Influence of the geographic location and house characteristics on the concentration of microplastics in indoor dust.

Microplastics (MPs) are known for their ubiquity, having been detected in virtually any environmental compartment. However, indoor MPs concentrations are poorly studied despite being closely related to human exposure. The present study aims to evaluate the presence of MPs in settled atmospheric dust in 60 houses distributed in 12 districts of the metropolitan city of Lima, Peru, and investigate the influence of their geographical location and house characteristics. MPs concentration ranged from 0.01 to 33.9 MPs per mg of dust. Fibers and blue were the most frequent shape and color (98 % and 69 %, respectively). Also, 82 % of the particles were between 500 µm - 5 mm in size. A higher concentration of MPs was identified in the center-south of the city. The houses located on the highest floors (levels 4 to 13 to ground) displayed higher concentrations. MPs were primarily composed of polyester (PET), polypropylene (PP), and ethylene-vinyl acetate (EVA), among others. The polymers identified suggest that MPs derived from the fragmentation of components frequently found in houses, such as synthetic clothing, food storage containers, toys, carpets, floors, and curtains. The incorporation of MPs from the outside into dwellings is not ruled out. Future studies should evaluate the influence of consumption habits and housing characteristics on the abundance of MPs.

Plastic and paint debris in marine protected areas of Peru.

Contamination with anthropogenic debris, such as plastic and paint particles, has been widely investigated in the global marine environment. However, there is a lack of information regarding their presence in marine protected areas (MPAs). In the present study, the abundance, distribution, and chemical characteristics of microplastics (MPs; <5 mm), mesoplastics (MePs; 5-25 mm), and paint particles were investigated in multiple environmental compartments of two MPAs from Peru. The characteristics of MPs across surface water, bottom sediments, and fish guts were similar, primarily dominated by blue fibers. On the other hand, MePs and large MPs (1-5 mm) were similar across sandy beaches. Several particles were composite materials consisting of multiple layers confirmed as alkyd resins by Fourier-transformed infrared spectroscopy, which were typical indicators of marine coatings. The microstructure of paint particles showed differentiated topography across layers, as well as different elemental compositions. Some layers displayed amorphous structures with Ba-, Cr-, and Ti-based additives. However, the leaching and impact of potentially toxic additives in paint particles require further investigation. The accumulation of multiple types of plastic and paint debris in MPAs could pose a threat to conservation goals. The current study contributed to the knowledge regarding anthropogenic debris contamination in MPAs and further elucidated the physical and chemical properties of paint particles in marine environments. While paint particles may look similar to MPs and MePs, more attention should be given to these contaminants in places where intense maritime activity takes place.

Rajids ovipositing on marine litter: A potential threat to their survival.

Marine litter is a complex environmental issue threatening the well-being of multiple organisms. In the present study, we present an overlooked pathway by which marine litter interaction with certain ovigerous skates (Family: Rajidae) communities could compromise their survival. We propose that skates from the genus Sympterygia deposit their egg capsules on marine litter substrates by accident, which are then washed ashore still unhatched. We conducted 10 monitoring surveys on three beaches of La Libertad Region, on the north coast of Peru, looking for marine litter conglomerates to determine the presence of egg capsules. We registered a total of 75 marine litter conglomerates, containing 1595 egg capsules, out of which only 15.9 % were presumably hatched, and 15.8 % were still fresh. Fishing materials were identified as the main item in marine litter conglomerates. We conclude that this behavior could contribute to the decline of Sympterygia communities, although further research is needed.

Degradation of plastics associated with the COVID-19 pandemic.

The ongoing COVID-19 pandemic has resulted in an unprecedented form of plastic pollution: personal protective equipment (PPE). Numerous studies have reported the occurrence of PPE in the marine environment. However, their degradation in the environment and consequences are poorly understood. Studies have reported that face masks, the most abundant type of PPE, are significant sources of microplastics due to their fibrous microstructure. The fibrous material (mostly consisting of polypropylene) exhibits physical changes in the environment, leading to its fracture and detachment of microfibers. Most studies have evaluated PPE degradation under controlled laboratory conditions. However, in situ degradation experiments, including the colonization of PPE, are largely lacking. Although ecotoxicological studies are largely lacking, the first attempts to understand the impact of MPs released from face masks showed various types of impacts, such as fertility and reproduction deficiencies in both aquatic and terrestrial organisms.

Binational survey of personal protective equipment (PPE) pollution driven by the COVID-19 pandemic in coastal environments: Abundance, distribution, and analytical characterization.

In the present contribution, two nationwide surveys of personal protective equipment (PPE) pollution were conducted in Peru and Argentina aiming to provide valuable information regarding the abundance and distribution of PPE in coastal sites. Additionally, PPE items were recovered from the environment and analyzed by Fourier transformed infrared (FTIR) spectroscopy, Scanning electron microscopy (SEM) with Energy dispersive X-ray (EDX), and X-ray diffraction (XRD), and compared to brand-new PPE in order to investigate the chemical and structural degradation of PPE in the environment. PPE density (PPE m-2) found in both countries were comparable to previous studies. FTIR analysis revealed multiple polymer types comprising common PPE, mainly polypropylene, polyamide, polyethylene terephthalate, and polyester. SEM micrographs showed clear weathering signs, such as cracks, cavities, and rough surfaces in face masks and gloves. EDX elemental mapping revealed the presence of elemental additives, such as Ca in gloves and face masks and AgNPs as an antimicrobial agent. Other metals found on the surface of PPE were Mo, P, Ti, and Zn. XRD patterns displayed a notorious decrease in the crystallinity of polypropylene face masks, which could alter its interaction with external contaminants and stability. The next steps in this line of research were discussed.

A baseline study of microplastics in the burrowing crab (Neohelice granulata) from a temperate southwestern Atlantic estuary.

Growing evidence has demonstrated that microplastics (MPs) are available for a wide range of marine organisms, with filter-feeding bivalves and crabs being especially vulnerable. The crab Neohelice granulata is considered a key and structuring species in the Bahía Blanca Estuary (BBE) (SW Atlantic) and its ecological role makes this species especially vulnerable to several pollutants. In this study, male specimens of N. granulata and water samples were collected at three sites in the BBE for the presence of MPs. Different types of MPs were found in all the crabs and the water column samples, although the most frequent were fibers <500-1500 µm, mainly blue. This is the first study to identify MPs in the gills and digestive tract of N. granulata. Moreover, gills presented higher total abundances of MPs than the digestive tract, which suggests that in this case the main uptake of MPs would be by adherence to the gills.

MicroRNA dynamics during human embryonic stem cell differentiation to pancreatic endoderm.

MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as critical regulators of human embryonic stem cell (hESC) pluripotency and differentiation. Despite the wealth of information about the role individual that miRNAs play in these two processes, there has yet to be a large-scale temporal analysis of the dynamics of miRNA expression as hESCs move from pluripotency into defined lineages. In this report, we used Next Generation Sequencing (NGS) to map temporal expression of miRNAs over ten 24-hour intervals as pluripotent cells were differentiated into pancreatic endoderm. Of the 2042 known human miRNAs, 694 had non-zero expression on all 11 days. Of these 694 miRNAs, 494 showed statistically significant changes in expression during differentiation. Clusters of miRNAs were identified, each displaying unique expression profiles distributed over multiple days. Selected miRNAs associated with pluripotency/differentiation (miR-302/367 and miR-371/372/373) and development/growth (miR-21, miR-25, miR-103, miR-9, and miR-92a) were found to have distinct expression profiles correlated with changes in media used to drive the differentiation process. Taken together, the clustering of miRNAs to identify expression dynamics that occur over longer periods of time (days vs. hours) provides unique insight into specific stages of differentiation. Major shifts in defined stages of hESC differentiation appear to be heavily dependent upon changes in external environmental factors, rather than intrinsic conditions in the cells.

Isolation, culture, and imaging of human fetal pancreatic cell clusters.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation(1-9). However in vitro, genesis of insulin producing cells from human fetal ICCs is low(10); results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust(11-17). A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro(11-22), far fewer exist for ICCs(10,23,24). Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue(6). Although the examples presented here focus upon the pancreatic endoderm proliferation and ß cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

sRNA-seq analysis of human embryonic stem cells and definitive endoderm reveals differentially expressed microRNAs and novel IsomiRs with distinct targets.

MicroRNAs (miRNAs) are noncoding, regulatory RNAs expressed dynamically during differentiation of human embryonic stem cells (hESCs) into defined lineages. Mapping developmental expression of miRNAs during transition from pluripotency to definitive endoderm (DE) should help to elucidate the mechanisms underlying lineage specification and ultimately enhance differentiation protocols. In this report, next generation sequencing was used to build upon our previous analysis of miRNA expression in human hESCs and DE. From millions of sequencing reads, 747 and 734 annotated miRNAs were identified in pluripotent and DE cells, respectively, including 77 differentially expressed miRNAs. Among these, four of the top five upregulated miRNAs were previously undetected in DE. Furthermore, the stem-loop for miR-302a, an important miRNA for both hESCs self-renewal and endoderm specification, produced several highly expressed miRNA species (isomiRs). Overall, isomiRs represented >10% of sequencing reads in >40% of all detected stem-loop arms, suggesting that the impact of these abundant miRNA species may have been overlooked in previous studies. Because of their relative abundance, the role of differential isomiR targeting was studied using the miR-302 cluster as a model system. A miRNA mimetic for miR-302a-5p, but not miR-302a-5p(+3), decreased expression of orthodenticle homeobox 2 (OTX2). Conversely, isomiR 302a-5p(+3) selectively decreased expression of tuberous sclerosis protein 1, but not OTX2, indicating nonoverlapping specificity of miRNA processing variants. Taken together, our characterization of miRNA expression, which includes novel miRNAs and isomiRs, helps establish a foundation for understanding the role of miRNAs in DE formation and selective targeting by isomiRs.

The SDF-1α/CXCR4 axis is required for proliferation and maturation of human fetal pancreatic endocrine progenitor cells.

The chemokine receptor CXCR4 and ligand SDF-1α are expressed in fetal and adult mouse islets. Neutralization of CXCR4 has previously been shown to diminish ductal cell proliferation and increase apoptosis in the IFNγ transgenic mouse model in which the adult mouse pancreas displays islet regeneration. Here, we demonstrate that CXCR4 and SDF-1α are expressed in the human fetal pancreas and that during early gestation, CXCR4 colocalizes with neurogenin 3 (ngn3), a key transcription factor for endocrine specification in the pancreas. Treatment of islet like clusters (ICCs) derived from human fetal pancreas with SDF-1α resulted in increased proliferation of epithelial cells in ICCs without a concomitant increase in total insulin expression. Exposure of ICCs in vitro to AMD3100, a pharmacological inhibitor of CXCR4, did not alter expression of endocrine hormones insulin and glucagon, or the pancreatic endocrine transcription factors PDX1, Nkx6.1, Ngn3 and PAX4. However, a strong inhibition of ß cell genesis was observed when in vitro AMD3100 treatment of ICCs was followed by two weeks of in vivo treatment with AMD3100 after ICC transplantation into mice. Analysis of the grafts for human C-peptide found that inhibition of CXCR4 activity profoundly inhibits islet development. Subsequently, a model pancreatic epithelial cell system (CFPAC-1) was employed to study the signals that regulate proliferation and apoptosis by the SDF-1α/CXCR4 axis. From a selected panel of inhibitors tested, both the PI 3-kinase and MAPK pathways were identified as critical regulators of CFPAC-1 proliferation. SDF-1α stimulated Akt phosphorylation, but failed to increase phosphorylation of Erk above the high basal levels observed. Taken together, these results indicate that SDF-1α/CXCR4 axis plays a critical regulatory role in the genesis of human islets.

Generation of definitive endoderm from human embryonic stem cells cultured in feeder layer-free conditions.

PURPOSE: The purpose of these studies was twofold: to reduce the level of nonhuman, potentially immunogenic sialic acid N-glycolylneuraminic acid (Neu5Gc) in human embryonic stem cells (hESCs) through culture of the cells in the absence of feeder layers; and to determine whether directed differentiation was preserved under these conditions, that is, using exclusively human-derived products. METHODS: Using a technique developed in our laboratory to culture hESCs in the absence of feeder layers, all nonhuman cell culture reagents were replaced with recombinant or human-derived reagents. The level of the nonhuman sialic acid (Neu5Gc) was measured by high-performance liquid chromatography and monitored over many passages. Subsequently, the cells were subjected to in vitro differentiation into definitive endoderm by lowering the serum concentrations and elevating the amount of activin A. RESULTS: Under standard tissue culture conditions using mouse and other animal products, the basal levels of Neu5Gc were measured between 7 and 10%. After the cell culture reagents were changed to all human products, Neu5Gc levels decreased steadily before leveling below 2%. Upon initiation of the differentiation protocol under these cell culture conditions, we observed robust endoderm formation, as measured by fluorescence-activated cell sorting analysis and the appearance of mRNA for markers of definitive endoderm (Sox17, CXCR4, Goosecoid and FoxA2). CONCLUSION: Consistent with other findings, elimination of nonhuman products in cell culture of hESCs decreases the levels of nonhuman and potentially immunogenic sialic acid levels. Furthermore, our studies demonstrate that in this feeder layer-free system, hESCs undergo directed differentiation into definitive endoderm.

Limited capacity of human adult islets expanded in vitro to redifferentiate into insulin-producing beta-cells.

Limited organ availability is an obstacle to the widespread use of islet transplantation in type 1 diabetic patients. To address this problem, many studies have explored methods for expanding functional human islets in vitro for diabetes cell therapy. We previously showed that islet cells replicate after monolayer formation under the influence of hepatocyte growth factor and selected extracellular matrices. However, under these conditions, senescence and loss of insulin expression occur after >15 doublings. In contrast, other groups have reported that islet cells expanded in monolayers for months progressed through a reversible epithelial-to-mesenchymal transition, and that on removal of serum from the cultures, islet-like structures producing insulin were formed (1). The aim of the current study was to compare the two methods for islet expansion using immunostaining, real-time quantitative PCR, and microarrays at the following time points: on arrival, after monolayer expansion, and after 1 week in serum-free media. At this time, cell aliquots were grafted into nude mice to study in vivo function. The two methods showed similar results in islet cell expansion. Attempts at cell differentiation after expansion by both methods failed to consistently recover a beta-cell phenotype. Redifferentiation of beta-cells after expansion is still a challenge in need of a solution.

Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers.

To date, all human embryonic stem cells (hESCs) available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Activation of STAT3 by leukemia inhibitory factor is required to maintain "stemness" in mouse embryonic stem cells, but not in hESCs, suggesting the existence of alternate signaling pathways for self-renewal and pluripotency in human cells. Here we show that activin A is secreted by mouse embryonic feeder layers (mEFs) and that culture medium enriched with activin A is capable of maintaining hESCs in the undifferentiated state for >20 passages without the need for feeder layers, conditioned medium from mEFs, or STAT3 activation. hESCs retained both normal karyotype and markers of undifferentiated cells, including Oct-4, nanog, and TRA-1-60 and remained pluripotent, as shown by the in vivo formation of teratomas.

Maintenance of pluripotency in human embryonic stem cells is STAT3 independent.

The preservation of "stemness" in mouse embryonic stem (mES) cells is maintained through a signal transduction pathway that requires the gp130 receptor, the interleukin-6 (IL-6) family of cytokines, and the Janus Kinase-signal transducer and activator (JAK/STAT) pathway. The factors and signaling pathways that regulate "stemness" in human embryonic stem (hES) cells remain to be elucidated. Here we report that STAT3 activation is not sufficient to block hES cell differentiation when the cells are grown on mouse feeder cells or when they are treated with conditioned media from feeder cells. Human ES cells differentiate in the presence of members of the IL-6 family of cytokines including leukemia inhibitory factor (LIF) and IL-6 or in the presence of the designer cytokine hyper-IL-6, which is a complex of soluble interleukin-6 receptor (IL-6R) and IL-6 with greatly enhanced bioactivity. Human ES cells express LIF, IL-6, and gp130 receptors, as well as the downstream signaling molecules. Stimulation of human and mouse ES cells with gp130 cytokines resulted in a robust phosphorylation of downstream ERK1, ERK2, and Akt kinases, as well as the STAT3 transcription factor. Loss of the pluripotency markers Nanog, Oct-4, and TRA-1-60 was observed in hES cells during gp130-dependent signaling, indicating that signaling through this pathway is insufficient to prevent the onset of differentiation. These data underscore a fundamental difference in requirements of murine versus hES cells. Furthermore, the data demonstrate the existence of an as-yet-unidentified factor in the conditioned media of mouse feeder layer cells that acts to maintain hES cell renewal in a STAT3-independent manner.

A novel approach to increase human islet cell mass while preserving beta-cell function.

Human islet expansion in monolayer culture leads to loss of function and senescence. By maintaining the 3-D configuration of islets in fibrin gels, it is feasible to expand beta-cells in response to hepatocyte growth factor (HGF) while preserving physiologic glucose responsiveness both in vitro and in vivo after transplantation into nude mice. Islets were cultured free floating with or without growth factors and nicotinamide and in fibrin gels with the same conditions. Proliferation was observed only in islets cultured in fibrin gels and the cocktail; total insulin increased by threefold, with a concomitant increase in beta-cell mass by morphometry. Insulin release after glucose challenge was also preserved. Islets in fibrin gels gave rise in vivo to large grafts rich in insulin and glucagon, and grafts from free-floating islets were smaller with fewer endocrine cells. Circulating human C-peptide levels were higher than in the mice receiving free-floating islets. In summary, fibrin allows for HGF-mediated cell proliferation while preserving glucose responsiveness in an environment that preserves cell-cell contacts. Limited islet ex vivo expansion under these conditions may improve recipient-donor tissue ratios to equal the functional results of whole-organ transplants.

Exendin 4 up-regulates expression of PDX 1 and hastens differentiation and maturation of human fetal pancreatic cells.

In addition to stimulating insulin secretion, glucagon-like peptide and its long-acting analog exendin 4 have been reported to increase beta-cell mass by both differentiation/neogenesis of precursor cells and enhanced replication of existing beta-cells. Here, we investigated the effect of exendin 4 in the growth and differentiation of beta-cells from undifferentiated precursors in islet-like cell clusters (ICCs) derived from human fetal pancreases. Our results show that the addition of exendin 4 to the culture media stimulates PDX 1 expression in ICCs as shown by immunofluorescence staining. The up-regulation of PDX 1 was not accompanied by changes in insulin expression because we did not find a significant difference in the number of insulin-positive cells in the exendin 4-treated ICCs, compared with controls. We also tested the effects of exendin 4 in the glucose-induced insulin secretion of human ICCs transplanted under the kidney capsule of athymic rats. In the exendin 4-treated rats (given ip during 10 d) 8 wk after the beginning of the treatment, insulin was released in response to glucose as detected by the measurement of circulating human C-peptide. In control (saline-treated) rats, the basal levels of human C-peptide did not change significantly after glucose stimulation. Thus, exendin 4 induces functional maturation of fetal beta-cells in response to glucose. In these rats, serial sections of the kidney-bearing grafts were examined histologically for insulin containing cells. We found a significant increase in beta-cell number, compared with the control rats. Overall, these results show that in vivo exendin 4 causes growth and differentiation of human fetal beta-cells from undifferentiated precursor cells. It also accelerates the functional maturation of fetal beta-cells as evidenced by their glucose-stimulated insulin secretion.