A billion-year shift in the formation of Earth's largest ore deposits.

Banded iron formations (BIFs) archive the relationship between Earth's lithosphere, hydrosphere, and atmosphere through time. However, constraints on the origin of Earth's largest ore deposits, hosted by BIFs, are limited by the absence of direct geochronology. Without this temporal context, genetic models cannot be correlated with tectono-thermal and atmospheric drivers responsible for BIF upgrading through time. Utilizing in situ iron oxide U-Pb geochronology, we provide a direct timeline of events tracing development of all the giant BIF-hosted hematite deposits of the Hamersley Province (Pilbara Craton, Western Australia). Direct dating demonstrates that the major iron ore deposits in the region formed during 1.4 to 1.1 Ga. This is one billion to hundreds of millions of years later than previous age constraints based upon 1) the presence of hematite ore clasts in conglomerate beds deposited before ~1.84 Ga, and 2) phosphate mineral dating, which placed the onset of iron mineralization in the Province at ~2.2 to 2.0 Ga during the great oxidation event. Dating of the hematite clasts verified the occurrence of a ~2.2 to 2.0 Ga event, reflecting widespread, but now largely eroded iron mineralization occurring when the Pilbara and Kaapvaal cratons were proximal. No existing phosphate mineral dates overlap with obtained hematite dates and therefore cannot be related to hematite crystallization and ore formation. New geochronology conclusively links all major preserved hematite deposits to a far younger (1.4 to 1.1 Ga) formation period, correlated with the amalgamation of Australia following breakup of the Columbia supercontinent.

Nuclear interferon-stimulated gene product maintains heterochromatin on the herpes simplex viral genome to limit lytic infection.

Interferons (IFN) are expressed in and secreted from cells in response to virus infection, and they induce the expression of a variety of genes called interferon-stimulated genes (ISGs) in infected and surrounding cells to block viral infection and limit spread. The mechanisms of action of a number of cytoplasmic ISGs have been well defined, but little is known about the mechanism of action of nuclear ISGs. Constitutive levels of nuclear interferon-inducible protein 16 (IFI16) serve to induce innate signaling and epigenetic silencing of herpes simplex virus (HSV), but only when the HSV infected cell protein 0 (ICP0) E3 ligase, which promotes IFI16 degradation, is inactivated. In this study, we found that following IFN induction, the pool of IFI16 within the infected cell remains high and can restrict wild-type viral gene expression and replication due to both the induced levels of IFI16 and the IFI16-mediated repression of ICP0 levels. Restriction of viral gene expression is achieved by IFI16 promoting the maintenance of heterochromatin on the viral genome, which silences it epigenetically. These results indicate that a nuclear ISG can restrict gene expression and replication of a nuclear DNA virus by maintaining or preventing the removal of repressive heterochromatin associated with the viral genome.

Mössbauer and EPR detection of iron trafficking kinetics and possibly labile iron pools in whole Saccharomyces cerevisiae cells.

The kinetics of iron trafficking in whole respiring Saccharomyces cerevisiae cells were investigated using Mössbauer and EPR spectroscopies. The Mössbauer-active isotope 57Fe was added to cells growing under iron-limited conditions; cells were analyzed at different times post iron addition. Spectroscopic changes suggested that the added 57Fe initially entered the labile iron pool, and then distributed to vacuoles and mitochondria. The first spectroscopic feature observed, ∼ 3 min after adding 57Fe plus a 5 to 15 min processing dead time, was a quadrupole doublet typical of nonheme high-spin FeII. This feature likely arose from labile FeII pools in the cell. At later times (15-150 min), magnetic features due to S = 5/2 FeIII developed; these likely arose from FeIII in vacuoles. Corresponding EPR spectra were dominated by a g = 4.3 signal from the S = 5/2 FeIII ions that increased in intensity over time. Developing at a similar rate was a quadrupole doublet typical of S = 0 [Fe4S4]2+ clusters and low-spin FeII hemes; such centers are mainly in mitochondria, cytosol, and nuclei. Development of these features was simulated using a published mathematical model, and simulations compared qualitatively well with observations. In the five sets of experiments presented, all spectroscopic features developed within the doubling time of the cells, implying that the detected iron trafficking species are physiologically relevant. These spectroscopy-based experiments allow the endogenous labile iron pool within growing cells to be detected without damaging or altering the pool, as definitely occurs using chelator-probe detection and possibly occurs using chromatographic separations.

Impact of the interaction between herpes simplex virus 1 ICP22 and FACT on viral gene expression and pathogenesis.

Facilitates chromatin transcription (FACT) interacts with nucleosomes to promote gene transcription by regulating the dissociation and reassembly of nucleosomes downstream and upstream of RNA polymerase II (Pol II). A previous study reported that herpes simplex virus 1 (HSV-1) regulatory protein ICP22 interacted with FACT and was required for its recruitment to the viral DNA genome in HSV-1-infected cells. However, the biological importance of interactions between ICP22 and FACT in relation to HSV-1 infection is unclear. Here, we mapped the minimal domain of ICP22 required for its efficient interaction with FACT to a cluster of five basic amino acids in ICP22. A recombinant virus harboring alanine substitutions in this identified cluster led to the decreased accumulation of viral mRNAs from UL54, UL38, and UL44 genes, reduced Pol II occupancy of these genes in MRC-5 cells, and impaired HSV-1 virulence in mice following ocular or intracranial infection. Furthermore, the treatment of mice infected with wild-type HSV-1 with CBL0137, a FACT inhibitor currently being investigated in clinical trials, significantly improved the survival rate of mice. These results suggested that the interaction between ICP22 and FACT was required for efficient HSV-1 gene expression and pathogenicity. Therefore, FACT might be a potential therapeutic target for HSV-1 infection.IMPORTANCEICP22 is a well-known regulatory factor of HSV-1 gene expression, but its mechanism(s) are poorly understood. Although the interaction of FACT with ICP22 was reported previously, its significance in HSV-1 infection is unknown. Given that FACT is involved in gene transcription, it is of interest to investigate this interaction as it relates to HSV-1 gene expression. To determine a direct link between the interaction and HSV-1 infection, we mapped a minimal domain of ICP22 required for its efficient interaction with FACT and generated a recombinant virus carrying mutations in the identified domain. Using the recombinant virus, we obtained evidence suggesting that the interaction between ICP22 and FACT promoted Pol II transcription from HSV-1 genes and viral virulence in mice. In addition, CBL0137, an inhibitor of FACT, effectively protected mice from lethal HSV-1 infection, suggesting FACT might be a potential target for the development of novel anti-HSV drugs.

Aberrant RNA polymerase initiation and processivity on the genome of a herpes simplex virus 1 mutant lacking ICP27.

Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.

An examination of the metal ion content in the active sites of human endonucleases CPSF73 and INTS11.

Human cleavage and polyadenylation specificity factor (CPSF)73 (also known as CPSF3) is the endoribonuclease that catalyzes the cleavage reaction for the 3'-end processing of pre-mRNAs. The active site of CPSF73 is located at the interface between a metallo-ß-lactamase domain and a ß-CASP domain. Two metal ions are coordinated by conserved residues, five His and two Asp, in the active site, and they are critical for the nuclease reaction. The metal ions have long been thought to be zinc ions, but their exact identity has not been examined. Here we present evidence from inductively coupled plasma mass spectrometry and X-ray diffraction analyses that a mixture of metal ions, including Fe, Zn, and Mn, is present in the active site of CPSF73. The abundance of the various metal ions is different in samples prepared from different expression hosts. Zinc is present at less than 20% abundance in a sample expressed in insect cells, but the sample is active in cleaving a pre-mRNA substrate in a reconstituted canonical 3'-end processing machinery. Zinc is present at 75% abundance in a sample expressed in human cells, which has comparable endonuclease activity. We also observe a mixture of metal ions in the active site of the CPSF73 homolog INTS11, the endonuclease for Integrator. Taken together, our results provide further insights into the role of metal ions in the activity of CPSF73 and INTS11 for RNA 3'-end processing.

Discovery of an unusual copper homeostatic mechanism in yeast cells respiring on minimal medium and an unexpectedly diverse labile copper pool.

Copper is essential for all eukaryotic cells but many details of how it is trafficked within the cell and how it is homeostatically regulated remain uncertain. Here, we characterized the copper content of cytosol and mitochondria using liquid chromatography with ICP-MS detection. Chromatograms of cytosol exhibited over two dozen peaks due to copper proteins and coordination complexes. Yeast cells respiring on minimal media did not regulate copper import as media copper concentration increased; rather, they imported copper at increasing rates while simultaneously increasing the expression of metallothionein CUP1 which then sequestered most of the excessive imported copper. Peak intensities due to superoxide dismutase SOD1, other copper proteins, and numerous coordination complexes also increased, but not as drastically. The labile copper pool was unexpectedly diverse and divided into two groups. One group approximately comigrated with copper-glutathione, -cysteine, and -histidine standards; the other developed only at high media copper concentrations and at greater elution volumes. Most cytosolic copper arose from copper-bound proteins, especially CUP1. Cytosol contained an unexpectedly high percentage of apo-copper proteins and apo-coordination complexes. Copper-bound forms of non-CUP1 proteins and complexes coexisted with apo-CUP1 and with the chelator BCS. Both experiments suggest unexpectedly stable-binding copper proteins and coordination complexes in cytosol. COX17Δ cytosol chromatograms were like those of WT cells. Chromatograms of soluble mitochondrial extracts were obtained, and mitoplasting helped distinguish copper species in the intermembrane space versus in the matrix/inner membrane. Issues involving the yeast copperome, copper homeostasis, labile copper pool, and copper trafficking are discussed.

HSV-1 ICP0 dimer domain adopts a novel ß-barrel fold.

Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine ß-strands and two α-helices. Interestingly, two adjacent ß-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two ß-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two ß-strands of each dimer, creating a "stacking" of the ß-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.

Metallomic mapping of gut and brain in heavy metal exposed earthworms: A novel paradigm in ecotoxicology.

This study explored the uptake of lead in the epigeic earthworm Dendrobaena veneta exposed to 0, 1000, and 2500 µg Pb/g soil. The soil metal content was extracted using strong acid digestion and water leaching, and analysed by means of Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to estimate absolute and bioavailable concentrations of metals in the soil. The guts and heads of lead-exposed earthworms were processed into formalin-fixed and paraffin embedded sections for high-resolution multi-element metallomic imaging via Laser Ablation ICP-MS (LA-ICP-MS). Metallomic maps of phosphorus, zinc, and lead were produced at 15-µm resolution in the head and gut of D. veneta. Additional 4-µm resolution metallomic maps of the earthworm brains were taken, revealing the detailed localisation of metals in the brain. The Pb bioaccumulated in the chloragogenous tissues of the earthworm in a dose-dependent manner, making it possible to track the extent of soil contamination. The bioaccumulation of P and Zn in earthworm tissues was independent of Pb exposure concentration. This approach demonstrates the utility of LA-ICP-MS as a powerful approach for ecotoxicology and environmental risk assessments.

Effects on exocytosis by two HSV-1 mutants unable to block autophagy.

IMPORTANCE: Pathogens often hijack extracellular vesicle (EV) biogenesis pathways for assembly, egress, and cell-to-cell spread. Herpes simplex virus 1 (HSV-1) infection stimulated EV biogenesis through a CD63 tetraspanin biogenesis pathway and these EVs activated antiviral responses in recipient cells restricting the infection. HSV-1 inhibits autophagy to evade the host, and increased CD63 exocytosis could be a coping mechanism, as CD63 is involved in both cargo delivery to lysosomes during autophagy and exocytosis. We analyzed exocytosis after infection with two HSV-1 mutants, a ΔICP34.5 and a ΔICP0, that could not inhibit autophagy. Unlike HSV-1(F), neither of these viruses stimulated increased EV biogenesis through the CD63 pathway. ΔICP34.5 stimulated production of microvesicles and apoptotic bodies that were CD63-negative, while ΔICP0 displayed an overall reduced production of EVs. These EVs activated innate immunity gene expression in recipient cells. Given the potential use of these mutants for therapeutic purposes, the immunomodulatory properties of EVs associated with them may be beneficial.

Herpes Simplex Virus-1 ICP27 Nuclear Export Signal Mutants Exhibit Cell Type-Dependent Deficits in Replication and ICP4 Expression.

Herpes simplex virus type-1 (HSV-1) protein ICP27 is an essential immediate early (IE) protein that promotes the expression of viral early (E) and late (L) genes via multiple mechanisms. Our understanding of this complex regulatory protein has been greatly enhanced by the characterization of HSV-1 mutants bearing engineered alterations in the ICP27 gene. However, much of this analysis has been performed in interferon-deficient Vero monkey cells. Here, we assessed the replication of a panel of ICP27 mutants in several other cell types. Our analysis shows that mutants lacking ICP27's amino (N)-terminal nuclear export signal (NES) display a striking cell type-dependent growth phenotype, i.e., they grow semi-permissively in Vero and some other cells but are tightly blocked for replication in primary human fibroblasts and multiple human cell lines. This tight growth defect correlates with a failure of these mutants to replicate viral DNA. We also report that HSV-1 NES mutants are deficient in expressing the IE protein ICP4 at early times postinfection. Analysis of viral RNA levels suggests that this phenotype is due, at least in part, to a defect in the export of ICP4 mRNA to the cytoplasm. In combination, our results (i) show that ICP27's NES is critically important for HSV-1 replication in many human cells, and (ii) suggest that ICP27 plays a heretofore unappreciated role in the expression of ICP4. IMPORTANCE HSV-1 IE proteins drive productive HSV-1 replication. The major paradigm of IE gene induction, developed over many years, involves the parallel activation of the five IE genes by the viral tegument protein VP16, which recruits the host RNA polymerase II (RNAP II) to the IE gene promoters. Here, we provide evidence that ICP27 can enhance ICP4 expression early in infection. Because ICP4 is required for transcription of viral E and L genes, this finding may be relevant to understanding how HSV-1 enters and exits the latent state in neurons.

The expression and function of HSV ICP47 and its promoter in mice.

IMPORTANCE: Immune evasion and latency are key mechanisms that underlie the success of herpesviruses. In each case, interactions between viral and host proteins are required and due to co-evolution, not all mechanisms are preserved across host species, even if infection is possible. This is highlighted by the herpes simplex virus (HSV) protein immediate early-infected cell protein (ICP)47, which inhibits the detection of infected cells by killer T cells and acts with high efficiency in humans, but poorly, if at all in mouse cells. Here, we show that ICP47 retains modest but detectable function in mouse cells, but in an in vivo model we found no role during acute infection or latency. We also explored the activity of the ICP47 promoter, finding that it could be active during latency, but this was dependent on genome location. These results are important to interpret HSV pathogenesis work done in mice.

Quantification of snake venom proteomes by mass spectrometry-considerations and perspectives.

The advent of soft ionization mass spectrometry-based proteomics in the 1990s led to the development of a new dimension in biology that conceptually allows for the integral analysis of whole proteomes. This transition from a reductionist to a global-integrative approach is conditioned to the capability of proteomic platforms to generate and analyze complete qualitative and quantitative proteomics data. Paradoxically, the underlying analytical technique, molecular mass spectrometry, is inherently nonquantitative. The turn of the century witnessed the development of analytical strategies to endow proteomics with the ability to quantify proteomes of model organisms in the sense of "an organism for which comprehensive molecular (genomic and/or transcriptomic) resources are available." This essay presents an overview of the strategies and the lights and shadows of the most popular quantification methods highlighting the common misuse of label-free approaches developed for model species' when applied to quantify the individual components of proteomes of nonmodel species (In this essay we use the term "non-model" organisms for species lacking comprehensive molecular (genomic and/or transcriptomic) resources, a circumstance that, as we detail in this review-essay, conditions the quantification of their proteomes.). We also point out the opportunity of combining elemental and molecular mass spectrometry systems into a hybrid instrumental configuration for the parallel identification and absolute quantification of venom proteomes. The successful application of this novel mass spectrometry configuration in snake venomics represents a proof-of-concept for a broader and more routine application of hybrid elemental/molecular mass spectrometry setups in other areas of the proteomics field, such as phosphoproteomics, metallomics, and in general in any biological process where a heteroatom (i.e., any atom other than C, H, O, N) forms integral part of its mechanism.

Online Isotope Analysis of Sulfur in Proteins via Capillary Electrophoresis Coupled With Multicollector ICP-MS (CE/MC-ICP-MS): A Proof of Concept Study.

Isotope ratio analysis of sulfur in biological samples using inductively coupled plasma-mass spectrometry (ICP-MS) has gained significant interest for applications in quantitative proteomics. Advancements like coupling separation techniques with multicollector ICP-MS (MC-ICP-MS) enhance the throughput of species-specific sulfur isotope ratio measurements, fostering new avenues for studying sulfur metabolism in complex biological matrices. This proof-of-concept study investigates the feasibility of online CE/MC-ICP-MS for directly analyzing sulfur isotope ratios in proteins (albumin). Leveraging our previous work on the applicability of CE/ICP-MS for quantifying sulfur-containing biological molecules, we explore its potential for sulfur isotope analysis. Our results demonstrate that direct analysis of sulfur isotopes in albumin protein using online capillary electrophoresis MC-ICP-MS (CE/MC-ICP-MS) eliminates the need for laborious pretreatment steps, while yielding isotope ratios comparable to the reference values. Although initial precision can be improved through further system optimization and protein injection techniques, this approach paves the way for future analysis of mixtures of various biological compounds in, for example, clinical diagnosis studies.

Novel Applications of CE-ICP-MS/MS: Monitoring of Antiaging GHK-Cu Cosmetic Component Encapsulation in Liposomes.

The hyphenation of the separation technique with the high-sensitive mass spectrometry detection is one of the driving forces of modern analysis enabling measurements in complex matrices. In particular, capillary electrophoresis coupled to inductively coupled plasma tandem mass spectrometry allows for speciation analysis of selected analytes with a superior resolution. The mild, physiological-friendly conditions of this separation technique offer the unique advantage of analyzing chemical entities in their intact form, which has been successfully exploited in various areas. Herein, we report the pioneering application of such a hyphenated technique in the cosmetic field to investigate the encapsulation of copper tripeptide complex (GHK-Cu) in liposomes. By monitoring copper and phosphorus signals, the formation of liposomes via a simple ethanol injection method was confirmed, and the concentration of GHK-Cu in the liposomes was assessed. The application of coupling of capillary electrophoresis with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS/MS) in cosmetic studies could lead to the development of diverse liposomal formulations with preferential properties and expand their accessibility.

A CH4-Driven Ion Cloud-Stretched Approach Enables ICP-qMS for Multiplex Single-Cell Analysis.

In the last 40 years, inductively coupled plasma quadrupole (q) mass spectrometry (ICP-qMS) has been recognized as one of the best tools for the quantification of multiple elements/isotopes and even the biomolecules they labeled in a homogeneous solution sample. However, it meets a tough challenge when acquiring multi-m/z signals from an intact single-cell dispersed in a cell suspension, since the single-cell ion cloud generated in ICP presents an intermittently transient event with a duration time of hundreds of microseconds while the dwell time plus settling time of the q is at the similar time scale when peak-hopping between different m/z. Herein, we report CH4 is able to stretch the single-cell ion cloud duration time to more than 7,000 µs in collision-reaction-cell (CRC), allowing multi-m/z signals acquisition by ICP-qMS. Quantification of single-cell's multiple phenotype protein markers can thus be achieved on ICP-(CH4-CRC)-qMS, not only revealing the heterogeneity between the single cells but also enabling an unambiguous cell-classification of their subtypes. CH4-driven ion cloud-stretched approach breaks through the long-standing bottleneck limited single-cell multiplex analysis on ICP-qMS, paving a path for more important applications of ICP-qMS in the fields related to single-cell analysis.

ICP-MS based detection method combined with Au NP and Ag NP labeling for bacteremia diagnosis.

Bacteremia, as a serious infectious disease, has an increasing incidence and a high mortality rate. Early diagnosis and early treatment are crucial for improving the cure rate. In this work, we proposed an inductively coupled plasma mass spectrometry (ICP-MS)-based detection method combined with gold nanoparticle (Au NP) and silver nanoparticle (Ag NP) labeling for the simultaneous detection of Salmonella and Escherichia coli (E. coli O157:H7) in human blood samples. Salmonella and E. coli O157:H7 were captured by magnetic beads coupled with anti-8G3 and anti-7C2, and then specifically labeled by Au NP-anti-5H12 and Ag NP-anti-8B1 respectively, which were used as signal probes for ICP-MS detection. Under the optimal experimental conditions, the limits of detection of 164 CFU mL-1 for Salmonella, 220 CFU mL-1for E. coli O157:H7 and the linear ranges of 400-80,000 CFU mL-1Salmonella, 400-60,000 CFU mL-1 E. coli O157:H7 were obtained. The proposed method can realize the simultaneous detection of two types of pathogenic bacteria in human whole blood in 3.5 h, showing great potential for the rapid diagnosis of bacteremia in clinic.

Comparing nitric acid treatment and microwave digestion for efficiency of metal extraction from bioprocess samples.

Metal ions may act as enzyme cofactors and influence the kinetics of biochemical reactions that may also influence the biological production of therapeutic proteins and quality attributes such as glycosylation. Because sample preparation is a significant step in the reliable analysis of metals, we compared two sample preparation procedures for metal analysis of bioreactor culture media samples by ICP-MS: (i) samples were diluted in 2 % nitric acid (treatment with nitric acid, TNA); and (ii) samples were mixed with equal volume of 5 % nitric acid and closed vessel digestion was performed in a microwave (closed vessel digestion, CVD). In the comparison of extraction efficiencies between TNA and CVD procedures, CVD showed better extraction for Ca and Cu among bulk metals (∼30 %) and for Ni among the trace metals (∼65 %) for the bioreactor broth supernatant samples. For the cell pellet samples, the CVD procedure was found to be better for extraction of Fe (∼65 % more) among bulk metals, Zn (∼20 % more) among minor metals and Co (∼60 % more) and Ni (∼45 % more) among trace metals. Differences between the two procedures were less than 10 % and TNA was better for all other metals quantified from both supernatant samples and cell pellet samples. The current study helps bring more clarity to the methodology on comprehensive metal analysis to monitor and maintain trace metal content for biologics production.

The relationship between lymphocyte DNA damage, coronary artery disease, and blood trace elements.

Somatic DNA damage and causative factors (occupational exposures, foods, habits, etc.) are thought to contribute to the pathogenesis of atherosclerosis, although knowledge about their role in coronary artery disease (CAD) is still insufficient. This study aimed to determine the effects of lymphocyte-DNA damage and blood trace element concentrations on CAD. The single-cell alkaline comet was used in the measuring of the lymphocyte DNA damage in blood samples obtained from patients (n = 99) whose CAD grade was determined by the syntax score while the angiographic intervention was carried out. Blood trace element (n = 14) concentrations were monitored by the inductively coupled plasma-optical emission spectroscopy (ICP-OES) after microwave digestion. The relationship between the DNA damage frequencies of the participants and their syntax scores, blood trace element concentrations, and other demographic and clinic parameters were statistically analyzed. Significant correlations were detected between comet data and syntax score (r = 0.858, P < .001), age (r = 0.337, P < .001), blood-urea (r = 0.360, P < .001), creatinine (r = 0.388, P < .001), HbA1c (0.218, P < .05), ECG-QRS time (r = 0.286, P < .01), ECHO-EF (r = -0.377, P < .001), and platelet (r = -0.222, P < .05). The DNA damage frequencies of the groups formed according to their CAD scores were significantly different from the control group (P < .001) and also each other (P ≤ .01). Comet frequencies and CAD grades were found to be correlated with aging (P < .05). DNA damage frequency and syntax score values were significantly (P < .05) higher in males compared to females. Syntax scores were correlated with aging (r = 0.348, P < .01), ECHO-EF (r = 0.374, P < .001), blood-urea (r = 0.398, P < .001), creatinine (r = 0.433, P < .001), glucose (0.218, P < .05), and HbA1c (r = 0.200, P < .05). Significant correlations were observed between trace elements and demographic values, blood parameters, diseases, angio parameters, ECHO, and ECG parameters. It was observed that the concentrations of trace elements detected in the blood were 93.4% correlated with each other. Lymphocyte DNA damage is a strong biomarker for the atherosclerotic indicator of CAD. Aging is an effective factor both in the DNA damage frequency and CAD risk index. Creatinine and urea are factors that have the power to change the CAD risk index and DNA damage frequency. The higher DNA damage and CAD risk were monitored in males compared to females. The relationship between some biomarkers and blood trace element concentrations showed that further studies are needed to more accurately evaluate the relationship between trace elements, DNA damage frequencies, and CAD.

Objective causal predictions from observational data.

Many recent articles in public health risk assessment have stated that causal conclusions drawn from observational data must rely on inherently untestable assumptions. They claim that such assumptions ultimately can only be evaluated by informed human judgments. We call this the subjective approach to causal interpretation of observational results. Its theoretical and conceptual foundation is a potential outcomes model of causation in which counterfactual outcomes cannot be observed. It risks depriving decision-makers and the public of the key benefits of traditional objective science, which invites scrutiny and independent verification through testable causal models and interventional hypotheses. We introduce an alternative objective approach to causal analysis of exposure-response relationships in observational data. This is designed to be more objective in the specific sense that it is independently verifiable (or refutable) and data-driven, requiring no inherently untestable assumptions. This approach uses empirically testable interventional causal models, specifically causal Bayesian networks (CBNs), instead of untestable potential outcomes models. It enables empirical validation of causal claims through Invariant Causal Prediction (ICP) tests across multiple studies. We explain how to use CBNs and individual conditional expectation (ICE) plots to quantify the effects on health risks of changing exposures while taking into account realistic complexities such as imperfectly controlled confounding, missing data, and measurement error. By ensuring that all causal assumptions are explicit and empirically testable, our framework may help to improve the reliability and transparency of causal inferences in health risk assessments.