control group, asymptomatic individuals, patients with symptomatic disease, and cohabitants of infected individuals. HLA typing was performed for all participants, followed by an association analysis with infection status and disease progression. Our findings indicate that the HLA-B*38 and HLA-C*03 alleles are associated with protection against L. infantum infection. These results contribute to a better understanding of the disease's progression, offer potential for new therapeutic approaches such as vaccines, and expand the existing knowledge in the literature.

Posterior reversible encephalopathy syndrome (PRES) in post liver transplantation.

PRES (posterior reversible encephalopathy syndrome) is a neurological condition characterised by epileptic seizures, altered consciousness, visual disturbances and/or headache with typical neuroimaging showing reversible subcortical vasogenic oedema mainly in parieto-occipital regions. The pathophysiological mechanism is not fully understood. We present a clinical case in the field of liver transplantation where tacrolimus neurotoxicity may play a relevant role in the development of this syndrome.

Major Histocompatibility Complex Class I Chain-Related α (MICA) STR Polymorphisms in COVID-19 Patients.

The SARS-CoV-2 disease presents different phenotypes of severity. Comorbidities, age, and being overweight are well established risk factors for severe disease. However, innate immunity plays a key role in the early control of viral infections and may condition the gravity of COVID-19. Natural Killer (NK) cells are part of innate immunity and are important in the control of virus infection by killing infected cells and participating in the development of adaptive immunity. Therefore, we studied the short tandem repeat (STR) transmembrane polymorphisms of the major histocompatibility complex class I chain-related A (MICA), an NKG2D ligand that induces activation of NK cells, among other cells. We compared the alleles and genotypes of MICA in COVID-19 patients versus healthy controls and analyzed their relation to disease severity. Our results indicate that the MICA*A9 allele is related to infection as well as to symptomatic disease but not to severe disease. The MICA*A9 allele may be a risk factor for SARS-CoV-2 infection and symptomatic disease.

Diversity of the green macroalgal genus Ulva (Ulvophyceae, Chlorophyta) from the east and gulf coast of the United States based on molecular data.

Ulva (Ulvophyceae, Chlorophyta) is common in intertidal environments and can also be found in freshwater ecosystems. The difficulty to morphologically identify Ulva species due to cryptic diversity and morphological plasticity has caused a taxonomic conundrum. Fortunately, molecular data have begun to unravel a better understanding of its diversity. Here, we present a molecular analysis with 247 samples of Ulva from the Gulf of Mexico and Atlantic USA based on chloroplast (rbcL and tufA) and nuclear (ITS1-5.8S-ITS2) molecular markers. Twenty-four Ulva taxa had previously been reported for this area based on morphology and earlier molecular studies mostly from Northeastern USA and Canada. In this study, sixteen Ulva clades were identified representing 13 named clades and putatively three undescribed species. Only nine of the 24 taxa previously reported for the Western Atlantic were confirmed. Four species were identified for the first time in the U.S. East and Gulf Coast (U. aragoënsis, U. californica, U. meridionalis, and U. tepida). This study provides a foundation for future research on Ulva in this area and reiterates the necessity of using molecular-assisted identifications for this group.

Assessment of littoral algal diversity from the northern Gulf of Mexico using environmental DNA metabarcoding.

Traditional methods for algal biodiversity monitoring are costly and time inefficient because they rely on high-level taxonomic expertise to address species identity problems involving phenotypic plasticity and morphological convergence. These problems are exacerbated in regions such as the Gulf of Mexico, that has a limited history of phycological exploration, but that are economically important or threatened by numerous anthropogenic stressors. Given the high pace of disturbance to natural systems, there is a critical need for expedient and cost-effective tools for the study of benthic algal communities. Here we document the use of environmental DNA metabarcoding, using the partial LSU rDNA and 23S rDNA plastid molecular markers, to elucidate littoral algal diversity in the Northern Gulf of Mexico. We assigned 73.7% of algal OTUs to genus and 59.6% to species ranks. Our current study detected molecular signals for 35 algal/protist species with no previous reports in the Gulf of Mexico, thus providing an important, molecular-validated, baseline of species richness for this region. We also make several bioinformatic recommendations for the efficient use of high-throughput sequence data to assess biological communities.

Spectrum of Movement Disorders in two Movement Disorders Centers in the Philippines.

OBJECTIVE: Presently, there are no epidemiologic data on the prevalence of movement disorders in the Philippines. We aim to describe the most common phenomenologies and movement disorders in two specialty centers in Metro Manila dedicated to movement disorders. METHODS: We investigated the clinical spectrum and etiologies of movement disorders referred to our centers from January 2007-December 2019 using a standardized collection form. RESULTS: A total of 1438 patients presenting with complaints relating to movement disorders were evaluated between 2007 to 2019. There were 770 (53.5%) men. The mean age was 57.1 ± 17.9 years. The most common movement disorders were parkinsonism (n=677, 47.1%), myoclonus (n=212, 14.7%) and tremor (n=208, 14.5%). The least common was restless legs syndrome (n=4, 0.3%). There were 78 (37.7% of total dystonia cases) X-linked dystonia-parkinsonism patients referred to our clinic. Majority of the botulinum toxin injections were for hemifacial spasms (n=206). A small number of patients (n=41) were also seen at the center for deep brain stimulation programming. CONCLUSION: The most common movement disorders managed were parkinsonism, myoclonus and tremor. The most common diagnoses were Parkinson's disease, hemifacial spasm and essential tremor. This study highlights the spectrum of movement disorders encountered in two specialty clinics in two Philippine tertiary hospitals. Given these varied cases, there is also a need for more movement specialists and centers dedicated to movement disorders to manage these cases.

Reassessment of the classification of Bryopsidales (Chlorophyta) based on chloroplast phylogenomic analyses.

The Bryopsidales is a morphologically diverse group of mainly marine green macroalgae characterized by a siphonous structure. The order is composed of three suborders - Ostreobineae, Bryopsidineae, and Halimedineae. While previous studies improved the higher-level classification of the order, the taxonomic placement of some genera in Bryopsidineae (Pseudobryopsis and Lambia) as well as the relationships between the families of Halimedineae remains uncertain. In this study, we re-assess the phylogeny of the order with datasets derived from chloroplast genomes, drastically increasing the taxon sampling by sequencing 32 new chloroplast genomes. The phylogenies presented here provided good support for the major lineages (suborders and most families) in Bryopsidales. In Bryopsidineae, Pseudobryopsis hainanensis was inferred as a distinct lineage from the three established families allowing us to establish the family Pseudobryopsidaceae. The Antarctic species Lambia antarctica was shown to be an early-branching lineage in the family Bryopsidaceae. In Halimedineae, we revealed several inconsistent phylogenetic positions of macroscopic taxa, and several entirely new lineages of microscopic species. A new classification scheme is proposed, which includes the merger of the families Pseudocodiaceae, Rhipiliaceae and Udoteaceae into a more broadly circumscribed Halimedaceae, and the establishment of tribes for the different lineages found therein. In addition, the deep-water genus Johnson-sea-linkia, currently placed in Rhipiliopsis, was reinstated based on our phylogeny.

Response of a coastal Baltic Sea diatom-dominated phytoplankton community to experimental heat shock and changing salinity.

Climate change has been altering the ocean environment, affecting as a consequence the biological communities including microorganisms. We performed a mesocosm experiment to test whether biodiversity loss caused by one stressor would influence plankton community sensitivity to a subsequent stressor, as envisioned in Vinebrooke's multiple stressor concept. A natural Baltic Sea diatom-dominated phytoplankton assemblage was used as a model system where we examined whether a preceding heat shock would affect the community's response to changing salinity. Initially, the community was treated by a short-term temperature increase of 6 °C, which resulted in a loss of species compared to the control. Thereafter, the control and the heat-shocked communities were subject to a salinity change (- 5 psu, control, + 5 psu). The species Skeletonema dohrnii, Thalassiosira anguste-lineata, Thalassiosira nordenskioeldii, Chaetoceros socialis and Ditylum brightwellii were major components of the control and heat-shocked assemblages (> 80% of the total biomass). We examined the effect on species composition and biodiversity (morphospecies and operational taxonomic units (OTUs) related to phytoplankton) and on phytoplankton biomass. In addition, we explored the single species response of five dominant diatoms on these environmental perturbations. Our results showed that increased salinity significantly reduced the OTUs richness both in the control and the less diverse heated community as well as the phytoplankton biomass in the heated community. On the other hand, decreased salinity significantly increased species richness and phytoplankton biomass in both communities and OTUs richness in the control community. The five dominant diatoms reached their highest biomass under decreased salinity and responded negatively to increased salinity (lower biomass than ambient salinity). Contrary to Vinebrooke's multiple stressor concept, there was no indication that the heat treatment had altered the community's sensitivity to the salinity stress in our study system.

Natural Extracts Abolished Lipid Accumulation in Cells Harbouring non-favourable PNPLA3 genotype.

Background & aims. G-allele of PNPLA3 (rs738409) favours triglycerides accumulation and steatosis. In this study, we examined the effect of quercetin and natural extracts from mushroom and artichoke on reducing lipid accumulation in hepatic cells. MATERIAL AND METHODS: Huh7.5 cells were exposed to oleic acid (OA) and treated with quercetin and extracts to observe the lipid accumulation, the intracellular-TG concentration and the LD size. Sterol regulatory element binding proteins-1 (SREBP-1), peroxisome proliferator-activated receptor (PPARα-γ) and cholesterol acyltransferase (ACAT) gene expression levels were analysed. RESULTS: Quercetin decreased the intracellular lipids, LD size and the levels of intracellular-TG through the down-regulation of SREBP-1c, PPARγ and ACAT1 increasing PPARα. The natural-extracts suppressed OA-induced lipid accumulation and the intracellular-TG. They down-regulate the hepatic lipogenesis through SREBP-1c, besides the activation of lipolysis through the increasing of PPARα expression. CONCLUSIONS: Quercetin and the aqueous extracts decrease intracellular lipid accumulation by down-regulation of lipogenesis and up-regulation of lipolysis.

The chloroplast genomes of Bryopsis plumosa and Tydemania expeditiones (Bryopsidales, Chlorophyta): compact genomes and genes of bacterial origin.

BACKGROUND: Species of Bryopsidales form ecologically important components of seaweed communities worldwide. These siphonous macroalgae are composed of a single giant tubular cell containing millions of nuclei and chloroplasts, and harbor diverse bacterial communities. Little is known about the diversity of chloroplast genomes (cpDNAs) in this group, and about the possible consequences of intracellular bacteria on genome composition of the host. We present the complete cpDNAs of Bryopsis plumosa and Tydemania expeditiones, as well as a re-annotated cpDNA of B. hypnoides, which was shown to contain a higher number of genes than originally published. Chloroplast genomic data were also used to evaluate phylogenetic hypotheses in the Chlorophyta, such as monophyly of the Ulvophyceae (the class in which the order Bryopsidales is currently classified). RESULTS: Both DNAs are circular and lack a large inverted repeat. The cpDNA of B. plumosa is 106,859 bp long and contains 115 unique genes. A 13 kb region was identified with several freestanding open reading frames (ORFs) of putative bacterial origin, including a large ORF (>8 kb) closely related to bacterial rhs-family genes. The cpDNA of T. expeditiones is 105,200 bp long and contains 125 unique genes. As in B. plumosa, several regions were identified with ORFs of possible bacterial origin, including genes involved in mobile functions (transposases, integrases, phage/plasmid DNA primases), and ORFs showing close similarity with bacterial DNA methyltransferases. The cpDNA of B. hypnoides differs from that of B. plumosa mainly in the presence of long intergenic spacers, and a large tRNA region. Chloroplast phylogenomic analyses were largely inconclusive with respect to monophyly of the Ulvophyceae, and the relationship of the Bryopsidales within the Chlorophyta. CONCLUSIONS: The cpDNAs of B. plumosa and T. expeditiones are amongst the smallest and most gene dense chloroplast genomes in the core Chlorophyta. The presence of bacterial genes, including genes typically found in mobile elements, suggest that these have been acquired through horizontal gene transfer, which may have been facilitated by the occurrence of obligate intracellular bacteria in these siphonous algae.

Oxidative stress and renal status of farmers exposed to pesticides in Seville (Spain).

The aim was to determine whether indirect exposure to pesticides, specifically a copper-based fungicide, induces alterations in oxidative stress and subclinical and early kidney biomarkers in male farmers tasked with olives harvesting. Furthermore, we tested whether sex influences the susceptibility to pesticide-induced renal damage by comparing the results of this study with those obtained previously. The study focused on olive farmers (n = 41) indirectly exposed to copper-based fungicides in Estepa (Sevilla, Spain), comparing them with a control group (n = 32). Blood samples were analyzed for metal concentrations (Cu, Mn, Se, and Zn), lipid peroxidation (MDA), protein oxidation (carbonyl groups), and antioxidant enzyme activities (SOD and CAT) while urine samples were assessed for biomarkers of early kidney damage (NGAL, KIM-1, transferrin, IGFBP7, TIMP-2). Although no significant, a tendency to increase lipid and protein oxidation was observed, together with the activity of antioxidant enzymes SOD and CAT, and a decrease in total antioxidants. Moreover, an increase in urinary NGAL and IGFBP7 among pesticide-exposed farmers suggests potential underdiagnosis of kidney damage. Farmers exhibit a subtle tendency to oxidative stress compared to control, while metal levels are significantly lower in farmers, suggesting potential compensatory responses. Furthermore, biomarkers for early kidney damage are elevated, emphasizing their vulnerability in both sexes. These findings highlight the need for investigations of renal health in pesticide-exposed farmers for preventative measures and regular health monitoring.

Comparison of an Enzyme Linked-Immunosorbent Assay and a Chemiluminescent Immunoassay with an Immunofluorescence Assay for Detection of Phase II IgM and IgG Antibodies to Coxiella burnetii.

In this study, we have compared the detection of IgM and IgG against C. burnetii phase II of an enzyme-linked immunosorbent assay (ELISA) (Euroimmun) and a chemiluminescent immunoassay (CLIA) (VIRCLIA, Vircell). In addition, an indirect immunofluorescence assay (IFA) was used as a reference test. One hundred forty-eight sera were used for IgG evaluation, and eighty-eight for IgM. The sensitivity of ELISA and CLIA in detecting phase II IgM was excellent. On the other hand, the CLIA IgM showed better specificity than the ELISA IgM. As for phase II IgG, the specificity of ELISA and CLIA was similar, while the ELISA technique showed a higher sensitivity. In conclusion, the best system to detect phase II IgM antibodies against C. burnetii is the CLIA from Vircell, which is characterized by high sensitivity and specificity. For the detection of phase II IgG, the Euroimmun ELISA and Vircell CLIA assays are suitable for the determination of this marker in the laboratory, although the IgG ELISA has greater sensitivity.

Role of diabetes mellitus on hepatic encephalopathy.

Hepatic encephalopathy is the main cognitive dysfunction in cirrhotic patients associated with impaired prognosis. Hyperammonemia plus inflammatory response do play a crucial role on hepatic encephalopathy. However, in some patients HE appeared without hyperammonemia and patients with increased levels of ammonia could not show cognitive dysfunction. This has led to investigate other factors that could act in a synergistic way. Diabetes mellitus and insulin resistance are characterized by releasing and enhancing these pro-inflammatory cytokines and, additionally, has been related to hepatic encephalopathy. Indeed, patients with diabetes showed raised risk of over hepatic encephalopathy in comparison with non-cirrhotics. Type 2 diabetes mellitus could impair hepatic encephalopathy by different mechanisms that include: a) increasing glutaminase activity; b) impairing gut motility and promoting constipation, intestinal bacterial overgrowth and bacterial translocation. Despite of insufficient clarity about the practicability of anti-diabetic therapy and the most efficacious therapy, we would have to pay a special attention to the management of type 2 diabetes mellitus and insulin resistance in cirrhotic patients.

Site-specific variability in the chemical diversity of the Antarctic red alga Plocamium cartilagineum.

Plocamium cartilagineum is a common red alga on the benthos of Antarctica and can be a dominant understory species along the western Antarctic Peninsula. Algae from this region have been studied chemically, and like "P. cartilagineum" from other worldwide locations where it is common, it is rich in halogenated monoterpenes, some of which have been implicated as feeding deterrents toward sympatric algal predators. Secondary metabolites are highly variable in this alga, both qualitatively and quantitatively, leading us to probe individual plants to track the possible link of variability to genetic or other factors. Using cox1 and rbcL gene sequencing, we find that the Antarctic alga divides into two closely related phylogroups, but not species, each of which is further divided into one of five chemogroups. The chemogroups themselves, defined on the basis of Bray-Curtis similarity profiling of GC/QqQ chromatographic analyses, are largely site specific within a 10 km² area. Thus, on the limited geographical range of this analysis, P. cartilagineum displays only modest genetic radiation, but its secondary metabolome was found to have experienced more extensive radiation. Such metabogenomic divergence demonstrated on the larger geographical scale of the Antarctic Peninsula, or perhaps even continent-wide, may contribute to the discovery of cryptic speciation.

Synthesis and structural characterization of fluorinated thiosemicarbazones.

Six new fluorinated thiosemicarbazones R­C(R')=N-NH-C(S)NH(2) (R = 2,4-C(6)H(3)F(2), R' = H (1); R = 2,5-C(6)H(3)F(2), R' = H (2); R = 2,6-C(6)H(3)F(2), R' = H (3); R = 3,4-C(6)H(3)F(2), R' = H (4); R = 3,5-C(6)H(3)F(2), R' = H (5) and R = 4-C(6)H(4)F, R' = C(6)H(5), (6)) have been prepared. The molecular structures of compounds 1 to 6 have been determined.

Guillain-Barré Syndrome Following the Administration of Adenovirus Vector-Based COVID-19 Vaccine.

As countries worldwide deployed their respective coronavirus disease 2019 (COVID-19) vaccination programs to mitigate the risk of COVID-19 to their citizens, several side effects and complications from the use of the various types of COVID-19 vaccines were documented and are continued to be monitored to further study the safety and efficacy of these vaccines. One such complication, the Guillain-Barré syndrome (GBS), was reported in some individuals who received a COVID-19 adenovirus vector-based vaccine. In this particular report, we describe one such case. The patient had diarrhea and fever one day after vaccination, which was a triggering event. Seven days post vaccination, the patient had bilateral symmetrical weakness with absent deep tendon reflexes. However, causality between the vaccine administered and the suspected adverse reaction cannot be readily assumed. The benefits and risk profiles of each available vaccine should be assessed continuously for it to be improved and truly useful in this pandemic. Thorough post-vaccination surveillance, along with national reporting mechanisms are needed to help establish and confirm possible links between GBS and adenovirus vector-based COVID-19 vaccines. This link needs to be probed further in prospective studies and clinical trials.

Adaptation and Validation of a Serious Game for Motor Learning Training in Children with Cerebral Palsy.

Objective: Children with cerebral palsy (CP) present motor learning disorders and somatosensory dysfunction. Although many protocols use videogames in children with CP, few apply or examine motor learning principles. This study aims at (1) implementing therapist-user-designer collaboration in adapting a videogame to the principles of motor learning and the characteristics of users with CP, and (2) piloting the effectiveness of these adaptations by analyzing the achievement of motor learning parameters (learning rate acquisition, retention, and transfer to motor and somatosensory function). Materials and Methods: Periodical interprofessional meetings conducted to the adaptation of a videogame, requiring the control of a joystick for traveling through a maze, to motor learning principles. In a pilot validation, effects in unilateral upper limb function, gross manual dexterity, and somatosensory thresholds were assessed before and after 10-week training in 13 children with CP. Results: After 10-week training with the adapted serious game, children showed learning rates above 90% and improvement in motor learning parameters along the sessions. Manual dexterity and pronation-supination of the dominant hand improved after training. No significant effects were found on somatosensory thresholds. Conclusion: Serious games are useful as motor learning tools for improving motor function in children with PC. Cooperative work among professionals and users is advisable for designing efficient videogames according to rehabilitation best practices.

Assessment of the oxidative status in mother-child couples from Seville (Spain): A prospective cohort study.

Pregnancy requires a high demand of energy, which leads to an increase of oxidative stress. The aim of this study was to assess the oxidative status in 200 couples of pregnant women-newborns at the time of delivery, for the first time, who gave birth in two University Hospitals from the province of Seville. Recruited women filled an epidemiological questionnaire with their demographic characteristics and dietary habits during pregnancy. At the time of delivery, both maternal and cord blood samples were collected. Protein oxidation, superoxide dismutase, and catalase levels were measured to assess the oxidative status of these women, together with the levels of vitamins D, B12, Zn, Se, and Cu. Our results showed a tendency for all biomarkers measured to be higher in cord blood than in maternal blood. For the correlations established between the OS markers and sociodemographic characteristics, only significant differences for carbonyl groups values were found on both maternal and cord blood, relating these higher values to the use of insecticides in the women's homes. For newborns, only a significant correlation was detected between antioxidant enzymes and the newborn's weight, specifically for superoxide dismutase activity. Additionally, the higher values obtained in cord blood might suggest metabolization, while a higher production of ROS and antioxidant enzymes might be required to maintain the balance. Measured levels for Se were similar in both maternal and cord blood, unlike Cu and Zn, where higher levels were found for maternal blood than cord blood, indicating a correlation between maternal Se values and SOD as OS biomarker. Furthermore, vitamin D levels were around the optimum values established, finding a relationship between vitamin D and new-born's height, unlike for vitamin B12 values, where a correlation with maternal food consumption characteristics was established. Overall values were inside normal ranges and consistent for our population.

Monitoring of metals and metalloids from maternal and cord blood samples in a population from Seville (Spain).

Nowadays there is an increasing concern about exposition during prenatal stage to environmental pollutants such as metals, that make pregnant women a vulnerable group of population. Numerous studies have shown associations between the prenatal exposition to some metals and an impact on cognitive, motor and intellectual development of the child. Metals and metalloid are ubiquitous in the environment and pregnant women are exposed to them though their diet, lifestyle factors or occupational and environmental sources. One hundred of maternal and one hundred of cord blood samples were obtained at delivery from pregnant women after signing of the informed consent to determine simultaneously levels of Al, As, Cd, Cr, Cu, Hg, Mn, Ni, Pb, Se and Zn by ICP-MS. Among these metals, essential ones (Cu, Mn, Se and Zn) can have health beneficial effects at low levels, however, in high concentration are potentially toxic. On the other hand, elements such as Al, As, Cd, Hg, Pb are classified as toxic metals, no matter what its concentration was. The aim of this study was to find the potential relationships between these metals and metalloid levels, newborn's parameters, pregnancy details and the epidemiologic information obtained using a questionnaire data from the participant pregnant women from Seville (Spain). A n = 100 of participants have been enrolled, 15.6 % of the women from Virgen del Rocio Hospital were smokers during pregnancy but only 11.1 % from Virgen de Valme had the habit. Dietary habits of all participants from both hospital were quite similar in average rice, fish and canned food consumption. The characteristics of newborns were also quite similar for both hospitals. A positive correlation between maternal and cord blood was found between all metals except for Cr and Cu. The strongest correlation was found for Hg (r = 0.779, p < 0.005). Positive but weaker correlations between maternal blood and lifestyle habits were also established.