Immunoanalytical Approach for Detecting and Identifying Ancestral Peptide Biomarkers in Early Earth Analogue Environments.

Several mass spectrometry and spectroscopic techniques have been used in the search for molecular biomarkers on Mars. A major constraint is their capability to detect and identify large and complex compounds such as peptides or other biopolymers. Multiplex immunoassays can detect these compounds, but antibodies must be produced for a large number of sequence-dependent molecular targets. Ancestral Sequence Reconstruction (ASR) followed by protein "resurrection" in the lab can help to narrow the selection of targets. Herein, we propose an immunoanalytical method to identify ancient and universally conserved protein/peptide sequences as targets for identifying ancestral biomarkers in nature. We have developed, tested, and validated this approach by producing antibodies to eight previously described ancestral resurrected proteins (three ß-lactamases, three thioredoxins, one Elongation Factor Tu, and one RuBisCO, all of them theoretically dated as Precambrian), and used them as a proxy to search for any potential feature of them that could be present in current natural environments. By fluorescent sandwich microarray immunoassays (FSMI), we have detected positive immunoreactions with antibodies to the oldest ß-lactamase and thioredoxin proteins (ca. 4 Ga) in samples from a hydrothermal environment. Fine epitope mapping and inhibitory immunoassays allowed the identification of well-conserved epitope peptide sequences that resulted from ASR and were present in the sample. We corroborated these results by metagenomic sequencing and found several genes encoding analogue proteins with significant matches to the peptide epitopes identified with the antibodies. The results demonstrated that peptides inferred from ASR studies have true counterpart analogues in Nature, which validates and strengthens the well-known ASR/protein resurrection technique and our immunoanalytical approach for investigating ancient environments and metabolisms on Earth and elsewhere.

Spatial coherence from Nd3+ quantum emitters mediated by a plasmonic chain.

Controlling the coherence properties of rare earth emitters in solid-state platforms in the absence of an optical cavity is highly desirable for quantum light-matter interfaces and photonic networks. Here, we demonstrate the possibility of generating directional and spatially coherent light from Nd3+ ions coupled to the longitudinal plasmonic mode of a chain of interacting Ag nanoparticles. The effect of the plasmonic chain on the Nd3+ emission is analyzed by Fourier microscopy. The results reveal the presence of an interference pattern in which the Nd3+ emission is enhanced at specific directions, as a distinctive signature of spatial coherence. Numerical simulations corroborate the need of near-field coherent coupling of the emitting ions with the plasmonic chain mode. The work provides fundamental insights for controlling the coherence properties of quantum emitters at room temperature and opens new avenues towards rare earth based nanoscale hybrid devices for quantum information or optical communication in nanocircuits.

Maternal and perinatal outcomes and placental pathologic examination of 29 SARS-CoV-2 infected patients in the third trimester of gestation.

AIM: On December 31, 2019, an unknown outbreak of pulmonary disease was reported in China. The novel coronavirus SARS-CoV-2 was the etiologic agent of this disease, and responsible of the current pandemic of COVID-19. Accumulated evidence on placental features is based most on case-reports and small case-series, with differing results. METHODS: We gathered a cohort of 29 infected pregnant mothers who delivered 32 newborns, and had placentas available for pathologic examination. Placentas were compared with a control group. RESULTS: Of the 29 mothers, clinical and radiological features were similar to what was already described in COVID-19. Pregnancy modified some analytical parameters. One of the mothers succumbed to the disease. Of the 32 newborns, 1 developed an early infection, with positive reverse-transcriptase polymerase chain reaction (RT-PCR) at 48 h of life, with an initial RT-PCR negative. SARS-CoV-2 presence was assessed on placental tissue with immunohistochemistry and RT-PCR, both were negative. All newborns had good clinical outcomes. No differences in morphological placental findings were found among both groups. CONCLUSION: Lack of statistically significant differences among case and control groups suggest that placentas from SARS-CoV-2 infected mothers represent a cohort of normal placentas only submitted because of maternal SARS-CoV-2 status. To the best of our knowledge, no irrefutable cases of vertical transmission have been yet described. Other authors have failed to demonstrate presence of viral RNA in placental tissue. Accumulated knowledge suggests that if vertical transmission is possible, it is a rare event.

Engineering Protein Nanoparticles Out from Components of the Human Microbiome.

Nanoscale protein materials are highly convenient as vehicles for targeted drug delivery because of their structural and functional versatility. Selective binding to specific cell surface receptors and penetration into target cells require the use of targeting peptides. Such homing stretches should be incorporated to larger proteins that do not interact with body components, to prevent undesired drug release into nontarget organs. Because of their low interactivity with human body components and their tolerated immunogenicity, proteins derived from the human microbiome are appealing and fully biocompatible building blocks for the biofabrication of nonreactive, inert protein materials within the nanoscale. Several phage and phage-like bacterial proteins with natural structural roles are produced in Escherichia coli as polyhistidine-tagged recombinant proteins, looking for their organization as discrete, nanoscale particulate materials. While all of them self-assemble in a variety of sizes, the stability of the resulting constructs at 37 °C is found to be severely compromised. However, the fine adjustment of temperature and Zn2+ concentration allows the formation of robust nanomaterials, fully stable in complex media and under physiological conditions. Then, microbiome-derived proteins show promise for the regulatable construction of scaffold protein nanomaterials, which can be tailored and strengthened by simple physicochemical approaches.

Evaluation of specific quality metrics to assess the performance of a specialised newborn transport programme.

There is a lack of consensus on quality indicators suitable for neonatal transport. The aim of this study is to make a proposal for specific quality indicators for newborn transport. A retrospective descriptive study was performed (2009 to 2015) where twenty-four indicators were selected, evaluated and classified according to the 6 dimensions of quality of the Institute of Medicine. Among the 24 evaluated quality metrics, there were 3 of them which needed a correction when evaluating neonatal transport performance, because they were significantly correlated with gestational age. They were (a) stabilisation time, (b) prevalence of newborn arterial hypotension (defined by gestational age) and (c) unnoticed hypothermia at referral hospital.Conclusion: Quality evaluation through the definition of specific metrics in newborn transport is feasible. These indicators should be defined or adjusted for newborn population to measure the actual performance of the transport service.What is Known:• Quality indicators may help in defining metrics for clinical practice, promoting benchmarking and defining areas of improvement.• Newborn characteristics call for a specialised care, and quality measure during newborn transport require specific metrics. Quality metrics for paediatric transport have been defined using Delphi method. Some of these measures need to be specific for newborn, due to their intrinsic characteristics.What is New:• Using evidence-based literature and our newborn transport experience, specific quality indicators for newborn transport are suggested.• Data analysis shows how some indicators need to be adjusted for gestational age.

Recruiting potent membrane penetrability in tumor cell-targeted protein-only nanoparticles.

The membrane pore-forming activities of the antimicrobial peptide GWH1 have been evaluated in combination with the CXCR4-binding properties of the peptide T22, in self-assembling protein nanoparticles with high clinical potential. The resulting materials, of 25 nm in size and with regular morphologies, show a dramatically improved cell penetrability into CXCR4+ cells (more than 10-fold) and enhanced endosomal escape (the lysosomal degradation dropping from 90% to 50%), when compared with equivalent protein nanoparticles lacking GWH1. These data reveal that GWH1 retains its potent membrane activity in form of nanostructured protein complexes. On the other hand, the specificity of T22 in the CXCR4 receptor binding is subsequently minimized but, unexpectedly, not abolished by the presence of the antimicrobial peptide. The functional combination T22-GWH1 results in 30% of the nanoparticles entering cells via CXCR4 while also exploiting pore-based uptake. Such functional materials are capable to selectively deliver highly potent cytotoxic drugs upon chemical conjugation, promoting CXCR4-dependent cell death. These data support the further development of GWH1-empowered cell-targeted proteins as nanoscale drug carriers for precision medicines. This is a very promising approach to overcome lysosomal degradation of protein nanostructured materials with therapeutic value.

Selective CXCR4+ Cancer Cell Targeting and Potent Antineoplastic Effect by a Nanostructured Version of Recombinant Ricin.

Under the unmet need of efficient tumor-targeting drugs for oncology, a recombinant version of the plant toxin ricin (the modular protein T22-mRTA-H6) is engineered to self-assemble as protein-only, CXCR4-targeted nanoparticles. The soluble version of the construct self-organizes as regular 11 nm planar entities that are highly cytotoxic in cultured CXCR4+ cancer cells upon short time exposure, with a determined IC50 in the nanomolar order of magnitude. The chemical inhibition of CXCR4 binding sites in exposed cells results in a dramatic reduction of the cytotoxic potency, proving the receptor-dependent mechanism of cytotoxicity. The insoluble version of T22-mRTA-H6 is, contrarily, moderately active, indicating that free, nanostructured protein is the optimal drug form. In animal models of acute myeloid leukemia, T22-mRTA-H6 nanoparticles show an impressive and highly selective therapeutic effect, dramatically reducing the leukemia cells affectation of clinically relevant organs. Functionalized T22-mRTA-H6 nanoparticles are then promising prototypes of chemically homogeneous, highly potent antitumor nanostructured toxins for precise oncotherapies based on self-mediated intracellular drug delivery.

Conformational Conversion during Controlled Oligomerization into Nonamylogenic Protein Nanoparticles.

Protein materials are rapidly gaining interest in materials sciences and nanomedicine because of their intrinsic biocompatibility and full biodegradability. The controlled construction of supramolecular entities relies on the controlled oligomerization of individual polypeptides, achievable through different strategies. Because of the potential toxicity of amyloids, those based on alternative molecular organizations are particularly appealing, but the structural bases on nonamylogenic oligomerization remain poorly studied. We have applied spectrofluorimetry and spectropolarimetry to identify the conformational conversion during the oligomerization of His-tagged cationic stretches into regular nanoparticles ranging around 11 nm, useful for tumor-targeted drug delivery. We demonstrate that the novel conformation acquired by the proteins, as building blocks of these supramolecular assemblies, shows different extents of compactness and results in a beta structure enrichment that enhances their structural stability. The conformational profiling presented here offers clear clues for understanding and tailoring the process of nanoparticle formation through the use of cationic and histidine rich stretches in the context of protein materials usable in advanced nanomedical strategies.

Switching cell penetrating and CXCR4-binding activities of nanoscale-organized arginine-rich peptides.

Arginine-rich protein motifs have been described as potent cell-penetrating peptides (CPPs) but also as rather specific ligands of the cell surface chemokine receptor CXCR4, involved in the infection by the human immunodeficiency virus (HIV). Polyarginines are commonly used to functionalize nanoscale vehicles for gene therapy and drug delivery, aimed to enhance cell penetrability of the therapeutic cargo. However, under which conditions these peptides do act as either unspecific or specific ligands is unknown. We have here explored the cell penetrability of differently charged polyarginines in two alternative presentations, namely as unassembled fusion proteins or assembled in multimeric protein nanoparticles. By this, we have observed that arginine-rich peptides switch between receptor-mediated and receptor-independent mechanisms of cell penetration. The relative weight of these activities is determined by the electrostatic charge of the construct and the oligomerization status of the nanoscale material, both regulatable by conventional protein engineering approaches.

Engineering multifunctional protein nanoparticles by in vitro disassembling and reassembling of heterologous building blocks.

The engineering of protein self-assembling at the nanoscale allows the generation of functional and biocompatible materials, which can be produced by easy biological fabrication. The combination of cationic and histidine-rich stretches in fusion proteins promotes oligomerization as stable protein-only regular nanoparticles that are composed by a moderate number of building blocks. Among other applications, these materials are highly appealing as tools in targeted drug delivery once empowered with peptidic ligands of cell surface receptors. In this context, we have dissected here this simple technological platform regarding the controlled disassembling and reassembling of the composing building blocks. By applying high salt and imidazole in combination, nanoparticles are disassembled in a process that is fully reversible upon removal of the disrupting agents. By taking this approach, we accomplish here the in vitro generation of hybrid nanoparticles formed by heterologous building blocks. This fact demonstrates the capability to generate multifunctional and/or multiparatopic or multispecific materials usable in nanomedical applications.

Plasmonic enhancement of second harmonic generation from nonlinear RbTiOPO4 crystals by aggregates of silver nanostructures.

We demonstrate a 60-fold enhancement of the second harmonic generation (SHG) response at the nanoscale in a hybrid metal-dielectric system. By using complex silver nanostructures photochemically deposited on the polar surface of a ferroelectric crystal, we tune the plasmonic resonances from the visible to the near-infrared (NIR) spectral region, matching either the SH or the fundamental frequency. In both cases the SHG signal at the metal-dielectric interface is enhanced, although with substantially different enhancement values: around 5 times when the plasmonic resonance is at the SH frequency or up to 60 times when it matches the fundamental NIR radiation. The results are consistent with the more spatially-extended near-field response of complex metallic nanostructures and can be well explained by taking into account the quadratic character of the SHG process. The work points out the potential of aggregates of silver nanostructures for enhancing optical nonlinearities at the nanoscale and provides an alternative approach for the development of nanometric nonlinear photonic devices in a scalable way.

Recombinant pharmaceuticals from microbial cells: a 2015 update.

Diabetes, growth or clotting disorders are among the spectrum of human diseases related to protein absence or malfunction. Since these pathologies cannot be yet regularly treated by gene therapy, the administration of functional proteins produced ex vivo is required. As both protein extraction from natural producers and chemical synthesis undergo inherent constraints that limit regular large-scale production, recombinant DNA technologies have rapidly become a choice for therapeutic protein production. The spectrum of organisms exploited as recombinant cell factories has expanded from the early predominating Escherichia coli to alternative bacteria, yeasts, insect cells and especially mammalian cells, which benefit from metabolic and protein processing pathways similar to those in human cells. Up to date, around 650 protein drugs have been worldwide approved, among which about 400 are obtained by recombinant technologies. Other 1300 recombinant pharmaceuticals are under development, with a clear tendency towards engineered versions with improved performance and new functionalities regarding the conventional, plain protein species. This trend is exemplified by the examination of the contemporary protein-based drugs developed for cancer treatment.

Physical and chemical characterization of cerium(IV) oxide nanoparticles.

Chemical composition, size and structure of the nanoparticle are required to describe nanoceria. Nanoparticles of similar size and Ce(III) content might exhibit different chemical behaviour due to their differences in structure. A simple and direct procedure based on affordable techniques for all the laboratories is presented in this paper. The combination of Raman and UV-vis spectroscopy and particle impact coulometry (PIC) allows the characterization of nanoceria of small size from 4 to 65 nm at a concentration from micromolar to nanomolar, a concentration range suitable for the analysis of lab-prepared or commercial nanoparticle suspensions, but too high for most analytical purposes aimed at nanoparticle monitoring. While the PIC limits of size detection are too high to observe small nanoparticles unless catalytic amplification is used, the method provides a simple means to study aggregation of nanoparticles in the media they are needed to be dispersed for each application. Raman spectroscopy provided information about structure of the nanoparticle, and UV-vis about their chemical behaviour against some common reducing and oxidizing agents. Graphical Abstract To characterize nanoceria it is necessary to provide information about the shape, size and structure of the nanoparticles as well as the chemical composition.

Controlling solid state gain media by deposition of silver nanoparticles: from thermally- quenched to plasmon-enhanced Nd(3+) luminescence.

We show the possibility of controlling the optical properties of Nd(3+) laser ions by using different configurations of metallic nanoparticles (NPs) deposited on a solid state gain medium. In particular, we analyze the effect of two different silver NP arrangements on the optical properties of Nd(3+) ions in LiNbO(3): a two-dimensional (2D) high density and disordered Ag NP distribution and a one-dimensional (1D) long single chain of Ag NPs. We demonstrate that while the 2D disordered distribution produces a thermal quenching of the Nd(3+) luminescence, the 1D single chain leads to the enhancement of the fluorescence from the (4)F(3/2) metastable state. The experimental data are theoretically interpreted by taking into account the different character, radiative or non-radiative, of the localized surface plasmonic modes supported by the Ag nanoparticle distributions at the excitation wavelength. The results point out the capabilities of rare earth ions as optical tools to probe the local plasmonic fields and are relevant to determine the optimal configuration of metallic arrays to improve the performance of potential rare earth ion based sub-micrometer lasers.

Expanding the recombinant protein quality in Lactococcus lactis.

BACKGROUND: Escherichia coli has been a main host for the production of recombinant proteins of biomedical interest, but conformational stress responses impose severe bottlenecks that impair the production of soluble, proteolytically stable versions of many protein species. In this context, emerging Generally Recognized As Safe (GRAS) bacterial hosts provide alternatives as cell factories for recombinant protein production, in which limitations associated to the use of Gram-negative microorganisms might result minimized. Among them, Lactic Acid Bacteria and specially Lactococcus lactis are Gram-positive GRAS organisms in which recombinant protein solubility is generically higher and downstream facilitated, when compared to E. coli. However, deep analyses of recombinant protein quality in this system are still required to completely evaluate its performance and potential for improvement. RESULTS: We have explored here the conformational quality (through specific fluorescence emission) and solubility of an aggregation-prone GFP variant (VP1GFP) produced in L. lactis. In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism. CONCLUSIONS: Metabolic regime and cultivation temperature greatly influence solubility and conformational quality of an aggregation-prone protein in L. lactis. Specifically, the present study proves that anaerobic growth is the optimal condition for recombinant protein production purposes. Besides, growth temperature plays an important role regulating both protein solubility and conformational quality. Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species. These findings not only confirm L. lactis as an excellent producer of recombinant proteins but also reveal room for significant improvement by the exploitation of external protein quality modulators.

Serpentinization-associated travertines as spatio-temporal archives for lipid biomarkers key for the search for life on Mars.

Serpentinization is a well-known aqueous alteration process that may have played important roles in the origins and early evolution of life on Earth, and perhaps Mars, but there are still aspects related to biomarker distribution, partitioning, and preservation that merit further study. To assess the role that precipitation of carbonate phases in serpentinization settings may have on biomarker preservation, we search for life signs in one of the world's largest outcrops of subcontinental peridotites (Ronda, South Spain). We investigate the organic record of groundwater and associated carbonate deposits (travertines) in seven hyperalkaline springs, and reconstruct the biological activity and metabolic interactions of the serpentinization-hosted ecosystem. We identified lipid biomarkers and isotopic evidences of life, whose concentration and variety were much lower in groundwater than travertine deposits (ppb/ppt versus ppm level). Groundwater carried organics of abiotic (n-alkanes with values of CPI âˆ¼ 1) and/or biotic origin, of fresher (e.g. acids or alcohols) or more diagenetized (mature hopanes and n-alkanes) nature. In contrast, associated travertines held a more prolific record of biomarkers incorporating (molecular and isotopic) fingerprints of surface (mostly phototrophs) and subsurface (chemolithotrophs, methanogens and/or methanotrophs) life. Serpentinization-associated travertines seem to act as biomolecule archives over time fed by autochthonous and allochthonous sources, hence amplifying the dim biological signal of groundwater. These results illustrate the relevance of serpentinization-associated surface mineral deposits in searching for traces of life on analogous environments on Mars. We highlight the diversity of lipids produced in serpentinizing land environments and emphasize the potential of these geostable biomolecules to preserve fingerprints of life.

Lipid-based paleoecological and biogeochemical reconstruction of Store Saltsø, an extreme lacustrine system in SW Greenland.

Polar lakes harbour a unique biogeochemistry that reflects the implications of climatic fluctuations against a susceptible yet extreme environment. In addition to polar, Store Saltsø (Kangerlussuaq, southwestern Greenland) is an endorheic lake with alkaline and oligotrophic waters that host a distinctive ecology adapted to live in such particular physico-chemical and environmental conditions. By exploring the sedimentary record of Store Saltsø at a molecular and compound-specific isotopic level, we were able to understand its ecology and biogeochemical evolution upon climate change. We employed lipid biomarkers to identify biological sources and metabolic traits in different environmental samples (shore terrace, sediment core, and white precipitates at the shore), and their succession over time to reconstruct the lake paleobiology. Different molecular ratios and geochemical proxies provided further insights toward the evolution of environmental conditions in the frame of the deglaciation history of Kangerlussuaq. The relative abundance of terrestrial (i.e., plant derived) biomarkers (odd long-chain n-alkanes, even long-chain n-alkanols, and phytosterols) in the upper half of the shore terrace versus the relatively more present aquatic biomarkers (botryococcenes and long-chain alkenones) in its lower half revealed higher lake water levels in the past. Moreover, the virtual absence of organics in the deepest section of the sediment core (32-29 cm depth) suggested that the lake did not yet exist at the northwestern shore of Store Saltsø ∼5100 years ago. According to the relative abundance of lipid biomarkers detected in the adjacent section above (29-25 cm depth), we hypothesize that the northwestern shore of Store Saltsø formed ∼4900 years ago. By combining the molecular and compound-specific isotopic analysis of lipids in a ∼360 cm sedimentary sequence, we recreated the paleobiology and evolution of an extreme lacustrine environment suitable for the study of the limits of life and the effects of climate warming.

Specific IgA, But Not IgG, in Human Milk From COVID-19-Infected Mothers Neutralizes SARS-CoV-2.

This study highlights the importance of human milk in providing anti-severe acute respiratory syndrome coronavirus 2 immunity to newborns. The highest protective activity of human milk against COVID-19 was found in colostrum from infected mothers. Neutralizing activity was associated with high levels of specific IgA. Depletion of IgA, but not IgG, from milk samples completely abolished the ability of human milk to neutralize severe acute respiratory syndrome coronavirus 2.

The Molecular Profile of Soil Microbial Communities Inhabiting a Cambrian Host Rock.

The process of soil genesis unfolds as pioneering microbial communities colonize mineral substrates, enriching them with biomolecules released from bedrock. The resultant intricate surface units emerge from a complex interplay among microbiota and plant communities. Under these conditions, host rocks undergo initial weathering through microbial activity, rendering them far from pristine and challenging the quest for biomarkers in ancient sedimentary rocks. In addressing this challenge, a comprehensive analysis utilizing Gas Chromatography Mass Spectrometry (GC-MS) and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) was conducted on a 520-Ma-old Cambrian rock. This investigation revealed a diverse molecular assemblage with comprising alkanols, sterols, fatty acids, glycerolipids, wax esters, and nitrogen-bearing compounds. Notably, elevated levels of bacterial C16, C18 and C14 fatty acids, iso and anteiso methyl-branched fatty acids, as well as fungal sterols, long-chained fatty acids, and alcohols, consistently align with a consortium of bacteria and fungi accessing complex organic matter within a soil-type ecosystem. The prominence of bacterial and fungal lipids alongside maturity indicators denotes derivation from heterotrophic activity rather than ancient preservation or marine sources. Moreover, the identification of long-chain (>C22) n-alkanols, even-carbon-numbered long chain (>C20) fatty acids, and campesterol, as well as stigmastanol, provides confirmation of plant residue inputs. Furthermore, findings highlight the ability of contemporary soil microbiota to inhabit rocky substrates actively, requiring strict contamination controls when evaluating ancient molecular biosignatures or extraterrestrial materials collected.

Biosignature Detection and MinION Sequencing of Antarctic Cryptoendoliths After Exposure to Mars Simulation Conditions.

In the search for life in our Solar System, Mars remains a promising target based on its proximity and similarity to Earth. When Mars transitioned from a warmer, wetter climate to its current dry and freezing conditions, any putative extant life probably retreated into habitable refugia such as the subsurface or the interior of rocks. Terrestrial cryptoendolithic microorganisms (i.e., those inhabiting rock interiors) thus represent possible modern-day Mars analogs, particularly those from the hyperarid McMurdo Dry Valleys in Antarctica. As DNA is a strong definitive biosignature, given that there is no known abiotic chemistry that can polymerize nucleobases, we investigated DNA detection with MinION sequencing in Antarctic cryptoendoliths after an ∼58-sol exposure in MARTE, a Mars environmental chamber capable of simulating martian temperature, pressure, humidity, ultraviolet (UV) radiation, and atmospheric composition, in conjunction with protein and lipid detection. The MARTE conditions resulted in changes in community composition and DNA, proteins, and cell membrane-derived lipids remained detectable postexposure. Of the multitude of extreme environmental conditions on Mars, UV radiation (specifically UVC) is the most destructive to both cells and DNA. As such, we further investigated if a UVC exposure corresponding to ∼278 martian years would impede DNA detection via MinION sequencing. The MinION was able to successfully detect and sequence DNA after this UVC radiation exposure, suggesting its utility for life detection in future astrobiology missions focused on finding relatively recently exposed biomarkers inside possible martian refugia.