Brain high-throughput multi-omics data reveal molecular heterogeneity in Alzheimer's disease.

Unbiased data-driven omic approaches are revealing the molecular heterogeneity of Alzheimer disease. Here, we used machine learning approaches to integrate high-throughput transcriptomic, proteomic, metabolomic, and lipidomic profiles with clinical and neuropathological data from multiple human AD cohorts. We discovered 4 unique multimodal molecular profiles, one of them showing signs of poor cognitive function, a faster pace of disease progression, shorter survival with the disease, severe neurodegeneration and astrogliosis, and reduced levels of metabolomic profiles. We found this molecular profile to be present in multiple affected cortical regions associated with higher Braak tau scores and significant dysregulation of synapse-related genes, endocytosis, phagosome, and mTOR signaling pathways altered in AD early and late stages. AD cross-omics data integration with transcriptomic data from an SNCA mouse model revealed an overlapping signature. Furthermore, we leveraged single-nuclei RNA-seq data to identify distinct cell-types that most likely mediate molecular profiles. Lastly, we identified that the multimodal clusters uncovered cerebrospinal fluid biomarkers poised to monitor AD progression and possibly cognition. Our cross-omics analyses provide novel critical molecular insights into AD.

Rapid tumor DNA analysis of cerebrospinal fluid accelerates treatment of central nervous system lymphoma.

ABSTRACT: Delays and risks associated with neurosurgical biopsies preclude timely diagnosis and treatment of central nervous system (CNS) lymphoma and other CNS neoplasms. We prospectively integrated targeted rapid genotyping of cerebrospinal fluid (CSF) into the evaluation of 70 patients with CNS lesions of unknown cause. Participants underwent genotyping of CSF-derived DNA using a quantitative polymerase chain reaction-based approach for parallel detection of single-nucleotide variants in the MYD88, TERT promoter, IDH1, IDH2, BRAF, and H3F3A genes within 80 minutes of sample acquisition. Canonical mutations were detected in 42% of patients with neoplasms, including cases of primary and secondary CNS lymphoma, glioblastoma, IDH-mutant brainstem glioma, and H3K27M-mutant diffuse midline glioma. Genotyping results eliminated the need for surgical biopsies in 7 of 33 cases (21.2%) of newly diagnosed neoplasms, resulting in significantly accelerated initiation of disease-directed treatment (median, 3 vs 12 days; P = .027). This assay was then implemented in a Clinical Laboratory Improvement Amendments environment, with 2-day median turnaround for diagnosis of CNS lymphoma from 66 patients across 4 clinical sites. Our study prospectively demonstrates that targeted rapid CSF genotyping influences oncologic management for suspected CNS tumors.

Genetic architecture of plasma Alzheimer disease biomarkers.

Genome-wide association studies (GWAS) of cerebrospinal fluid (CSF) Alzheimer's Disease (AD) biomarker levels have identified novel genes implicated in disease risk, onset and progression. However, lumbar punctures have limited availability and may be perceived as invasive. Blood collection is readily available and well accepted, but it is not clear whether plasma biomarkers will be informative for genetic studies. Here we perform genetic analyses on concentrations of plasma amyloid-ß peptides Aß40 (n = 1,467) and Aß42 (n = 1,484), Aß42/40 (n = 1467) total tau (n = 504), tau phosphorylated (p-tau181; n = 1079) and neurofilament light (NfL; n = 2,058). GWAS and gene-based analysis was used to identify single variant and genes associated with plasma levels. Finally, polygenic risk score and summary statistics were used to investigate overlapping genetic architecture between plasma biomarkers, CSF biomarkers and AD risk. We found a total of six genome-wide significant signals. APOE was associated with plasma Aß42, Aß42/40, tau, p-tau181 and NfL. We proposed 10 candidate functional genes on the basis of 12 single nucleotide polymorphism-biomarker pairs and brain differential gene expression analysis. We found a significant genetic overlap between CSF and plasma biomarkers. We also demonstrate that it is possible to improve the specificity and sensitivity of these biomarkers, when genetic variants regulating protein levels are included in the model. This current study using plasma biomarker levels as quantitative traits can be critical to identification of novel genes that impact AD and more accurate interpretation of plasma biomarker levels.

The relationship between freight train length and the risk of derailment.

In recent years, longer and heavier trains have become more common, primarily driven by efficiency and cost-saving measures in the railroad industry. Regulation of train length is currently under consideration in the United States at both the federal and state levels, because of concerns that longer trains may have a higher risk of derailment, but the relationship between train length and risk of derailment is not yet well understood. In this study, we use data on freight train accidents during the 2013-2022 period from the Federal Railroad Administration (FRA) Rail Equipment Accident and Highway-Rail Grade Crossing Accident databases to estimate the relationship between freight train length and the risk of derailment. We determine that longer trains do have a greater risk of derailment. Based on our analysis, running 100-car trains is associated with 1.11 (95% confidence interval: 1.10-1.12) times the derailment odds of running 50-car trains (or a 11% increase), even accounting for the fact that only half as many 100-car trains would need to run. For 200-car trains, the odds increase by 24% (odds ratio 1.24, 95% confidence interval: 1.20-1.28), again accounting for the need for fewer trains. Understanding derailment risk is an important component for evaluating the overall safety of the rail system and for the future development and regulation of freight rail transportation. Given the limitations of the current data on freight train length, this study provides an important step toward such an understanding.

Multi-ancestry GWAS reveals excitotoxicity associated with outcome after ischaemic stroke.

During the first hours after stroke onset, neurological deficits can be highly unstable: some patients rapidly improve, while others deteriorate. This early neurological instability has a major impact on long-term outcome. Here, we aimed to determine the genetic architecture of early neurological instability measured by the difference between the National Institutes of Health Stroke Scale (NIHSS) within 6 h of stroke onset and NIHSS at 24 h. A total of 5876 individuals from seven countries (Spain, Finland, Poland, USA, Costa Rica, Mexico and Korea) were studied using a multi-ancestry meta-analyses. We found that 8.7% of NIHSS at 24 h of variance was explained by common genetic variations, and also that early neurological instability has a different genetic architecture from that of stroke risk. Eight loci (1p21.1, 1q42.2, 2p25.1, 2q31.2, 2q33.3, 5q33.2, 7p21.2 and 13q31.1) were genome-wide significant and explained 1.8% of the variability suggesting that additional variants influence early change in neurological deficits. We used functional genomics and bioinformatic annotation to identify the genes driving the association from each locus. Expression quantitative trait loci mapping and summary data-based Mendelian randomization indicate that ADAM23 (log Bayes factor = 5.41) was driving the association for 2q33.3. Gene-based analyses suggested that GRIA1 (log Bayes factor = 5.19), which is predominantly expressed in the brain, is the gene driving the association for the 5q33.2 locus. These analyses also nominated GNPAT (log Bayes factor = 7.64) ABCB5 (log Bayes factor = 5.97) for the 1p21.1 and 7p21.1 loci. Human brain single-nuclei RNA-sequencing indicates that the gene expression of ADAM23 and GRIA1 is enriched in neurons. ADAM23, a presynaptic protein and GRIA1, a protein subunit of the AMPA receptor, are part of a synaptic protein complex that modulates neuronal excitability. These data provide the first genetic evidence in humans that excitotoxicity may contribute to early neurological instability after acute ischaemic stroke.

The Early-Onset Alzheimer's Disease Whole-Genome Sequencing Project: Study design and methodology.

INTRODUCTION: Sequencing efforts to identify genetic variants and pathways underlying Alzheimer's disease (AD) have largely focused on late-onset AD although early-onset AD (EOAD), accounting for ∼10% of cases, is largely unexplained by known mutations, resulting in a lack of understanding of its molecular etiology. METHODS: Whole-genome sequencing and harmonization of clinical, neuropathological, and biomarker data of over 5000 EOAD cases of diverse ancestries. RESULTS: A publicly available genomics resource for EOAD with extensive harmonized phenotypes. Primary analysis will (1) identify novel EOAD risk loci and druggable targets; (2) assess local-ancestry effects; (3) create EOAD prediction models; and (4) assess genetic overlap with cardiovascular and other traits. DISCUSSION: This novel resource complements over 50,000 control and late-onset AD samples generated through the Alzheimer's Disease Sequencing Project (ADSP). The harmonized EOAD/ADSP joint call will be available through upcoming ADSP data releases and will allow for additional analyses across the full onset range. HIGHLIGHTS: Sequencing efforts to identify genetic variants and pathways underlying Alzheimer's disease (AD) have largely focused on late-onset AD although early-onset AD (EOAD), accounting for ∼10% of cases, is largely unexplained by known mutations. This results in a significant lack of understanding of the molecular etiology of this devastating form of the disease. The Early-Onset Alzheimer's Disease Whole-genome Sequencing Project is a collaborative initiative to generate a large-scale genomics resource for early-onset Alzheimer's disease with extensive harmonized phenotype data. Primary analyses are designed to (1) identify novel EOAD risk and protective loci and druggable targets; (2) assess local-ancestry effects; (3) create EOAD prediction models; and (4) assess genetic overlap with cardiovascular and other traits. The harmonized genomic and phenotypic data from this initiative will be available through NIAGADS.

Metabolomic and lipidomic signatures in autosomal dominant and late-onset Alzheimer's disease brains.

INTRODUCTION: The identification of multiple genetic risk factors for Alzheimer's disease (AD) suggests that many pathways contribute to AD onset and progression. However, the metabolomic and lipidomic profiles in carriers of distinct genetic risk factors are not fully understood. The metabolome can provide a direct image of dysregulated pathways in the brain. METHODS: We interrogated metabolomic signatures in the AD brain, including carriers of pathogenic variants in APP, PSEN1, and PSEN2 (autosomal dominant AD; ADAD), APOE ɛ4, and TREM2 risk variant carriers, and sporadic AD (sAD). RESULTS: We identified 133 unique and shared metabolites associated with ADAD, TREM2, and sAD. We identified a signature of 16 metabolites significantly altered between groups and associated with AD duration. DISCUSSION: AD genetic variants show distinct metabolic perturbations. Investigation of these metabolites may provide greater insight into the etiology of AD and its impact on clinical presentation. HIGHLIGHTS: APP/PSEN1/PSEN2 and TREM2 variant carriers show distinct metabolic changes. A total of 133 metabolites were differentially abundant in AD genetic groups. ß-citrylglutamate is differentially abundant in autosomal dominant, TREM2, and sporadic AD. A 16-metabolite profile shows differences between Alzheimer's disease (AD) genetic groups. The identified metabolic profile is associated with duration of disease.

Genetic associations with age at dementia onset in the PSEN1 E280A Colombian kindred.

INTRODUCTION: Genetic associations with Alzheimer's disease (AD) age at onset (AAO) could reveal genetic variants with therapeutic applications. We present a large Colombian kindred with autosomal dominant AD (ADAD) as a unique opportunity to discover AAO genetic associations. METHODS: A genetic association study was conducted to examine ADAD AAO in 340 individuals with the PSEN1 E280A mutation via TOPMed array imputation. Replication was assessed in two ADAD cohorts, one sporadic early-onset AD study and four late-onset AD studies. RESULTS: 13 variants had p<1×10-7 or p<1×10-5 with replication including three independent loci with candidate associations with clusterin including near CLU. Other suggestive associations were identified in or near HS3ST1, HSPG2, ACE, LRP1B, TSPAN10, and TSPAN14. DISCUSSION: Variants with suggestive associations with AAO were associated with biological processes including clusterin, heparin sulfate, and amyloid processing. The detection of these effects in the presence of a strong mutation for ADAD reinforces their potentially impactful role.

Clarification of large-volume bacterial cultures using a centrifuge-free protocol.

AIMS: To provide a reliable, reproducible and centrifuge-free filtration protocol for clarification of large volumes of bacterial cultures. METHODS AND RESULTS: Four experiments were designed to compare different techniques enabling clarification of Escherichia coli cultures using as a benchmark the concentration and quality of bacterial outer membrane vesicles (OMVs). The experiments were designed to examine the performance of different extraction methods on large volume (≥1 L) filtrations of bacterial culture media. Performance parameters included filtration flow rates, sterility testing and characterization of the filtrates by: (i) SDS-PAGE, (ii) cryogenic transmission electron microscopy, (iii) nanoparticle tracking analysis and (iv) Qubit protein quantification. The experiments revealed that: (i) addition of the filter aid Diatomaceous Earth to the bacterial cultures improved filtration flow rates significantly and eliminated the need for centrifugation prior to filtration; (ii) sterile filtration was successful as no bacterial passage was identified through the membrane filter; (iii) centrifuge-free filtrates contained an increased amount of OMVs compared to centrifuged filtrates. CONCLUSIONS: In comparison to conventional centrifuge-based protocols, the clarification method presented has universal applicability for a broad range of microbial extraction procedures, regardless of the volume of culture harvested. Moreover, the decreased amount of OMVs presented in the filtrates following centrifugation step provides an additional argument in favour of a centrifuge-free approach. SIGNIFICANCE AND IMPACT OF THE STUDY: Sterile filtration is a universal method for the clarification of bacterial cultures. Common challenges related to filtration include filter clogging and long processing times, due to limited centrifugation capacity, which can affect product quality. The proposed protocol is likely to ensure a highly effective filtration process and could be a novel approach in improving the filtrate products without the need of centrifugation.

Schizophrenia Outside the Brain.

Schizophrenia is a multifactorial mental disorder, characterized by positive symptoms (delusions, hallucinations), negative symptoms (anhedonia, social withdraw), and cognitive symptoms (impairment of memory, learning, and executive functions). Despite the classic symptoms being related to the central nervous system, schizophrenia has been described by recent studies as a systemic disease, affecting other organs, tissues, and systems out of the brain. In this chapter, we summarize the main tissues and systems found affected in schizophrenic patients, both before and after antipsychotic administration. We offer an overview of the recent findings in the field about musculoskeletal system, metabolism, and immune system dysfunctions found in patients as well in models in vitro. We also discuss some of the side effects of certain antipsychotics often related to increased risk of comorbidities in patients with schizophrenia during the treatment.

The TMEM106B FTLD-protective variant, rs1990621, is also associated with increased neuronal proportion.

Apart from amyloid ß deposition and tau neurofibrillary tangles, Alzheimer's disease (AD) is a neurodegenerative disorder characterized by neuronal loss and astrocytosis in the cerebral cortex. The goal of this study is to investigate genetic factors associated with the neuronal proportion in health and disease. To identify cell-autonomous genetic variants associated with neuronal proportion in cortical tissues, we inferred cellular population structure from bulk RNA-Seq derived from 1536 individuals. We identified the variant rs1990621 located in the TMEM106B gene region as significantly associated with neuronal proportion (p value = 6.40 × 10-07) and replicated this finding in an independent dataset (p value = 7.41 × 10-04) surpassing the genome-wide threshold in the meta-analysis (p value = 9.42 × 10-09). This variant is in high LD with the TMEM106B non-synonymous variant p.T185S (rs3173615; r2 = 0.98) which was previously identified as a protective variant for frontotemporal lobar degeneration (FTLD). We stratified the samples by disease status, and discovered that this variant modulates neuronal proportion not only in AD cases, but also several neurodegenerative diseases and in elderly cognitively healthy controls. Furthermore, we did not find a significant association in younger controls or schizophrenia patients, suggesting that this variant might increase neuronal survival or confer resilience to the neurodegenerative process. The single variant and gene-based analyses also identified an overall genetic association between neuronal proportion, AD and FTLD risk. These results suggest that common pathways are implicated in these neurodegenerative diseases, that implicate neuronal survival. In summary, we identified a protective variant in the TMEM106B gene that may have a neuronal protection effect against general aging, independent of disease status, which could help elucidate the relationship between aging and neuronal survival in the presence or absence of neurodegenerative disorders. Our findings suggest that TMEM106B could be a potential target for neuronal protection therapies to ameliorate cognitive and functional deficits.

Overlap in the Genetic Architecture of Stroke Risk, Early Neurological Changes, and Cardiovascular Risk Factors.

Background and Purpose- The genetic relationships between stroke risk, stroke severity, and early neurological changes are complex and not completely understood. Genetic studies have identified 32 all stroke risk loci. Polygenic risk scores can be used to compare the genetic architecture of related traits. In this study, we compare the genetic architecture of stroke risk, stroke severity, and early neurological changes with that of 2 stroke risk factors: type 2 diabetes mellitus (T2DM) and hypertension. Methods- We assessed the degree of overlap in the genetic architecture of stroke risk, T2DM, hypertension, and 2 acute stroke phenotypes based on the National Institutes of Health Stroke Scale (NIHSS), which ranges from 0 for no stroke symptoms to 21 to 42 for a severe stroke: baseline (within 6 hours after onset) and change in NIHSS (ΔNIHSS=NIHSS at baseline-NIHSS at 24 hours). This was done by (1) single-nucleotide polymorphism by single-nucleotide polymorphism comparison, (2) weighted polygenic risk scores with sentinel variants, and (3) whole-genome polygenic risk scores using multiple P thresholds. Results- We found evidence of genetic architecture overlap between stroke risk and T2DM ( P=2.53×10-169), hypertension ( P=3.93×10-04), and baseline NIHSS ( P=0.03). However, there was no evidence of overlap between ΔNIHSS and stroke risk, T2DM, or hypertension. Conclusions- The genetic architecture of stroke risk is correlated with that of T2DM, hypertension, and initial stroke severity (NIHSS within 6 hours of stroke onset). However, the genetic architecture of early neurological change after stroke (ΔNIHSS) is not correlated with that of ischemic stroke risk, T2DM, or hypertension. Thus, stroke risk and early neurological change after stroke have distinct genetic architectures.

Energy-Degeneracy-Driven Covalency in Actinide Bonding.

Evaluating the nature of chemical bonding for actinide elements represents one of the most important and long-standing problems in actinide science. We directly address this challenge and contribute a Cl K-edge X-ray absorption spectroscopy and relativistic density functional theory study that quantitatively evaluates An-Cl covalency in AnCl62- (AnIV = Th, U, Np, Pu). The results showed significant mixing between Cl 3p- and AnIV 5f- and 6d-orbitals (t1u*/t2u* and t2 g*/eg *), with the 6d-orbitals showing more pronounced covalent bonding than the 5f-orbitals. Moving from Th to U, Np, and Pu markedly changed the amount of M-Cl orbital mixing, such that AnIV 6d - and Cl 3p-mixing decreased and metal 5f - and Cl 3p-orbital mixing increased across this series.

Plate-based diversity subset screening generation 2: an improved paradigm for high-throughput screening of large compound files.

High-throughput screening (HTS) is an effective method for lead and probe discovery that is widely used in industry and academia to identify novel chemical matter and to initiate the drug discovery process. However, HTS can be time consuming and costly and the use of subsets as an efficient alternative to screening entire compound collections has been investigated. Subsets may be selected on the basis of chemical diversity, molecular properties, biological activity diversity or biological target focus. Previously, we described a novel form of subset screening: plate-based diversity subset (PBDS) screening, in which the screening subset is constructed by plate selection (rather than individual compound cherry-picking), using algorithms that select for compound quality and chemical diversity on a plate basis. In this paper, we describe a second-generation approach to the construction of an updated subset: PBDS2, using both plate and individual compound selection, that has an improved coverage of the chemical space of the screening file, whilst only selecting the same number of plates for screening. We describe the validation of PBDS2 and its successful use in hit and lead discovery. PBDS2 screening became the default mode of singleton (one compound per well) HTS for lead discovery in Pfizer.

Prognosis and patterns of failure for the extubation of patients who remain intubated after head and neck surgery.

OBJECTIVE: This study aimed to analyze the rate of failure, patterns of failure, and prognostic factors for patients who remain intubated after head and neck surgery and then undergo delayed extubation. METHODS: Retrospective chart review of all otolaryngology patients who remained intubated after head and neck surgery and then underwent delayed extubation between 2006 and 2013. The incidence and patterns of extubation failure were analyzed. Univariable logistic regression analysis was performed to identify risk factors for postextubation failure. RESULTS: Fifteen of the 129 patients (12%) who remained intubated after head and neck surgery and underwent delayed extubation subsequently failed and required either repeat intubation or an emergency surgical airway. The most common reasons for failure were hemorrhage (47%) and upper airway edema (33%). Failure typically occurred within 6 hours of extubation. Twenty-seven percent of the patients who failed extubation (4/15) required an emergency surgical airway. On univariable logistic regression analysis, ligation of a major neck vessel predicted extubation failure (odds ratio=5.20; 95% confidence interval, 1.48-18.23). CONCLUSION: Postextubation failure in carefully selected patients undergoing delayed extubation after head and neck surgery is infrequent and most commonly due to postoperative bleeding. Prospective data are required to facilitate safe and quality care for these patients.

Radical surgical resection with molecular margins is associated with improved survival in IDH wild-type glioblastoma.

BACKGROUND: Survival is variable in patients with glioblastoma IDH wild-type (GBM), even after comparable surgical resection of radiographically detectable disease, highlighting the limitations of radiographic assessment of infiltrative tumor anatomy. The majority of postsurgical progressive events are failures within 2 cm of the resection margin, motivating supramaximal resection strategies to improve local control. However, which patients benefit from such radical resections remains unknown. METHODS: We developed a predictive model to identify which IDH wild-type GBMs are amenable to radiographic gross-total resection (GTR). We then investigated whether GBM survival heterogeneity following GTR is correlated with microscopic tumor burden by analyzing tumor cell content at the surgical margin with a rapid qPCR-based method for detection of TERT promoter mutation. RESULTS: Our predictive model for achievable GTR, developed on retrospective radiographic and molecular data of GBM patients undergoing resection, had an area under the curve of 0.83, sensitivity of 62%, and specificity of 90%. Prospective analysis of this model in 44 patients found that 89% of patients were correctly predicted to achieve a residual volume (RV) < 4.9cc. Of the 44 prospective patients undergoing rapid qPCR TERT promoter mutation analysis at the surgical margin, 7 had undetectable TERT mutation, of which 5 also had a GTR (RV < 1cc). In these 5 patients at 30 months follow-up, 75% showed no progression, compared to 0% in the group with TERT mutations detected at the surgical margin (P = .02). CONCLUSIONS: These findings identify a subset of patients with GBM that may derive local control benefits from radical resection to undetectable molecular margins.

Novel early-onset Alzheimer-associated genes influence risk through dysregulation of glutamate, immune activation, and intracell signaling pathways.

Alzheimer Disease (AD) is a highly polygenic disease that presents with relatively earlier onset (≤70yo; EOAD) in about 5% of cases. Around 90% of these EOAD cases remain unexplained by pathogenic mutations. Using data from EOAD cases and controls, we performed a genome-wide association study (GWAS) and trans-ancestry meta-analysis on non-Hispanic Whites (NHW, NCase=6,282, NControl=13,386), African Americans (AA NCase=782, NControl=3,663) and East Asians (NCase=375, NControl=838 CO). We identified eight novel significant loci: six in the ancestry-specific analyses and two in the trans-ancestry analysis. By integrating gene-based analysis, eQTL, pQTL and functional annotations, we nominate four novel genes that are involved in microglia activation, glutamate production, and signaling pathways. These results indicate that EOAD, although sharing many genes with LOAD, harbors unique genes and pathways that could be used to create better prediction models or target identification for this type of AD.

Glioblastoma-Infiltrating CD8+ T Cells Are Predominantly a Clonally Expanded GZMK+ Effector Population.

Recent clinical trials have highlighted the limited efficacy of T cell-based immunotherapy in patients with glioblastoma (GBM). To better understand the characteristics of tumor-infiltrating lymphocytes (TIL) in GBM, we performed cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing with paired V(D)J sequencing, respectively, on TILs from two cohorts of patients totaling 15 patients with high-grade glioma, including GBM or astrocytoma, IDH-mutant, grade 4 (G4A). Analysis of the CD8+ TIL landscape reveals an enrichment of clonally expanded GZMK+ effector T cells in the tumor compared with matched blood, which was validated at the protein level. Furthermore, integration with other cancer types highlights the lack of a canonically exhausted CD8+ T-cell population in GBM TIL. These data suggest that GZMK+ effector T cells represent an important T-cell subset within the GBM microenvironment and may harbor potential therapeutic implications. SIGNIFICANCE: To understand the limited efficacy of immune-checkpoint blockade in GBM, we applied a multiomics approach to understand the TIL landscape. By highlighting the enrichment of GZMK+ effector T cells and the lack of exhausted T cells, we provide a new potential mechanism of resistance to immunotherapy in GBM. This article is featured in Selected Articles from This Issue, p. 897.

Covalency in metal-oxygen multiple bonds evaluated using oxygen K-edge spectroscopy and electronic structure theory.

Advancing theories of how metal-oxygen bonding influences metal oxo properties can expose new avenues for innovation in materials science, catalysis, and biochemistry. Historically, spectroscopic analyses of the transition metal MO(4)(x-) anions have formed the basis for new M-O bonding theories. Herein, relative changes in M-O orbital mixing in MO(4)(2-) (M = Cr, Mo, W) and MO(4)(-) (M = Mn, Tc, Re) are evaluated for the first time by nonresonant inelastic X-ray scattering, X-ray absorption spectroscopy using fluorescence and transmission (via a scanning transmission X-ray microscope), and time-dependent density functional theory. The results suggest that moving from Group 6 to Group 7 or down the triads increases M-O e* (π*) mixing; for example, it more than doubles in ReO(4)(-) relative to CrO(4)(2-). Mixing in the t(2)* orbitals (σ* + π*) remains relatively constant within the same Group, but increases on moving from Group 6 to Group 7. These unexpected changes in orbital energy and composition for formally isoelectronic tetraoxometalates are evaluated in terms of periodic trends in d orbital energy and radial extension.