Bringing proteomics to bear on male fertility: key lessons.

INTRODUCTION: Male infertility is a major public health concern globally. Proteomics has revolutionized our comprehension of male fertility by identifying potential infertility biomarkers and reproductive defects. Studies comparing sperm proteome with other male reproductive tissues have the potential to refine fertility diagnostics and guide infertility treatment development. AREAS COVERED: This review encapsulates literature using proteomic approaches to progress male reproductive biology. Our search methodology included systematic searches of databases such as PubMed, Scopus, and Web of Science for articles up to 2023. Keywords used included 'male fertility proteomics,' 'spermatozoa proteome,' 'testis proteomics,' 'epididymal proteomics,' and 'non-hormonal male contraception.' Inclusion criteria were robust experimental design, significant contributions to male fertility, and novel use of proteomic technologies. EXPERT OPINION: Expert analysis shows a shift from traditional research to an integrative approach that clarifies male reproductive health's molecular intricacies. A gap exists between proteomic discoveries and clinical application. The expert opinions consolidated here not only navigate the current findings but also chart the future proteomic applications for scientific and clinical breakthroughs. We underscore the need for continued investment in proteomic research - both in the technological and collaborative arenas - to further unravel the secrets of male fertility, which will be central to resolving fertility issues in the coming era.

Rapid screening of phenolic compounds in extracts of photosynthetic organisms separated using a C18 monolithic column based HPLC-UV method.

The sourcing of novel bioactive compounds from bioresources has become a sustainability priority for several industrial sectors. Reliable methods are hence needed for the screening and identification of desired molecules in complex extracts. As such, phenolic compounds have attracted increasing interests due to their antioxidant potential. Here, a HPLC-UV based method was developed for the determination of phenolic compounds using a monolithic column. This allowed the separation of 23 phenolic compounds within 11 min. The method was ground proofed in terms of chromatographic parameters and applied to identify polyphenols in extracts of blueberries, basil and marine microalgae. The dominant polyphenols in blueberries and basil were chlorogenic acid (26.0 mg. kg -1) and kaempferol (61.4 mg. kg -1), respectively, while the microalgae used only contained trace amounts of polyphenols. This study demonstrates the value of our new HPLC method to rapidly screen extracts and identify polyphenols from biological matrices.

Influence of exogenous phytohormone supplementation on the pigment and fatty acid content of three marine diatoms.

Diatoms are ubiquitous photosynthetic microorganisms with great potential for biotechnological applications. However, their commercialisation is hampered by production costs, requiring hence optimisation of cultivation methods. Phytohormones are plant growth regulators which may be used to influence physiological processes in microalgae, including diatoms. In this study, the model species Phaeodactylum tricornutum (Phaeodactylaceae) and two Irish isolates of Stauroneis sp. (Stauroneidaceae) and Nitzschia sp. (Bacillariaceae) were grown with varying amounts of the phytohormones indoleacetic acid (IAA), gibberellic acid (GA3), methyl jasmonate (MJ), abscisic acid (ABA) or salicylic acid (SA), and their influence on pigment and fatty acid profiles was monitored. The application of GA3 (200 mg/l) stimulated the growth of P. tricornutum which accumulated 52% more dry biomass compared to the control and concomitantly returned the highest eicosapentaenoic acid (EPA) yield (0.6 mg/l). The highest fucoxanthin yield (0.18 mg/l) was obtained for P. tricornutum cultivated with GA3 (2 mg/l) supplementation. In Stauroneis sp., SA (1 mg/l) had the most positive effect on EPA, the content of which was enhanced up to 45.7 µg/mg (4.6% of total dry weight). The SA (1 mg/l) treatment also boosted carotenogenesis in Nitzschia sp., leading to 1.7- and 14-fold increases in fucoxanthin and ß-carotene compared to the control, respectively. Of note, MJ (0.5 mg/l) increased the EPA content of all diatom species compared to their controls. These results indicate that phytohormone-based treatments can be used to alter the pigment and lipid content of microalgae, which tend to respond in dose- and species-specific manners to individual compounds.Key points• Response to phytohormones was investigated in diatoms from distinct families.• MJ (0.5 mg/l) caused an increase in EPA cellular content in all three diatoms.• Phytohormones mostly caused dose-dependent and species-specific responses.

Antioxidant Activity and Carotenoid Content Responses of Three Haematococcus sp. (Chlorophyta) Strains Exposed to Multiple Stressors.

There has been increasing demands worldwide for bioactive compounds of natural origins, especially for the nutraceutical and food-supplement sectors. In this context, microalgae are viewed as sustainable sources of molecules with an array of health benefits. For instance, astaxanthin is a xanthophyll pigment with powerful antioxidant capacity produced by microalgae such as the chlorophyte Haematococcus sp., which is regarded as the most suitable organism for the mass production of this pigment. In this study, three Haematococcus sp. strains were cultivated using a batch mode under favourable conditions to promote vegetative growth. Their environment was altered in a second phase using a higher and constant illumination regime combined with either exposure to blue LED light, an osmotic shock (with NaCl addition) or supplementation with a phytohormone (gibberellic acid, GA3), a plant extract (ginger), an herbicide (molinate) or an oxidant reagent (hydrogen peroxide). The effects of these stressors were evaluated in terms of antioxidant response and astaxanthin and ß-carotene accumulation. Overall, strain CCAP 34/7 returned the highest Trolox Equivalent Antioxidant Capacity (TEAC) response (14.1-49.1 µmoL Trolox eq. g- 1 of DW), while the highest antioxidant response with the Folin-Ciocalteu (FC) was obtained for strain RPFW01 (62.5-155 µmoL Trolox eq. g- 1 of DW). The highest ß-ß-carotene content was found in strain LAFW15 when supplemented with the ginger extract (4.8 mg. g- 1). Strain RPFW01 exposed to blue light returned the highest astaxanthin yield (2.8 mg. g- 1), 5-fold that of strain CCAP 34/7 on average. This study documents the importance of screening several strains when prospecting for species with potential to produce high-value metabolites. It highlights that strain-specific responses can ensue from exposure of cells to a variety of stressors, which is important for the adequate tailoring of a biorefinery pipeline.

Antibacterial Activity and Amphidinol Profiling of the Marine Dinoflagellate Amphidinium carterae (Subclade III).

Microalgae have received growing interest for their capacity to produce bioactive metabolites. This study aimed at characterising the antimicrobial potential of the marine dinoflagellate Amphidinium carterae strain LACW11, isolated from the west of Ireland. Amphidinolides have been identified as cytotoxic polyoxygenated polyketides produced by several Amphidinium species. Phylogenetic inference assigned our strain to Amphidinium carterae subclade III, along with isolates interspersed in different geographic regions. A two-stage extraction and fractionation process of the biomass was carried out. Extracts obtained after stage-1 were tested for bioactivity against bacterial ATCC strains of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa. The stage-2 solid phase extraction provided 16 fractions, which were tested against S. aureus and E. faecalis. Fractions I, J and K yielded minimum inhibitory concentrations between 16 µg/mL and 256 µg/mL for both Gram-positive. A targeted metabolomic approach using UHPLC-HRMS/MS analysis applied on fractions G to J evidenced the presence of amphidinol type compounds AM-A, AM-B, AM-22 and a new derivative dehydroAM-A, with characteristic masses of m/z 1361, 1463, 1667 and 1343, respectively. Combining the results of the biological assays with the targeted metabolomic approach, we could conclude that AM-A and the new derivative dehydroAM-A are responsible for the detected antimicrobial bioactivity.

Comparative Response of Marine Microalgae to H2O2-Induced Oxidative Stress.

There have been growing interests in the biorefining of bioactive compounds from marine microalgae, including pigments, omega-3 fatty acids or antioxidants for use in the nutraceutical and cosmetic sectors. This study focused on the comparative responses of five marine microalgal species from different lineages, including the dinoflagellate Amphidinium carterae, chlorophyte Brachiomonas submarina, diatom Stauroneis sp., haptophyte Diacronema sp. and rhodophyte Rhodella violacea, to exposure during their batch growth to hydrogen peroxide (H2O2). A. carterae returned an enhanced signal with the DPPH assay (8.8 µmol Trolox eq/g DW) when exposed to H2O2, which was associated with reduced pigment yields and increased proportions in saturated C16 and C18 fatty acids. B. submarina showed enhanced antioxidant response upon exposure to H2O2 with the DPPH assay (10 µmol Trolox eq/g DW), a threefold decrease in lutein (from 2.3 to 0.8 mg/g) but a twofold increase in chlorophyll b (up to 30.0 mg/g). Stauroneis sp. showed a downward response for the antioxidant assays, but its pigment yields did not vary significantly from the control. Diacronema sp. showed reduced antioxidant response and fucoxanthin content (from 4.0 to 0.2 mg/g) when exposed to 0.5 mM H2O2. R. violacea exposed to H2O2 returned enhanced antioxidant activity and proportions of EPA but was not significantly impacted in terms of pigment content. Results indicate that H2O2 can be used to induce stress and initiate metabolic changes in microalgae. The responses were however species-specific, which would require further dosage optimisation to modulate the yields of specific metabolites in individual species.

The role of methyl jasmonate in enhancing biomass yields and bioactive metabolites in Stauroneis sp. (Bacillariophyceae) revealed by proteome and biochemical profiling.

The diatom Stauroneis sp. was previously identified as a promising source of fucoxanthin and omega-3 oils. Methyl jasmonate (MJ) supplementation is known to enhance metabolite yields in this species without impacting on growth or photosynthesis. Therefore, a label-free proteomics approach was undertaken to further evaluate the functional role of MJ on the diatom's physiology. Of the twenty cultivation regimes were screened, Uf/2 medium with green+white LED's induced the greatest metabolic response when exposed to 10 µM MJ treatment. These conditions significantly enhanced the pigment and total cellular lipids contents. The increase in fucoxanthin correlating with a 20% increase in Trolox reducing equivalent in the total antioxidant assay, indicating a non-enzymatic antioxidant role of fucoxanthin to mitigate the detrimental effects of a redox imbalance within chloroplasts. The proteomics identified 197 proteins up-regulated 48 h after MJ exposure including cell signalling cascades, photosynthetic processes, carbohydrate metabolism, lipid biosynthesis and chloroplast biogenesis. MJ strengthened the dark reactions of photosynthesis to support growth and metabolite fluxes. The MJ-induced ER stress protein triggered lipid body production, facilitating metabolite turnover and trafficking between cellular organelles. Plastid terminal oxidase and glutamate 1-semialdehyde 2,1-aminomutase may act as MJ-induced ROS responsive regulatory switch to support chloroplast biosynthesis. SIGNIFICANCE STATEMENT: Phytohormones represents a promising tool to enhance the high-value metabolite yields in plants and algae, however little is known of the role of methyl jasmonate in diatoms at a molecular level. A shotgun proteomics approach was undertaken to determine the influence of MJ on the diatom's cellular physiology in the marine diatom Stauroneis sp., revealing a signal transduction cascade leading to increased lipid and pigment content and identified promising targets for genetic engineering.

Differential responses in EPA and fucoxanthin production by the marine diatom Stauroneis sp. under varying cultivation conditions.

There has been an increasing drive toward better valorising raw biological materials in the context of the sustainability of bio-based industries and the circular economy. As such, microalgae hold the ability to biosynthesise valuable metabolites, which are sought after within the bioenergy, pharmaceuticals, cosmetics or nutrition sectors. Owing to their bioactivities, the xanthophyll pigment fucoxanthin and the omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) have fostered increasing interests in terms of sustainably refining them from natural sources, such as microalgae. Together with the suitability of individual species to industrial cultivation, a key challenge resides in optimizing the yields of these compounds within the microalgal biomass they are retrieved from. The marine diatom Stauroneis sp. LACW24 was batch cultivated into its stationary phase of growth prior to being subjected at high cell density (1 × 106 cells mL-1 ) to seven different regimes of light exposure in replenished medium and under nutritional limitation (silica and nitrate) for 12 days. The highest EPA proportions and yields were obtained under blue LED in f/2 medium (16.5% and 4.8 mg g-1 , respectively), double the values obtained under red LED illumination. The fucoxanthin yield was the highest when cells were subjected to blue LEDs (5.9 mg g-1 ), a fourfold increase compared to the nitrogen-limited treatment under white LEDs. These results indicate that a two-stage approach to the batch cultivation of this diatom can be used for enhancing the production of the high-value metabolites fucoxanthin and EPA post-stationary phase.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 µg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and ß-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

Antioxidant Bioprospecting in Microalgae: Characterisation of the Potential of Two Marine Heterokonts from Irish Waters.

Microalgae constitute a heterogeneous and diverse range of organisms capable of accumulating bioactive metabolites, making them promising feedstock for applications in the nutraceutical, functional food, animal feed, biofertilisation or biofuel sectors. There has been renewed interest in recent times in natural sources of antioxidants, particularly as health products and preserving agents. Microalgae strains isolated from aquatic habitats in Ireland were successfully brought into culture. The 91 strains were grown phototrophically in nutrient-enriched media to generate biomass, which was harvested and assessed for antioxidant potential. Extracts were screened for antioxidant activity using a modified volumetric Trolox-ABTS assay and the Folin-Ciocalteu method. Two heterokont marine strains of interest were further studied to ascertain variations in antioxidant capacity across different stages of batch culture growth. The antioxidant activity of extracts of bacillariophyte cf. Stauroneis sp. LACW24 and ocrophyte cf. Phaeothamnion sp. LACW34 increased during growth with a maximum being observed during the late stationary or early death phase (2.5- to 8-fold increases between days 20 and 27). Strains LACW24 and LACW34 contained 5.9 and 3.0 mg g-1 (DW) of the xanthophyll fucoxanthin, respectively. Extracts of strains also showed no cytotoxicity towards mouse cell lines. These results highlight the potential of these strains for biomass valorisation and cultivation upscaling and to be further considered as part of ongoing bioprospecting efforts towards identifying novel species to join the relatively narrow range of commercially exploited marine microalgae species.

Community Dentistry: A Service Evaluation Study for 'Looked After Children' in England and Wales.

PURPOSE: Historically, the healthcare needs of 'Looked After Children' (LAC) within the UK have been relatively neglected and there is no universally adopted designated dental care pathway (DDCP) in place to ensure their care. This paper aims to discover the contribution of the community dental service (CDS) to the dental health of LAC throughout England and Wales. MATERIALS AND METHODS: An electronic questionnaire was sent to CDS Clinical Directors in England and local health boards in Wales between November 2011 and January 2012. Ten questions were included with 5-point Likert scale responses and the option for free text comments. RESULTS: In Wales, 41 questionnaires were returned (35% response rate) of which 37 services provided care for LAC. In England, 67 questionnaires were returned (44% response rate), of which 59 services provided care for LAC. CONCLUSION: The study found that the majority of CDSs in the UK provide care for LAC, However, we cannot ascertain if the CDS is the most appropriate service to provide dental care for this group of patients. The use of DDCPs, non-attendance and other care policies varied markedly amongst respondents, which may suggest that provision of dental care for LAC within the CDS in England and Wales is inconsistent. The authors believe that the utilisation of an LAC-specific and universally accepted DDCP that has been developed could help to reduce these inconsistencies. Further surveys are planned in order to monitor the care of this vulnerable group of patients and assess the impact of these recommendations.

Early Tooth Loss in Children: A Warning Sign of Childhood Hypophosphatasia.

Premature exfoliation of primary teeth may be the first manifestation of this serious condition and the general dental practitioner plays an important role in recognizing dental anomalies and referring patients at an appropriate time. This is imperative to ensuring early diagnosis and good quality patient care. This article describes the case of a 4-year-old boy affected by childhood hypophosphatasia, who presented with premature exfoliation of his primary teeth as the first manifestation of this condition. An overview of the condition is outlined including a discussion of the likelihood of permanent dentition involvement. Clinical relevance: Oral manifestations of hypophosphatasia may be the first and the only signs of this condition and may be the basis upon which a diagnosis is made.