Botanicals as promising antimicrobial agents for enhancing oral health: a comprehensive review.

The mouth houses the second largest diversity of microorganisms in the body, harboring more than 700 bacterial species colonizing the soft mucosa and hard tooth surfaces. Microbes are the cause of several health-related problems, such as dental carries, gingivitis, periodontitis, etc., in the mouth across different age groups and socioeconomic/demographic groups. Oral infections are major health problems that affect the standard of living. Compromised oral health is related to chronic conditions and systemic disorders. Microbes responsible for dental caries are acid-producing and aciduric Gram-positive bacteria (Streptococci, Lactobacilli). Gram-negative bacteria (Porphyromonas, Prevotella, Actinobacillus, and Fusobacterium) capable of growing in anaerobic environments are responsible for periodontal diseases. Due to the high prevalence of oral diseases, negative effects associated with the use of antimicrobial agents and increased antibiotic resistance in oral pathogens, suitable alternative methods (effective, economical and safe) to suppress microbes disturbing oral health need to be adopted. Side effects associated with the chemical antimicrobial agents are vomiting, diarrhea and tooth staining. Several researchers have studied the antimicrobial properties of plant extracts and phytochemicals and have used them as indigenous practices to control several infections. Therefore, phytochemicals extracted from plants can be suitable alternatives. This review focuses on the various phytochemical/plant extracts suppressing the growth of oral pathogens either by preventing their attachment to the surfaces or by preventing biofilm formation or other mechanisms.

Factors affecting the distribution of serum anti-müllerian hormone levels among infertile Asian women: a multi-nation, multi-centre, and multi-ethnicity prospective cohort study.

STUDY QUESTION: How do age, ethnicity, and other characteristics affect serum anti-mullerian hormone (AMH) levels in Asian women undergoing fertility treatment? SUMMARY ANSWER: Age, ethnicity, obesity (BMI ≥ 30 kg/m2), and polycystic ovarian syndrome (PCOS) significantly impacted serum AMH levels, with the rate of decrease accelerating as age increased; a concentration of 4.0 ng/ml was the optimal cut-off for diagnosis of PCOS. WHAT IS KNOWN ALREADY: There are significant differences in ovarian reserve among women from different races and ethnicities, and Asian women often have poorer reproductive outcomes during assisted reproductive treatment cycles. STUDY DESIGN, SIZE, DURATION: A population-based multi-nation, multi-centre, multi-ethnicity prospective cohort study of 4613 women was conducted from January 2020 to May 2021. Infertile women of 20-43 years of age were enrolled. The exclusion criteria included: age <20 or >43, non-Asian ethnicity, and missing critical data. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were Asian women of Chinese, Japanese, Korean, Thai, Vietnamese, Malay, Indian, and Indonesian ethnicities from 12 IVF centres across Asia. These women were all naïve to ovarian stimulation cycles and attended IVF centres for fertility assessment. The AMH measurement was performed using an AMH automated assay on a clinically validated platform. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 4556 infertile Asian women were included in the final analyses. The mean ± SD for serum AMH concentrations (ng/ml) across specific age groups were: overall, 3.44 ± 2.93; age <30, 4.58 ± 3.16; 30-31, 4.23 ± 3.23; 32-33, 3.90 ± 3.06; 34-35, 3.21 ± 2.65; 36-37, 2.74 ± 2.44; 38-39, 2.30 ± 1.91; 40 and above, 1.67 ± 2.00. The rate of AMH decrease was ∼0.13 ng/ml/year in patients aged 25-33 and 0.31 ng/ml/year in women aged 33-43. The highest rates of PCOS were found in Indians (18.6%), Malays (18.9%), and Vietnamese (17.7%). Age (P < 0.001), ethnicity (P < 0.001), obesity (P = 0.007), PCOS (P < 0.001), and a history of endometrioma cystectomy (P = 0.01) were significantly associated with serum AMH values. Smoking status, pretreatment with GnRH agonist (GnRHa) or the oral contraceptive pill (OCP), freezing-thawing of blood samples, and sampling on Day 2 to Day 5 of the menstrual cycle or randomly did not appear to affect serum AMH levels. An AMH concentration of 4.0 ng/ml was the optimal cut-off for PCOS diagnosis with a sensitivity of 71.7% and specificity of 75.8% (AUC = 0.81, CI 95%: 0.79-0.83; P < 0.001). LIMITATIONS, REASONS FOR CAUTION: The incidence of PCOS was supposedly high in this cohort as some IVF clinics were tertiary referral centres for managing specific fertility issues encountered by women with PCOS. Treatment with GnRHa or OCP before AMH testing was regionally and ethnically confined, mostly in Hong Kong SAR and Japan. Moreover, this reference for serum AMH value is limited to Asian women of the ethnicities examined and may not apply to other ethnicities not included in the study. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to collate and construct age-specific reference ranges for serum AMH levels using the same bioassay on Asian women of different ethnicities. The findings of this investigation can assist clinicians to counsel and prognosticate about Asian women's ovarian reserve and reproductive potential, thus providing better strategies for personalized fertility interventions. STUDY FUNDING/COMPETING INTEREST(S): This study was technically supported by Ferring Pharmaceuticals and received no specific grant from any funding agency. All authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER: NCT04203355.

Black soybean (Glycine max (L.) Merr.): paving the way toward new nutraceutical.

Black soybean (BS) is a nutritious legume that is high in proteins, essential amino acids, dietary fiber, vitamins, minerals, anthocyanins, phenolic acids, isoflavones, and flavones. Traditional approaches for extracting BS bioactive compounds are commonly employed because they are simple and inexpensive, but they use toxic solvents and have lower yields. As a result, new extraction techniques have been developed, such as microwave, ultrasound, and enzyme-assisted extraction. Modern approaches are less harmful to the environment, are faster, and produce higher yields. The major anthocyanin in the BS seed coat was discovered as cyanidin-3-O-glucoside, accounting for nearly 75% of the total anthocyanins. BS and its seed coat also contains phenolic acids (p-hydroxybenzoic, gallic, vanillin, syringic acid), isoflavones (daidzein, glycitein and genistein), flavones, flavonols, flavanones, and flavanols. Bioactive compounds present in BS exhibit antioxidant, anti-cancerous, anti-diabetic, anti-obesity, anti-inflammatory, cardio and neuroprotective activities. The characterization and biological activity investigation of these bioactive compounds has provided researchers and food manufacturers with valuable information for developing functional food products and nutraceutical ingredients. In this review, the nutritional makeup of BS is reviewed, and the paper seeks to provide an insight of bioactive compound extraction methods as well as bioactive compounds identified by various researchers. The biological activities of BS extracts and their potential applications in food products (noodles), biodegradable films (pH sensitive film), and therapeutic applications (wound healing and anti-inflammation) are also discussed in the study. Therefore, BS have enormous potential for use in developing functional foods and nutraceutical components. This is the first review of its sort to describe and explain various extraction methodologies and characterization of bioactives, as well as their biological activity recorded in diverse works of literature, making it possible for food manufacturers and scientists to get a quick overview.

Assessment of health status and its correlation with lung function in patients with chronic obstructive pulmonary disease: a study from a tertiary care center in north India.

The Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2023 recommends a comprehensive multidimensional assessment for patients with chronic obstructive pulmonary disease (COPD) and stresses the need for evaluation of their health status and quality of life (QOL). The COPD assessment test (CAT), clinical COPD questionnaire (CCQ), and St. George respiratory questionnaire (SGRQ) are recommended by GOLD for such assessments. However, their correlation with spirometry in the Indian population is not known. Other similar questionnaires like the COPD and sleep impact scale (CASIS), functional performance inventory-short form (FPI-SF), and COPD and asthma fatigue scale (CAFS), though used internationally as a research tool, are still in the offspring stage and have never been used in India. A cross-sectional study was hence conducted in the Department of Pulmonary Medicine, Government Medical College, Patiala, Punjab, India, on 100 COPD patients. Patients were assessed for health status and QOL by CAT, CCQ, SGRQ, CASIS, FPI-SF, and CAFS. The relationship between these questionnaires and airflow limitations was investigated. The majority of the patients were males (n=97), >50 years of age (n=83), illiterate (n=72), had moderate/severe COPD, and belonged to group B (n=66). The mean value of forced expiratory volume in one second (FeV1) decreased with a deterioration in CAT and CCQ score grouping (p<0.001). Patients with poorer CAT and CCQ scores belonged to higher GOLD grades (k=0.33, p<0.001). The correlation of health-related quality of life (HRQL) questionnaires among each other, with FEV1 predicted and with GOLD grade, was strong to very strong in most of the comparisons (p<0.01 in the majority). On comparison of GOLD grade with mean scores of HRQL questionnaires, it was seen that with the increase in GOLD grading from 1 to 4, the mean values of CAT, CCQ, SGRQ, CASIS, FPI-SF, and CAFS also deteriorated (p<0.001, p<0.001, p<0.001, p<0.005, p<0.001 and p<0.001, respectively). Various easy-to-use HRQL scores should be routinely used in outpatient departments for a comprehensive assessment of COPD patients. These questionnaires, in combination with clinical features, can help in providing a rough estimate of the severity of the disease in places where lung function assessments are not readily available.

Design of an ultra-sensitive bimetallic anisotropic PCF SPR biosensor for liquid analytes sensing.

In this research work, an anisotropic photonic crystal fiber (PCF) biosensor working on a refractive index (RI) variation and based on surface plasmon resonance (SPR) is presented. Liquid analytes (LA) having a RI within the range of 1.340 to 1.380 RIU are investigated from the proposed biosensor. Spectroscopy analysis of LA having RI values of 1.340 RIU, 1.360 RIU, and 1.380 RIU is performed from the developed sensing setup for modeling an ultrasensitive biosensor. The numerical analysis of the sensing parameters for the proposed sensor presents a maximum wavelength sensitivity (WS) of 20000 nm/RIU for x- polarization (x - pol.) and 18000 nm/RIU for y- polarization (y - pol.), respectively, using the wavelength interrogation technique. Maximum amplitude sensitivity (AS) of 2158 RIU-1 and 3167 RIU-1 is obtained for x - pol. and y - pol., respectively, using the amplitude interrogation technique. Maximum sensor resolution (SR) of 5.00 × 10-6RIU and 5.55 × 10-6RIU is obtained for x - pol. and y - pol., respectively. The linear relationship of the resonant wavelength (RW) with the RI produces R2 =  0.9972 and R2 = 0.9978, corresponding to a degree (2) for x - pol. and y - pol., respectively. The figure of merit (FOM) for x - pol. and y - pol. are 93.45 RIU-1 and 105.88 RIU-1, respectively. The sensing parameters have obtained the maximum value for the LA having a RI value of 1.375 RIU.

Contemporary beliefs surrounding the menarche: a pilot study of adolescent girls at a school in middle England.

Despite menarche affecting half of the population, there is limited research into its effects and limited dialogue within UK society. As Relationships and Sex Education (RSE), which encompasses issues like puberty, becomes compulsory in all schools in England for the first time, understanding the impact of menarche is vital. This study aimed to explore the beliefs about and experiences of menarche amongst adolescent girls. Semi-structured interviews were conducted with eleven participants aged 16-18 attending a secondary school in Warwickshire. A thematic analysis was carried out, with emerging themes discussed between two researchers.All participants had received some RSE teaching at school, although many felt that it did not provide sufficient detail. These sessions also occurred too late for some participants who had already reached menarche. A range of emotions were recalled at this milestone; while some girls felt prepared and mature, others reported disbelief and shame. Mothers were the most important person for guidance, while conversations with fathers about menstruation were generally avoided. GPs were not considered a source of advice, despite some participants experiencing significant symptoms and menstrual irregularity.It is clear that the impact of menarche should not be underestimated and stigma surrounding menstruation still exists today. To address these issues, suggested improvements include mixed gender teaching to promote open discussions and making the role of GPs more visible to the adolescent community.

Consensus Mutagenesis and Ancestral Reconstruction Provide Insight into the Substrate Specificity and Evolution of the Front-End Δ6-Desaturase Family.

Marine algae are a major source of ω-3 long-chain polyunsaturated fatty acids (ω3-LCPUFAs), which are conditionally essential nutrients in humans and a target for industrial production. The biosynthesis of these molecules in marine algae requires the desaturation of fatty acids by Δ6-desaturases, and enzymes from different species display a range of specificities toward ω3- and ω6-LCPUFA precursors. In the absence of a molecular structure, the structural basis for the variable substrate specificity of Δ6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the Δ6-desaturase family, focusing on the ω3-specific Δ6-desaturase from Micromonas pusilla (MpΔ6des) and the bispecific (ω3/ω6) Δ6-desaturase from Ostreococcus tauri (OtΔ6des). Our characterization of consensus amino acid substitutions in MpΔ6des revealed that residues in diverse regions of the protein, such as the N-terminal cytochrome b5 domain, can make important contributions to determining substrate specificity. Ancestral protein reconstruction also suggests that some extant Δ6-desaturases, such as OtΔ6des, could have adapted to different environmental conditions by losing specificity for ω3-LCPUFAs. This data set provides a map of regions within Δ6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology.

Engineering Trienoic Fatty Acids into Cottonseed Oil Improves Low-Temperature Seed Germination, Plant Photosynthesis and Cotton Fiber Quality.

Alpha-linolenic acid (ALA, 18:3Δ9,12,15) and γ-linolenic acid \ (GLA, 18:3Δ6,9,12) are important trienoic fatty acids, which are beneficial for human health in their own right, or as precursors for the biosynthesis of long-chain polyunsaturated fatty acids. ALA and GLA in seed oil are synthesized from linoleic acid (LA, 18:2Δ9,12) by the microsomal ω-3 fatty acid desaturase (FAD3) and Δ6 desaturase (D6D), respectively. Cotton (Gossypium hirsutum L.) seed oil composition was modified by transforming with an FAD3 gene from Brassica napus and a D6D gene from Echium plantagineum, resulting in approximately 30% ALA and 20% GLA, respectively. The total oil content in transgenic seeds remained unaltered relative to parental seeds. Despite the use of a seed-specific promoter for transgene expression, low levels of GLA and increased levels of ALA were found in non-seed cotton tissues. At low temperature, the germinating cottonseeds containing the linolenic acid isomers elongated faster than the untransformed controls. ALA-producing lines also showed higher photosynthetic rates at cooler temperature and better fiber quality compared to both untransformed controls and GLA-producing lines. The oxidative stability of the novel cottonseed oils was assessed, providing guidance for potential food, pharmaceutical and industrial applications of these oils.

Effect of Pulsed Electromagnetic Fields on Human Mesenchymal Stem Cells Using 3D Magnetic Scaffolds.

Alternative bone regeneration strategies that do not rely on harvested tissue or exogenous growth factors are needed. One of the major challenges in tissue reconstruction is recreating the bone tissue microenvironment using the appropriate combination of cells, scaffold, and stimulation to direct differentiation. This study presents a bone regeneration formulation that involves the use of human adipose-derived mesenchymal stem cells (hASCs) and a three-dimensional (3D) hydrogel scaffold based on self-assembled RADA16 peptides containing superparamagnetic iron oxide nanoparticles (NPs). Although superparamagnetic NPs could be used as stimulus to manipulate the cell proliferation and differentiation, in this paper their use is explored for assisting osteogenic differentiation of hASCs in conjunction with direct stimulation by extremely low-frequency pulsed electromagnetic fields (pEMFs). Cellular morphology, proliferation, and viability, as well as alkaline phosphatase activity, calcium deposition, and osteogenic capacity were monitored for cells cultured up to 21 days in the 3D construct. The results show that the pEMFs and NPs do not have any negative effect on cell viability, but instead distinctly induced early differentiation of hASCs to an osteoblastic phenotype, when compared with cells without biophysical stimulation. This effect is attributed to synergy between the pEMFs and NPs, which may have stimulated mechanotransduction pathways, which, in turn activated biochemical signals between cells to differentiate or proliferate. This approach may offer a safe and effective option for the treatment of non-union bone fractures. Bioelectromagnetics. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.

Redox-dependent interaction between thaumatin-like protein and ß-glucan influences malting quality of barley.

Barley is the cornerstone of the malting and brewing industry. It is known that 250 quantitative trait loci (QTLs) of the grain are associated with 19 malting-quality phenotypes. However, only a few of the contributing genetic components have been identified. One of these, on chromosome 4H, contains a major malting QTL, QTL2, located near the telomeric region that accounts, respectively, for 28.9% and 37.6% of the variation in the ß-glucan and extract fractions of malt. In the current study, we dissected the QTL2 region using an expression- and microsynteny-based approach. From a set of 22 expressed sequence tags expressed in seeds at the malting stage, we identified a candidate gene, TLP8 (thaumatin-like protein 8), which was differentially expressed and influenced malting quality. Transcript abundance and protein profiles of TLP8 were studied in different malt and feed varieties using quantitative PCR, immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The experiments demonstrated that TLP8 binds to insoluble (1, 3, 1, 4)-ß-D glucan in grain extracts, thereby facilitating the removal of this undesirable polysaccharide during malting. Further, the binding of TLP8 to ß-glucan was dependent on redox. These findings represent a stride forward in our understanding of the malting process and provide a foundation for future improvements in the final beer-making process.

What are general practitioners' views on the management of adolescents with mental health disorders? A qualitative study.

One in 10 children aged 11 to 16 years' have a mental health disorder. Whilst general practitioners (GPs) are considered ideal candidates to intervene and provide support, little research has focused on how this is managed, and the challenges they face, in the U.K. The aim of this work was to explore GPs' views on the management of adolescents with mental health disorders, and identify challenges they may face in current care. Eight GPs working in London were interviewed, using a semi-structured topic guide and a pre-prepared structured vignette. GPs with more clinical experience felt greater confidence in offering an intuitive approach to care. Many emphasised the therapeutic value of partnership with adolescents and discussed the complexity of identifying and labelling adolescents with a mental health disorder. Most GPs highlighted the need for a social picture: assessing their level of socio-economic deprivation, and the impact of parents. However, through this focus, some GPs felt adolescents from middle class backgrounds could be overlooked. This study offers some insight into the considerable impact GPs have on adolescents with mental health disorders, and challenges they face. To meet these challenges, proposals include a named contact within secondary care, and improved resources.

Exploring parents' perceptions on the importance and feasibility of child-centred consultations of 5-11 year olds in general practice.

BACKGROUND: The active involvement of children in their health care has been shown to increase compliance and improve outcomes. Despite this, children in the 6-12 year group have little meaningful involvement in General Practitioner (GP) consultations, contributing to less than 20% of interactions. AIM: To explore parents' perceptions on the importance and feasibility of child-centred consultations. METHODS: Purposive sampling was used to recruit parents from a primary school in London. Three audio recorded focus groups were conducted, transcribed verbatim, and subsequently thematically analysed. RESULTS: While most parents acknowledged the importance of child-centred consultations, they legitimately questioned their child's ability to make decisions. Parents attributed low child participation to several factors including the perceived approachability of the GP, whether their child had met the doctor before, their child's personality and the general lack of time during consultations. Parents described their own anxiety and worries surrounding their child's health care which lead to their role as their child's advocate, decision maker and protector during GP consultations. CONCLUSION: This study confirms the importance of child-centred consultations and highlights numerous barriers which need to be overcome to achieve greater child involvement in consultations. If the findings were to be replicated in future larger studies, then it could lead to changes in both training and, crucially, how child-parent-doctor consultations should be carried out in general practice.

Genotypic variability in nutritional and functional attributes of blueberry varieties grown in northern-western Himalayas.

Blueberry fruits are known for their high vitamin C, essential dietary fibre, antioxidant activity and anthocyanin pigments. Different blueberry varieties have been introduced in India but no attempt has been made for their nutritional profiling. Nutritional profiling of varieties helps us to know the unique varietal characters, which serves as a guideline for recommendation of a valuable variety for fresh consumption and/or processing. Therefore, the present study was conducted in eight different blueberry varieties such as 'Misty', 'Sharp Blue', 'Biloxi', 'Jewel', 'Gulf Coast', 'Blue Crop', 'Star', 'Legacy'. The results of the study revealed that all tested varieties differed significantly in physical attributes (10-berry weight, fruit firmness, roundness index, moisture content) and biochemical and functional attributes (ascorbic acid, total anthocyanin, total phenolic content, antioxidant activity, total sugars, organic acids) and mineral content. Regression analysis and Principal Component Analysis showed that antioxidant potential of blueberries was mainly contributed by phenolics followed by anthocyanins and ascorbic acid content. However for taste perception, fructose among sugars and succinic acid among sugars were the most influencing factors (p ≤ 0.05). Total phenolics and anthocyanins content were responsible for overall difference in functional attributes among the varieties. The attributes such as high fruit firmness, sensorial score, and appropriate shape and weight make 'Misty', the best variety for marketability and fresh consumption among all tested varieties.

Harnessing Biotechnology for the Development of New Seed Lipid Traits in Brassica.

The seed oil quality of Brassica oilseed species has been improved in the last few decades, using conventional breeding approaches. Modern biotechnology has enabled the significant development of new seed lipid traits in many oil crops. Alternation of seed lipid component with gene knockout by RNAi gene silencing, artificial microRNA or gene editing within the crop is relative straightforward. Introducing a new pathway from an exogenous source via biotechnology enables the creation of a new trait, where the biosynthetic pathway for such a new trait is not available in the host crop. This review updates the recent development of new seed lipid traits in six major Brassica species and highlights the capability of biotechnology to improve the composition of important fatty acids for both industrial and nutritional purposes.

Identification of Genes Involved in Lipid Biosynthesis through de novo Transcriptome Assembly from Cocos nucifera Developing Endosperm.

Cocos nucifera (coconut), a member of the Arecaceae family, is an economically important woody palm that is widely grown in tropical and subtropical regions. The coconut palm is well known for its ability to accumulate large amounts of oil, approximately 63% of the seed weight. Coconut oil varies significantly from other vegetable oils as it contains a high proportion of medium-chain fatty acids (MCFA; 85%). The unique composition of coconut oil raises interest in understanding how the coconut palm produces oil of a high saturated MCFA content, and if such an oil profile could be replicated via biotechnology interventions. Although some gene discovery work has been performed there is still a significant gap in the knowledge associated with coconut's oil production pathways. In this study, a de novo transcriptome was assembled for developing coconut endosperm to identify genes involved in the synthesis of lipids, particularly triacylglycerol. Of particular interest were thioesterases, acyltransferases and oleosins because of their involvement in the processes of releasing fatty acids for assembly, esterification of fatty acids into glycerolipids and protecting oils from degradation, respectively. It is hypothesized that some of these genes may exhibit a strong substrate preference for MCFA and hence may assist the future development of vegetable oils with an enriched MCFA composition. In this study, we identified and confirmed functionality of five candidate genes from the gene families of interest. This study will benefit future work in areas of increasing vegetable oil production and the tailoring of oil fatty acid compositions.

Up-regulation of lipid biosynthesis increases the oil content in leaves of Sorghum bicolor.

Synthesis and accumulation of the storage lipid triacylglycerol in vegetative plant tissues has emerged as a promising strategy to meet the world's future need for vegetable oil. Sorghum (Sorghum bicolor) is a particularly attractive target crop given its high biomass, drought resistance and C4 photosynthesis. While oilseed-like triacylglycerol levels have been engineered in the C3 model plant tobacco, progress in C4 monocot crops has been lagging behind. In this study, we report the accumulation of triacylglycerol in sorghum leaf tissues to levels between 3 and 8.4% on a dry weight basis depending on leaf and plant developmental stage. This was achieved by the combined overexpression of genes encoding the Zea mays WRI1 transcription factor, Umbelopsis ramanniana UrDGAT2a acyltransferase and Sesamum indicum Oleosin-L oil body protein. Increased oil content was visible as lipid droplets, primarily in the leaf mesophyll cells. A comparison between a constitutive and mesophyll-specific promoter driving WRI1 expression revealed distinct changes in the overall leaf lipidome as well as transitory starch and soluble sugar levels. Metabolome profiling uncovered changes in the abundance of various amino acids and dicarboxylic acids. The results presented here are a first step forward towards the development of sorghum as a dedicated biomass oil crop and provide a basis for further combinatorial metabolic engineering.

2D NMR Analysis of the Effect of Asparagine Deamidation Versus Methionine Oxidation on the Structure, Stability, Aggregation, and Function of a Therapeutic Protein.

Two of the most common forms of chemical modifications that compromise the efficacy of therapeutic proteins are the deamidation of asparagine residues and oxidation of methionine residues. We probed how deamidation affects the structure, stability, aggregation, and function of interferon alpha-2a (IFNA2a), and compared with our earlier results on methionine oxidation. Upon deamidation, no significant changes were observed in the global secondary structure of IFNA2a with minor changes in its tertiary structure. However, deamidation destabilized the protein, and increased its propensity to aggregate under accelerated stress conditions. Cytopathic inhibition and antiproliferation assays showed drastic decrease in the functionality of deamidated IFNA2a compared to the wild-type. 2D NMR measurements showed structural changes in local protein regions, with no effect on the overall global structure of IFNA2a. These local protein regions corresponded well with the aggregation hot-spots predicted by computational programs, and the functional hot-spots identified by site-directed mutagenesis. When compared to the effects of methionine oxidation, deamidation caused lesser aggregation, because of lesser structural unfolding observed in aggregation hot-spots by 2D NMR. In comparison to oxidation, deamidation showed larger decrease in function, because deamidation affected key amino acid residues in functional hot-spots as observed by 2D NMR and structural modeling. Such quantitative comparison between the effects of deamidation and oxidation on a pharmaceutical protein has not been done before, and the high-resolution structural information on local protein regions obtained by 2D NMR provided a better insight compared to low-resolution methods that probe global protein structure.

Gene Expression Profiling of Human Adipose Tissue Stem Cells during 2D versus 3D Adipogenesis.

Much of the current understanding on molecular and cellular events of adipose developmental biology comes from monolayer cell culture models using preadipocyte cell lines, although in vivo adipose tissue consists of a much more complex three-dimensional microenvironment of diverse cell types, extracellular network, and tissue-specific morphological and functional features. Added to this fact, the preadipocytes, on which the adipogenesis mechanisms are mostly explored, possess some serious limitations (e.g., time of initial subculture and adipogenic differentiation time), which, perhaps, can efficiently be replaced with progenitor cells such as adipose tissue-derived stem cells (ASCs). With the objective of developing a better in vitro model for adipose developmental biology, this project involves gene expression profiling of human ASCs (hASCs) during their differentiation to adipocytes in a 2D versus 3D culture model. This transcriptional-level analysis revealed that gene expression patterns of adipogenesis-induced hASCs in a 3D self-assembled polypeptide hydrogel are relatively different from the 2D monolayered cells on plastic hard substrate. Moreover, analysis of adipogenic lineage progression 9 days after adipogenic induction shows earlier differentiation of hASCs in 2D over their 3D counterparts. However, differentiation in 2D shows some unexpected behavior in terms of gene expression, which does not seem to be related to adipogenic lineage specification. Since hASCs are already being used in clinical trials due to their therapeutic potential, it is important to have a clear understanding of the molecular mechanisms in an in vivo model microenvironment like the one presented here.

Establishment of the First National Reference Standard for Insulin Lispro: Report of a collaborative study.

The present study was aimed to establish the First National Reference Standard (NRS) for Insulin lispro to allow stakeholders including manufacturer's laboratories, drug testing laboratories, drug regulatory authorities and academic institutions to demonstrate accuracy of the test results and to enable comparison and validation of analytical methods. The candidate standard for Insulin lispro was evaluated in a collaborative study to assign the vial content in order to serve it as NRS to support the Indian Pharmacopoeia (IP) monograph. The candidate standard was calibrated against the Ph. Eur. Insulin lispro reference standard by each of six participant laboratories in India using HPLC assay method as per the requirements of IP monograph. The results indicate that the candidate standard has an average content of 5.79 mg per vial with purity of 99.87%. Based on the study results the candidate standard was judged suitable to serve as the first NRS for Insulin lispro.

Comparison of the Substrate Preferences of ω3 Fatty Acid Desaturases for Long Chain Polyunsaturated Fatty Acids.

Omega-3 long chain polyunsaturated fatty acids (ω3 LC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5ω3) and docosahexaenoic acid (DHA; 22:6ω3) are important fatty acids for human health. These ω3 LC-PUFAs are produced from their ω3 precursors by a set of desaturases and elongases involved in the biosynthesis pathway and are also converted from ω6 LC-PUFA by omega-3 desaturases (ω3Ds). Here, we have investigated eight ω3-desaturases obtained from a cyanobacterium, plants, fungi and a lower animal species for their activities and compared their specificities for various C18, C20 and C22 ω6 PUFA substrates by transiently expressing them in Nicotiana benthamiana leaves. Our results showed hitherto unreported activity of many of the ω3Ds on ω6 LC-PUFA substrates leading to their conversion to ω3 LC-PUFAs. This discovery could be important in the engineering of EPA and DHA in heterologous hosts.