Multi-scale fractal Fourier Ptychographic microscopy to assess the dose-dependent impact of copper pollution on living diatoms.

Accumulation of bioavailable heavy metals in aquatic environment poses a serious threat to marine communities and human health due to possible trophic transfers through the food chain of toxic, non-degradable, exogenous pollutants. Copper (Cu) is one of the most spread heavy metals in water, and can severely affect primary producers at high doses. Here we show a novel imaging test to assay the dose-dependent effects of Cu on live microalgae identifying stress conditions when they are still capable of sustaining a positive growth. The method relies on Fourier Ptychographic Microscopy (FPM), capable to image large field of view in label-free phase-contrast mode attaining submicron lateral resolution. We uniquely combine FPM with a new multi-scale analysis method based on fractal geometry. The system is able to provide ensemble measurements of thousands of diatoms in the liquid sample simultaneously, while ensuring at same time single-cell imaging and analysis for each diatom. Through new image descriptors, we demonstrate that fractal analysis is suitable for handling the complexity and informative power of such multiscale FPM modality. We successfully tested this new approach by measuring how different concentrations of Cu impact on Skeletonema pseudocostatum diatom populations isolated from the Sarno River mouth.

Amphidinium spp. as a Source of Antimicrobial, Antifungal, and Anticancer Compounds.

Dinoflagellates make up the second largest marine group of marine unicellular eukaryotes in the world ocean and comprise both heterotrophic and autotrophic species, encompassing a wide genetic and chemical diversity. They produce a plethora of secondary metabolites that can be toxic to other species and are mainly used against predators and competing species. Dinoflagellates are indeed often responsible for harmful algal bloom, where their toxic secondary metabolites can accumulate along the food chain, leading to significant damages to the ecosystem and human health. Secondary metabolites from dinoflagellates have been widely investigated for potential biomedical applications and have revealed multiple antimicrobial, antifungal, and anticancer properties. Species from the genus Amphidinium seem to be particularly interesting for the production of medically relevant compounds. The present review aims at summarising current knowledge on the diversity and the pharmaceutical properties of secondary metabolites from the genus Amphidinium. Specifically, Amphidinium spp. produce a range of polyketides possessing cytotoxic activities such as amphidinolides, caribenolides, amphidinins, and amphidinols. Potent antimicrobial properties against antibiotic-resistant bacterial strains have been observed for several amphidinins. Amphidinols revealed instead strong activities against infectious fungi such as Candida albicans and Aspergillus fumigatus. Finally, compounds such as amphidinolides, isocaribenolide-I, and chlorohydrin 2 revealed potent cytotoxic activities against different cancer cell lines. Overall, the wide variety of antimicrobial, antifungal, and anticancer properties of secondary metabolites from Amphidinium spp. make this genus a highly suitable candidate for future medical applications, spanning from cancer drugs to antimicrobial products that are alternatives to currently available antibiotic and antimycotic products.

Emergency department visit patterns in the recently discharged, violently injured patient: Retrospective cohort review.

BACKGROUND: Analysis of the costs associated with emergency department (ED) visits after discharge for violent injury could highlight subgroups for the development of cost-effective interventions to support healing and prevent treatment failures in violently injured patients. METHODS: A retrospective cohort review was conducted of all patients with return ED visits within 90 days of discharge after treatment for a violent injury occurring between July 1, 2016, and June 30, 2018. Hospital costs were calculated for each incidence and analyzed against demographic and injury type variables to identify trends. RESULTS: 218 return ED visits were identified. Hospital costs showed a high frequency of low-cost visits. For more complex visits, distinct cost patterns were observed for Black and LatinX males compared to White males as a function of age. CONCLUSIONS: Analysis of hospital cost per visit identified trends among different subgroups. Underlying etiologies presumably vary between groups, but hypothesis-driven further investigation and needs assessment is required. Understanding the driving forces behind these cost trends may aid in developing effective interventions.

Identification of a Green Algal Strain Collected from the Sarno River Mouth (Gulf of Naples, Italy) and Its Exploitation for Heavy Metal Remediation.

Heavy metals (HMs) can induce both chronic and acute harmful effects on marine and freshwater biota. The environmental impact of HMs in freshwater, seawater, soil, and wastewater can be limited using microbes, including microalgae, that are able to remove metals from environmental matrices. Indeed, they can passively adsorb and actively accumulate these persistent pollutants within their organelles, limiting their detrimental effects on cellular metabolism. The Sarno River is a 30 km long freshwater stream located in Southern Italy, polluted by partially untreated municipal, agricultural, and industrial wastewaters. In spite of this, microalgal cultures from Sarno River or Sarno River Mouth have never been established. In the present study, we isolated a green algal strain from the Sarno River Mouth and determined its ability to grow in polluted seawater containing different concentrations of cadmium, lead, or zinc. This strain was found to be able to accumulate these elements within its biomass in a dose-dependent manner. Growth inhibition experiments confirm the relatively low toxicity of Cd and Pb below 50 µM, while algal growth was seriously affected in Zn-amended media. To the best of our knowledge, this is the first study focused on the ability of microalgae from Sarno River Mouth to tolerate and uptake HMs.

Toxic Effect of Metal Doping on Diatoms as Probed by Broadband Terahertz Time-Domain Spectroscopy.

The global marine environment is increasingly affected by human activities causing climate change, eutrophication, and pollution. These factors influence the metabolic mechanisms of phytoplankton species, such as diatoms. Among other pollutant agents, heavy metals can have dramatic effects on diatom viability. Detailed knowledge of the interaction of diatoms with metals is essential from both a fundamental and applicative point of view. To this aim, we assess terahertz time-domain spectroscopy as a tool for sensing the diatoms in aqueous systems which mimic their natural environment. Despite the strong absorption of terahertz radiation in water, we show that diatoms can be sensed by probing the water absorption enhancement in the terahertz range caused by the water-diatom interaction. We reveal that the addition of metal dopants affects this absorption enhancement, thus enabling the monitoring of the toxic effects of metals on diatoms using terahertz spectroscopy. We demonstrate that this technique can detect the detrimental effects of heavy metals earlier than conventional methods such as microscopy, enzymatic assays, and molecular analyses aimed at assessing the overexpression of genes involved in the heavy metal-stress response.

Copper Effect on Microalgae: Toxicity and Bioremediation Strategies.

Microalgae are increasingly recognised as suitable microorganisms for heavy metal (HM) removal, since they are able to adsorb them onto their cell wall and, in some cases, compartmentalise them inside organelles. However, at relatively high HM concentrations, they could also show signs of stress, such as organelle impairments and increased activities of antioxidant enzymes. The main aim of this review is to report on the mechanisms adopted by microalgae to counteract detrimental effects of high copper (Cu) concentrations, and on the microalgal potential for Cu bioremediation of aquatic environments. Studying the delicate balance between beneficial and detrimental effects of Cu on microalgae is of particular relevance as this metal is widely present in aquatic environments facing industrial discharges. This metal often induces chloroplast functioning impairment, generation of reactive oxygen species (ROS) and growth rate reduction in a dose-dependent manner. However, microalgae also possess proteins and small molecules with protective role against Cu and, in general, metal stress, which increase their resistance towards these pollutants. Our critical literature analysis reveals that microalgae can be suitable indicators of Cu pollution in aquatic environments, and could also be considered as components of eco-sustainable devices for HM bioremediation in association with other organisms.

Subthalamic nucleus deep brain stimulation programming settings do not correlate with Parkinson's disease severity.

BACKGROUND: Deep brain stimulation (DBS) is a well-established treatment for Parkinson's disease (PD). While the success of DBS is dependent on careful patient selection and accurate lead placement, programming parameters play a pivotal role in tailoring therapy on the individual level. Various algorithms have been developed to streamline the initial programming process, but the relationship between pre-operative patient characteristics and post-operative device settings is unclear. In this study, we investigated how PD severity correlates with DBS settings. METHODS: We conducted a retrospective review of PD patients who underwent DBS of the subthalamic nucleus at one US tertiary care center between 2014 and 2018. Pre-operative patient characteristics and post-operative programming data at various intervals were collected. Disease severity was measured using the Unified Parkinson's Disease Rating Scale score (UPDRS) as well as levodopa equivalent dose (LED). Correlation analyses were conducted looking for associations between pre-operative disease severity and post-operative programming parameters. RESULTS: Fifty-six patients were analyzed. There was no correlation between disease severity and any of the corresponding programming parameters. Pre-operative UPDRS scores on medication were similar to post-operative scores with DBS. Settings of amplitude, frequency, and pulse width increased significantly from 1 to 6 months post-operatively. Stimulation volume, inferred by the distance between contacts used, also increased significantly over time. CONCLUSIONS: Interestingly, we found that patients with more advanced disease responded to electrical stimulation similarly to patients with less advanced disease. These data provide foundational knowledge of DBS programming parameters used in a single cohort of PD patients over time.

Bioinformatic prediction of putative metallothioneins in non-ciliate protists.

Intracellular ligands that bind heavy metals (HMs) and thereby minimize their detrimental effects to cellular metabolism are attracting great interest for a number of applications including bioremediation and development of HM-biosensors. Metallothioneins (MTs) are short, cysteine-rich, genetically encoded proteins involved in intracellular metal-binding and play a key role in detoxification of HMs. We searched approximately 700 genomes and transcriptomes of non-ciliate protists for novel putative MTs by similarity and structural analyses and found 21 unique proteins playing a potential role as MTs. Most putative MTs derive from heterokonts and dinoflagellates and share common features such as (i) a putative metal-binding domain in proximity of the N-terminus, (ii) two putative MT-specific domains near the C-terminus and (iii) one to three CTCGXXCXCGXXCXCXXC patterns. Although the biological function of these proteins has not been experimentally proven, knowledge of their genetic sequences adds useful information on proteins that are potentially involved in HM-binding and can contribute to the design of future biomolecular assays on HM-microbe interactions and MT-based biosensors.

Kinematic analysis and visualization of Tetraselmis microalgae 3D motility by digital holography.

A study on locomotion in a 3D environment of Tetraselmis microalgae by digital holographic microscopy is reported. In particular, a fast and semiautomatic criterion is revealed for tracking and analyzing the swimming path of a microalga (i.e., Tetraselmis species) in a 3D volume. Digital holography (DH) in a microscope off-axis configuration is exploited as a useful method to enable fast autofocusing and recognition of objects in the field of view, thus coupling DH with appropriate numerical algorithms. Through the proposed method we measure, simultaneously, the tri-dimensional paths followed by the flagellate microorganism and the full set of the kinematic parameters that describe the swimming behavior of the analyzed microorganisms by means of a polynomial fitting and segmentation. Furthermore, the method is capable to furnish the accurate morphology of the microorganisms at any instant of time along its 3D trajectory. This work launches a promising trend having as the main objective the combined use of DH and motility microorganism analysis as a label-free and non-invasive environmental monitoring tool, employable also for in situ measurements. Finally, we show that the locomotion can be visualized intriguingly by different modalities to furnish marine biologists with a clear 3D representation of all the parameters of the kinematic set in order to better understand the behavior of the microorganism under investigation.

Highly Valuable Polyunsaturated Fatty Acids from Microalgae: Strategies to Improve Their Yields and Their Potential Exploitation in Aquaculture.

Microalgae have a great potential for the production of healthy food and feed supplements. Their ability to convert carbon into high-value compounds and to be cultured in large scale without interfering with crop cultivation makes these photosynthetic microorganisms promising for the sustainable production of lipids. In particular, microalgae represent an alternative source of polyunsaturated fatty acids (PUFAs), whose consumption is related to various health benefits for humans and animals. In recent years, several strategies to improve PUFAs' production in microalgae have been investigated. Such strategies include selecting the best performing species and strains and the optimization of culturing conditions, with special emphasis on the different cultivation systems and the effect of different abiotic factors on PUFAs' accumulation in microalgae. Moreover, developments and results obtained through the most modern genetic and metabolic engineering techniques are described, focusing on the strategies that lead to an increased lipid production or an altered PUFAs' profile. Additionally, we provide an overview of biotechnological applications of PUFAs derived from microalgae as safe and sustainable organisms, such as aquafeed and food ingredients, and of the main techniques (and their related issues) for PUFAs' extraction and purification from microalgal biomass.

Probing the Therapeutic Potential of Marine Phyla by SPE Extraction.

The marine environment is potentially a prolific source of small molecules with significant biological activities. In recent years, the development of new chromatographic phases and the progress in cell and molecular techniques have facilitated the search for marine natural products (MNPs) as novel pharmacophores and enhanced the success rate in the selection of new potential drug candidates. However, most of this exploration has so far been driven by anticancer research and has been limited to a reduced number of taxonomic groups. In this article, we report a test study on the screening potential of an in-house library of natural small molecules composed of 285 samples derived from 57 marine organisms that were chosen from among the major eukaryotic phyla so far represented in studies on bioactive MNPs. Both the extracts and SPE fractions of these organisms were simultaneously submitted to three different bioassays-two phenotypic and one enzymatic-for cytotoxic, antidiabetic, and antibacterial activity. On the whole, the screening of the MNP library selected 11 potential hits, but the distribution of the biological results showed that SPE fractionation increased the positive score regardless of the taxonomic group. In many cases, activity could be detected only in the enriched fractions after the elimination of the bulky effect due to salts. On a statistical basis, sponges and molluscs were confirmed to be the most significant source of cytotoxic and antimicrobial products, but other phyla were found to be effective with the other therapeutic targets.

Hyperthermiphile biofilms of Thermotoga neapolitana on different materials and electrostimulated: SEM micrographs and chemical data of the glucose fermentation in electrochemical reactors.

Hyperthermophile bacteria were seldom investigated in bioelectrochemical systems although they allow more effective control of the inoculum in comparison with mesophilic bacteria. Biofilm formed in hyperthermophilic conditions (>60 °C) also rarely was documented (d'Ippolito et al., 2020; Belkin et al., 1986, Pysz et al., 2004). Scanning Electron Microscopy (SEM) micrographs documenting biofilms formed by the Hyperthermophile bacterium Thermotoga neapolitana on different solid materials (ceramic carrier, stainless steel mesh, carbon felt, carbon paper, expanse graphite, and carbon cloth) are shown in this report. Also, micrographs of the biofilm formed on electrodes of carbon cloth under a dynamic polarization oscillating around ±1 V (±0.8 V and ±1.2 V) are reported. Two procedures of sample preparation for SEM analyses are described and used: 1) a fast drying of samples, which is enough to underline the biofilm shape that covers solids, and 2) a chemical treating of the samples with glutaraldehyde, which better preserves the shape of bacterial cell components in the biofilm, although this treatment might cause the detachment of pieces of the biofilm. The different effect of potentiostatic and potentiodynamic polarizations on the glucose metabolism of T. neapolitana has been screened and discussed in the associated article [1]. Here, data of Optical Densities (O.D.) of culture media are provided, indicating the presence or absence of bacteria growth in the bulk of the media. Data have been collected every 24 h from the differently polarized bioreactors. The electrodes set-up of small bioreactors is also illustrated. Chemical data, optical data and SEM images, accordingly, document a retard in the glucose fermentation process due to a settlement of T. Neapolitana in a stationary phase. The polarization of electrodes can modify the stationary condition, inducing a possible change of the bacteria metabolism.

Microalgal Metallothioneins and Phytochelatins and Their Potential Use in Bioremediation.

The persistence of heavy metals (HMs) in the environment causes adverse effects to all living organisms; HMs accumulate along the food chain affecting different levels of biological organizations, from cells to tissues. HMs enter cells through transporter proteins and can bind to enzymes and nucleic acids interfering with their functioning. Strategies used by microalgae to minimize HM toxicity include the biosynthesis of metal-binding peptides that chelate metal cations inhibiting their activity. Metal-binding peptides include genetically encoded metallothioneins (MTs) and enzymatically produced phytochelatins (PCs). A number of techniques, including genetic engineering, focus on increasing the biosynthesis of MTs and PCs in microalgae. The present review reports the current knowledge on microalgal MTs and PCs and describes the state of art of their use for HM bioremediation and other putative biotechnological applications, also emphasizing on techniques aimed at increasing the cellular concentrations of MTs and PCs. In spite of the broad metabolic and chemical diversity of microalgae that are currently receiving increasing attention by biotechnological research, knowledge on MTs and PCs from these organisms is still limited to date.

Diatoms synthesize sterols by inclusion of animal and fungal genes in the plant pathway.

Diatoms are ubiquitous microalgae that have developed remarkable metabolic plasticity and gene diversification. Here we report the first elucidation of the complete biosynthesis of sterols in the lineage. The study has been carried out on the bloom-forming species Skeletonema marinoi and Cyclotella cryptica that synthesise an ensemble of sterols with chemotypes of animals (cholesterol and desmosterol), plants (dihydrobrassicasterol and 24-methylene cholesterol), algae (fucosterol) and marine invertebrates (clionasterol). In both species, sterols derive from mevalonate through cyclization of squalene to cycloartenol by cycloartenol synthase. The pathway anticipates synthesis of cholesterol by enzymes of the phytosterol route in plants, as recently reported in Solanaceae. Major divergences stem from reduction of Δ24(28) and Δ24(25) double bonds which, in diatoms, are apparently dependent on sterol reductases of fungi, algae and animals. Phylogenetic comparison revealed a good level of similarity between the sterol biosynthetic genes of S. marinoi and C. cryptica with those in the genomes of the other diatoms sequenced so far.

Lipoxygenase Pathways in Diatoms: Occurrence and Correlation with Grazer Toxicity in Four Benthic Species.

Oxygenated derivatives of fatty acids, collectively called oxylipins, are a highly diverse family of lipoxygenase (LOX) products well described in planktonic diatoms. Here we report the first investigation of these molecules in four benthic diatoms, Cylindrotheca closterium, Nanofrustulum shiloi, Cocconeis scutellum, and Diploneis sp. isolated from the leaves of the seagrass Posidonia oceanica from the Gulf of Naples. Analysis by hyphenated MS techniques revealed that C. closterium, N. shiloi, and C. scutellum produce several polyunsaturated aldehydes (PUAs) and linear oxygenated fatty acids (LOFAs) related to the products of LOX pathways in planktonic species. Diploneis sp. also produced other unidentified fatty acid derivatives that are not related to LOX metabolism. The levels and composition of oxylipins in the benthic species match their negative effects on the reproductive success in the sea urchin Paracentrotus lividus. In agreement with this correlation, the most toxic species N. shiloi revealed the same LOX pathways of Skeletonema marinoi and Thalassiosira rotula, two bloom-forming planktonic diatoms that affect copepod reproduction. Overall, our data highlight for the first time a major role of oxylipins, namely LOFAs, as info-chemicals for benthic diatoms, and open new perspectives in the study of the structuring of benthic communities.

The Rise of Early Psychosis Care in California: An Overview of Community and University-Based Services.

OBJECTIVE: California's Mental Health Services Act Prevention and Early Intervention funds provide a unique opportunity for counties to initiate programs focused on early intervention in mental health, including early psychosis. To explain the configuration of early psychosis programs and plan for a statewide evaluation, this report provides an overview of California's early psychosis programming, including service composition, funding sources, inclusion criteria, and data collection practices. METHODS: Following a comprehensive identification process, early psychosis program representatives were contacted to complete the California Early Psychosis Assessment Survey (CEPAS). RESULTS: The response rate to the CEPAS was excellent (97%, 29 of 30 active programs across 24 of 58 counties). Most programs (N=27, 93%) serve individuals with first-episode psychosis between the ages of 12 and 25. Twenty-two programs (79%) provide more than half of the standard components of early psychosis care outlined in the First-Episode Psychosis Service Fidelity Scale. Sixty-four percent of programs collect client-level data at intake and follow up on five or more relevant outcome domains; however, these varied significantly across sites. CONCLUSIONS: Substantial variability in services, inclusion criteria, and data recorded was evident across programs. Prior to conducting any large-scale evaluation, these findings highlight the significant challenges in retrospectively evaluating program effectiveness, need to harmonize program data collection methods, and importance of assessing the impact of program variability on outcomes.

Immunostimulatory Phosphatidylmonogalactosyldiacylglycerols (PGDG) from the Marine Diatom Thalassiosira weissflogii: Inspiration for a Novel Synthetic Toll-Like Receptor 4 Agonist.

An unprecedented phosphatidylmonogalactosyldiacylglycerol pool (PGDG, 1) rich in polyunsaturated fatty acids was isolated from the marine diatoms Thalassiosira weissflogii. Here we report for the first time the NMR characterization of this rare lipid from marine organisms along with a synthetic strategy for the preparation of a PGDG analog (2). PGDG 1 exhibited immunostimulatory activity in human dendritic cells (DCs) and the synthetic PGDG 2 was prepared to explore its mechanism of action. A Toll-like receptor-4 (TLR-4) agonistic activity was evidenced in human and murine DCs underlying the antigen-specific T-cell activation of this class of molecules.

Which components of specialized early intervention for psychosis do senior providers see as most important?

AIM: Specialized early interventions improve outcomes in early psychosis (EP). Experts have proposed a number of essential treatment components. However, it is unclear whether these reflect the views of senior clinic staff charged with implementing this model in practice. METHOD: Twenty-Five senior EP clinic staff across California completed a survey indicating which features of EP treatment they considered most important. RESULTS: Components related to the service structure and the need for a prompt, comprehensive assessment and care planning were considered most important, despite the limited evidence base evaluating these aspects of care. Administration of clozapine to treatment-refractory patients and weight gain interventions were considered the least important, despite the relatively strong evidence base supporting these treatment components. CONCLUSION: The findings suggest a bi-directional dissemination gap, where components considered most important by senior providers receive limited research attention, while some areas with supporting evidence may be underappreciated in clinical practice.

The Marine Dinoflagellate Alexandrium minutum Activates a Mitophagic Pathway in Human Lung Cancer Cells.

Marine dinoflagellates are a valuable source of bioactive molecules. Many species produce cytotoxic compounds and some of these compounds have also been investigated for their anticancer potential. Here, we report the first investigation of the toxic dinoflagellate Alexandrium minutum as source of water-soluble compounds with antiproliferative activity against human lung cancer cells. A multi-step enrichment of the phenol⁻water extract yielded a bioactive fraction with specific antiproliferative effect (IC50 = 0.4 µg·mL-1) against the human lung adenocarcinoma cells (A549 cell line). Preliminary characterization of this material suggested the presence of glycoprotein with molecular weight above 20 kDa. Interestingly, this fraction did not exhibit any cytotoxicity against human normal lung fibroblasts (WI38). Differential gene expression analysis in A549 cancer cells suggested that the active fraction induces specific cell death, triggered by mitochondrial autophagy (mitophagy). In agreement with the cell viability results, gene expression data also showed that no mitophagic event was activated in normal cells WI38.

UPLC⁻MS/MS Identification of Sterol Sulfates in Marine Diatoms.

Diatoms are unicellular eukaryotic organisms that play a key ecological and biogeochemical role in oceans as major primary producers. Recently, these microalgae have also attracted interest as a promising source of functional products with widespread relevance. Progress in the knowledge of cell and molecular biology of diatoms is envisaged as a key step to understanding regulation of their life cycle in marine environments as well as facilitating their full and profitable exploitation by biotechnological platforms. Recently, we identified sterol sulfates (StS) as regulatory molecules of cell death in the diatom Skeletonema marinoi. As these compounds may have a general role in diatom physiology and chemical signals in aquatic systems, we investigated a suitable tool for their analysis in laboratory and field samples. Herein, we describe a sensitive, fast, and efficient ultra performance liquid chromatography⁻mass spectrometry (UPLC⁻MS) method for qualitative and quantitative analysis of StS from crude extract of diatoms and other microalgae. The method was applied to 13 different strains of our collection of marine protists. This first study suggested a species-specific distribution of StS and identified the sulfated derivatives of 24-methylene cholesterol and 24-methyl cholesterol as the most common members in diatoms.