A toolkit for stroke infarct volume estimation in rodents.

Stroke volume is a key determinant of infarct severity and an important metric for evaluating treatments. However, accurate estimation of stroke volume can be challenging, due to the often confined 2-dimensional nature of available data. Here, we introduce a comprehensive semi-automated toolkit to reliably estimate stroke volumes based on (1) whole brains ex-vivo magnetic resonance imaging (MRI) and (2) brain sections that underwent immunofluorescence staining. We located and quantified infarct areas from MRI three days (acute) and 28 days (chronic) after photothrombotic stroke induction in whole mouse brains. MRI results were compared with measures obtained from immunofluorescent histologic sections of the same brains. We found that infarct volume determined by post-mortem MRI was highly correlated with a deviation of only 6.6 % (acute) and 4.9 % (chronic) to the measurements as determined in the histological brain sections indicating that both methods are capable of accurately assessing brain tissue damage (Pearson r > 0.9, p < 0.001). The Dice similarity coefficient (DC) showed a high degree of coherence (DC > 0.8) between MRI-delineated regions of interest (ROIs) and ROIs obtained from histologic sections at four to six pre-defined landmarks, with histology-based delineation demonstrating higher inter-operator similarity compared to MR images. We further investigated stroke-related scarring and post-ischemic angiogenesis in cortical peri­infarct regions and described a negative correlation between GFAP+fluorescence intensity and MRI-obtained lesion size.

Association of central blood pressure with an exaggerated blood pressure response to exercise among elite athletes.

PURPOSE: The systolic blood pressure/workload (SBP/MET) slope was recently reported to be a reliable parameter to identify an exaggerated blood pressure response (eBPR) in the normal population and in athletes. However, it is unclear whether an eBPR correlates with central blood pressure (CBP) and vascular function in elite athletes. METHODS: We examined 618 healthy male elite athletes (age 25.8 ± 5.1 years) of mixed sports with a standardized maximum exercise test. CBP and vascular function were measured non-invasively with a validated oscillometric device. The SBP/MET slope was calculated and the threshold for an eBPR was set at > 6.2 mmHg/MET. Two groups were defined according to ≤ 6.2 and > 6.2 mmHg/MET, and associations of CBP and vascular function with the SBP/MET slope were compared for each group. RESULTS: Athletes with an eBPR (n = 180, 29%) displayed a significantly higher systolic CBP (102.9 ± 7.5 vs. 100 ± 7.7 mmHg, p = 0.001) but a lower absolute (295 ± 58 vs. 384 ± 68 W, p < 0.001) and relative workload (3.14 ± 0.54 vs. 4.27 ± 1.1 W/kg, p < 0.001) compared with athletes with a normal SBP/MET slope (n = 438, 71%). Systolic CBP was positively associated with the SBP/MET slope (r = 0.243, p < 0.001). In multiple logistic regression analyses, systolic CBP (odds ratio [OR] 1.099, 95% confidence interval [CI] 1.045-1.155, p < 0.001) and left atrial volume index (LAVI) (OR 1.282, CI 1.095-1.501, p = 0.002) were independent predictors of an eBPR. CONCLUSION: Systolic CBP and LAVI were independent predictors of an eBPR. An eBPR was further associated with a lower performance level, highlighting the influence of vascular function on the BPR and performance of male elite athletes.

Association of cardiorespiratory fitness level with vascular function and subclinical atherosclerosis in the elderly.

PURPOSE: Physical exercise is crucial for healthy aging and plays a decisive role in the prevention of atherosclerotic cardiovascular disease (ASCVD). A higher level of cardiorespiratory fitness (CRF) in the elderly is associated with lower cardiovascular and all-cause mortality. This study investigated the association of CRF level with vascular function and cardiovascular risk factors in the elderly. METHODS: We examined 79 apparently healthy and physically active subjects aged > 55 years (64 ± 4 years). Cardiovascular functional parameters assessed included brachial and central blood pressure (BP), pulse wave velocity (PWV), augmentation index (Aix), and ankle-brachial index. Sonography of the common carotid artery was performed. CRF level was determined by a cardiopulmonary exercise test, and everyday activity was quantified with an accelerometer. RESULTS: All participants had a higher CRF level than the reported age-specific normative values. Twenty-nine subjects had subclinical atherosclerosis of the common carotid artery. Compared with participants without atherosclerosis, they were older (p = 0.007), displayed higher brachial systolic BP (p = 0.006), and higher central systolic BP (p = 0.014). Lower brachial (p = 0.036) and central (p = 0.003) systolic BP, lower PWV (p = 0.004), lower Aix (p < 0.001), lower body fat percentage (< 0.001), and lower LDL cholesterol (p = 0.005) were associated with a higher CRF level. CONCLUSIONS: In this cohort of healthy and physically active individuals, subjects with subclinical atherosclerosis displayed higher systolic brachial and central BP. A higher CRF level was associated with enhanced vascular function, consistent with an influence of CRF on both BP and vascular function in the elderly.

The vascular gene Apold1 is dispensable for normal development but controls angiogenesis under pathological conditions.

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1-/- mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1-/- mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.

Stimulation of the cuneiform nucleus enables training and boosts recovery after spinal cord injury.

Severe spinal cord injuries result in permanent paraparesis in spite of the frequent sparing of small portions of white matter. Spared fibre tracts are often incapable of maintaining and modulating the activity of lower spinal motor centres. Effects of rehabilitative training thus remain limited. Here, we activated spared descending brainstem fibres by electrical deep brain stimulation of the cuneiform nucleus of the mesencephalic locomotor region, the main control centre for locomotion in the brainstem, in adult female Lewis rats. We show that deep brain stimulation of the cuneiform nucleus enhances the weak remaining motor drive in highly paraparetic rats with severe, incomplete spinal cord injuries and enables high-intensity locomotor training. Stimulation of the cuneiform nucleus during rehabilitative aquatraining after subchronic (n = 8 stimulated versus n = 7 unstimulated versus n = 7 untrained rats) and chronic (n = 14 stimulated versus n = 9 unstimulated versus n = 9 untrained rats) spinal cord injury re-established substantial locomotion and improved long-term recovery of motor function. We additionally identified a safety window of stimulation parameters ensuring context-specific locomotor control in intact rats (n = 18) and illustrate the importance of timing of treatment initiation after spinal cord injury (n = 14). This study highlights stimulation of the cuneiform nucleus as a highly promising therapeutic strategy to enhance motor recovery after subchronic and chronic incomplete spinal cord injury with direct clinical applicability.

Deep learning-based behavioral profiling of rodent stroke recovery.

BACKGROUND: Stroke research heavily relies on rodent behavior when assessing underlying disease mechanisms and treatment efficacy. Although functional motor recovery is considered the primary targeted outcome, tests in rodents are still poorly reproducible and often unsuitable for unraveling the complex behavior after injury. RESULTS: Here, we provide a comprehensive 3D gait analysis of mice after focal cerebral ischemia based on the new deep learning-based software (DeepLabCut, DLC) that only requires basic behavioral equipment. We demonstrate a high precision 3D tracking of 10 body parts (including all relevant joints and reference landmarks) in several mouse strains. Building on this rigor motion tracking, a comprehensive post-analysis (with >100 parameters) unveils biologically relevant differences in locomotor profiles after a stroke over a time course of 3 weeks. We further refine the widely used ladder rung test using deep learning and compare its performance to human annotators. The generated DLC-assisted tests were then benchmarked to five widely used conventional behavioral set-ups (neurological scoring, rotarod, ladder rung walk, cylinder test, and single-pellet grasping) regarding sensitivity, accuracy, time use, and costs. CONCLUSIONS: We conclude that deep learning-based motion tracking with comprehensive post-analysis provides accurate and sensitive data to describe the complex recovery of rodents following a stroke. The experimental set-up and analysis can also benefit a range of other neurological injuries that affect locomotion.

Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications.

BACKGROUND: Currently, there is no regenerative therapy for patients with neurological and neurodegenerative disorders. Cell-therapies have emerged as a potential treatment for numerous brain diseases. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. METHODS: We generated good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. NPCs were characterized in vitro for their differentiation potential and in vivo after transplantation into wild type as well as genetically immunosuppressed mice. RESULTS: Generated NPCs had a stable gene-expression over at least 15 passages and could be scaled for up to 1018 cells per initially seeded 106 cells. After withdrawal of growth factors in vitro, cells adapted a neural fate and mainly differentiated into active neurons. To ensure a pure NPC population for in vivo applications, we reduced the risk of iPSC contamination by applying micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted NPCs have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month, while no residual pluripotent cells were detectable. CONCLUSION: We describe the generation of transgene- and xeno-free NPCs. This simple differentiation protocol combined with the ability of in vivo cell tracking presents a valuable tool to develop safe and effective cell therapies for various brain injuries.

Nogo-A targeted therapy promotes vascular repair and functional recovery following stroke.

Stroke is a major cause of serious disability due to the brain's limited capacity to regenerate damaged tissue and neuronal circuits. After ischemic injury, a multiphasic degenerative and inflammatory response is coupled with severely restricted vascular and neuronal repair, resulting in permanent functional deficits. Although clinical evidence indicates that revascularization of the ischemic brain regions is crucial for functional recovery, no therapeutics that promote angiogenesis after cerebral stroke are currently available. Besides vascular growth factors, guidance molecules have been identified to regulate aspects of angiogenesis in the central nervous system (CNS) and may provide targets for therapeutic angiogenesis. In this study, we demonstrate that genetic deletion of the neurite outgrowth inhibitor Nogo-A or one of its corresponding receptors, S1PR2, improves vascular sprouting and repair and reduces neurological deficits after cerebral ischemia in mice. These findings were reproduced in a therapeutic approach using intrathecal anti-Nogo-A antibodies; such a therapy is currently in clinical testing for spinal cord injury. These results provide a basis for a therapeutic blockage of inhibitory guidance molecules to improve vascular and neural repair after ischemic CNS injuries.

An Assessment of the 10-Item Mental Health Recovery Measure in a Predominantly African American Sample of Adults with Serious Mental Illness.

Study objectives were to 1) assess the reliability and validity of the 10-item Mental Health Recovery Measure (MHRM-10) in sample of predominately African American participants with serious mental illness, and 2) evaluate differences in MHRM-10 scores between the present sample and two other samples of persons with serious mental illness with different racial compositions. Participants included 230 adults (85.7% African American) with chart diagnoses of schizophrenia-spectrum, bipolar-spectrum, and major depressive disorders receiving services from community mental health centers in Detroit, Michigan. In addition to the MHRM-10, participants completed measures of psychological symptoms (Brief Symptom Inventory (BSI)- General Severity Index (GSI) and depression subscale), well-being (12-Item World Health Organization Disability Assessment Schedule 2.0; WHODAS 2.0), and stress-related growth (Stress-Related Growth Scale - Short Form; SRGS-SF). Internal consistency and convergent validity of the MHRM-10 were examined. Differences in MHRM-10 scores between the present sample and other samples were characterized by effect sizes. The MHRM-10 demonstrated excellent internal consistency. Evidence for convergent validity of the MHRM-10 included moderate correlations with the BSI-GSI, BSI-depression subscale, SRGS-SF, and WHODAS 2.0. The present sample of predominately African American participants showed higher MHRM-10 scores than two other samples with smaller proportions of African American participants. The MHRM-10 demonstrates excellent internal consistency and good convergent validity among African Americans with serious mental illness. Although findings are promising, studies should further assess the psychometric properties of the MHRM-10 in African American samples. Additional research that examines racial differences in mental health recovery is needed.

Amyloid precursor protein dimerization and synaptogenic function depend on copper binding to the growth factor-like domain.

Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function.

Dataset on stroke infarct volume in rodents: A comparison of MRI and histological methods.

This dataset offers images of mouse brains impacted by photothrombotic stroke in the sensorimotor cortex published by Weber et al. NeuroImage (2024). Data is gathered using two primary techniques: (1) whole-brain ex-vivo magnetic resonance imaging (MRI) and (2) 40 µm thick coronal histological sections that undergo immunofluorescence staining with NeuroTrace. Infarct areas and volumes are assessed through MRI at two distinct time frames-three days (acute) and 28 days (chronic) following photothrombotic stroke induction. Subsequently, the brains are sectioned into 40 µm thick coronal slices, stained with NeuroTrace, and imaged as whole sections. The dataset holds considerable value for reuse, particularly for researchers focused on stroke volume estimation methods as well as those interested in comparing the efficacy of MRI and histological techniques.

Beneath the radar: immune-evasive cell sources for stroke therapy.

Stem cell therapy is an emerging treatment paradigm for stroke patients with remaining neurological deficits. While allogeneic cell transplants overcome the manufacturing constraints of autologous grafts, they can be rejected by the recipient's immune system, which identifies foreign cells through the human leukocyte antigen (HLA) system. The heterogeneity of HLA molecules in the human population would require a very high number of cell lines, which may still be inadequate for patients with rare genetic HLAs. Here, we outline key progress in genetic HLA engineering in pluripotent stem and derived cells to evade the host's immune system, reducing the number of allogeneic cell lines required, and examine safety measures explored in both preclinical studies and upcoming clinical trials.

Molecular and anatomical roadmap of stroke pathology in immunodeficient mice.

Background: Stroke remains a leading cause of disability and death worldwide. It has become apparent that inflammation and immune mediators have a pre-dominant role in initial tissue damage and long-term recovery. Still, different immunosuppressed mouse models are necessary in stroke research e.g., to evaluate therapies using human cell grafts. Despite mounting evidence delineating the importance of inflammation in the stroke pathology, it is poorly described to what extent immune deficiency influences overall stroke outcome. Methods: Here, we assessed the stroke pathology of popular genetic immunodeficient mouse models, i.e., NOD scid gamma (NSG) and recombination activating gene 2 (Rag2-/-) mice as well as pharmacologically immunosuppressed mice and compared them to immune competent, wildtype (WT) C57BL/6J mice three weeks after injury. We performed histology, gene expression, blood serum and behavioural analysis to identify the impact of immunosuppression on stroke progression. Results: We detected changes in microglia activation/macrophage infiltration, scar-forming and vascular repair in immune-suppressed mice three weeks after injury. Transcriptomic analysis of stroked tissue revealed the strongest deviation from WT was observed in NSG mice affecting immunological and angiogenic pathways. Pharmacological immunosuppression resulted in the least variation in gene expression compared with the WT. These anatomical and genetic changes did not affect functional recovery in a time course of three weeks. To determine whether timing of immunosuppression is critical, we compared mice with acute and delayed pharmacological immunosuppression after stroke. Mice with delayed immunosuppression (7d) showed increased inflammatory and scarring responses compared to animals acutely treated with tacrolimus, thus more closely resembling WT pathology. Transplantation of human cells in the brains of immunosuppressed mice led to prolonged cell survival in all immunosuppressed mouse models, which was most consistent in NSG and Rag2-/- mice. Conclusions: We detected distinct anatomical and molecular changes in the stroke pathology between individual immunosuppressed mouse models that should be considered when selecting an appropriate mouse model for stroke research.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain.

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Nutraceutical use in late-stage cancer.

Access to a wealth of information on the internet has led many cancer patients to use complementary methods as an adjunct to traditional therapy for cancer, with, and more often, without informing their primary caregiver. Of the common complementary modalities, the use of dietary supplements appears to be highly prevalent in patients in active treatment for cancer, and later in cancer survivors. Emerging research suggests that some plant-based agents may, indeed, impact late-stage cancer, influencing molecular processes corrupted by tumor cells to evade detection, expand clonally, and invade surrounding tissues. The intent of this article is to review some of the current science underpinning the use of nutraceuticals in the latter stages of cancer.

Empirical correlation methods for temporary anions.

A temporary anion is a short-lived radical anion that decays through electron autodetachment into a neutral molecule and a free electron. The energies of these metastable species are often predicted using empirical correlation methods because ab initio predictions are computationally very expensive. Empirical correlation methods can be justified in the framework of Weisskopf-Fano-Feshbach theory but tend to work well only within closely related families of molecules or within a restricted energy range. The reason for this behavior can be understood using an alternative theoretical justification in the framework of the Hazi-Taylor stabilization method, which suggests that the empirical parameters do not so much correct for the coupling of the computed state to the continuum but for electron correlation effects and that therefore empirical correlation methods can be improved by using more accurate electronic structure methods to compute the energy of the confined electron. This idea is tested by choosing a heterogeneous reference set of temporary states and comparing empirical correlation schemes based on Hartree-Fock orbital energies, Kohn-Sham orbital energies, and attachment energies computed with the equation-of-motion coupled-cluster method. The results show that using more reliable energies for the confined electron indeed enhances the predictive power of empirical correlation schemes and that useful correlations can be established beyond closely related families of molecules. Certain types of σ* states are still problematic, and the reasons for this behavior are analyzed. On the other hand, preliminary results suggest that the new scheme can even be useful for predicting energies of bound anions at a fraction of the computational cost of reliable ab initio calculations. It is then used to make predictions for bound and temporary states of the furantrione and croconic acid radical anions.

Astrocytes for brain repair: More than just a neuron's sidekick.

Transplantation of glial enriched progenitors provides therapeutic effects on axonal damage, cognitive and motor function following white matter stroke.

Physiological Health and Survival of Captive-Reared and Released Juvenile Blanding's Turtles.

AbstractConservation translocations are important in maintaining viable wildlife populations of vulnerable species within their indigenous ranges. To be effective, population restoration efforts (e.g., head start programs) must consider the species' life history, regional ecology, and physiology and the health status of wild and translocated populations. The decline of Blanding's turtles (Emydoidea blandingii) has prompted the initiation of head start programs, but the health and short-term survival of head-started juveniles released to the wild is largely unknown. From May to October 2016 and 2017, we radio tracked captive-reared, recently released juvenile Blanding's turtles and monitored their survivorship and monthly physiological health. We aimed to (1) compare physiological metrics of juveniles before and after release from captivity and between head-started cohorts, (2) identify seasonal trends in physiological metrics of recently released juveniles, (3) compare physiological metrics of recently released and formerly released juveniles, and (4) identify predictors of juvenile survivorship after release from captivity. Juvenile short-term survival was low compared with other studies. Most physiological metrics did not change after release from captivity, negating significant juvenile stress before or after release. Physiological metrics for recently released cohorts varied seasonally, suggesting that these juveniles were likely in good health. Some physiological metrics differed between recently released and formerly released juveniles, demonstrating a potential postrelease acclimatization period. Finally, no physiological metrics significantly predicted survival, but surviving juveniles had a higher percentage of fat. In all, juvenile deaths were not due to poor turtle health but rather to predation from human-subsidized mesocarnivores. Therefore, head-started juvenile Blanding's turtles released in suburban areas may benefit from antipredator training and mesocarnivore control at release sites.

Blood Pressure Response and Vascular Function of Professional Athletes and Controls.

Workload-indexed blood pressure response (wiBPR) to exercise has been shown to be superior to peak systolic blood pressure (SBP) in predicting mortality in healthy men. Thus far, however, markers of wiBPR have not been evaluated for athletes and the association with vascular function is unclear. We examined 95 male professional athletes (26±5 y) and 30 male controls (26±4 y). We assessed vascular functional parameters at rest and wiBPR with a graded bicycle ergometer test and compared values for athletes with those of controls. Athletes had a lower pulse wave velocity (6.4±0.9 vs. 7.2±1.5 m/s, p=0.001) compared to controls. SBP/Watt slope (0.34±0.13 vs. 0.44±0.12 mmHg/W), SBP/MET slope (6.2±1.8 vs. 7.85±1.8 mmHg/MET) and peak SBP/Watt ratio (0.61±0.12 vs. 0.95±0.17 mmHg/W) were lower in athletes than in controls (p<0.001). The SBP/Watt and SBP/MET slope in athletes were comparable to the reference values, whereas the peak SBP/Watt-ratio was lower. All vascular functional parameters measured were not significantly correlated to the wiBPR in either athletes or controls. In conclusion, our findings indicate the potential use of the SBP/Watt and SBP/MET slope in pre-participation screening of athletes. Further, vascular functional parameters, measured at rest, were unrelated to the wiBPR in athletes and controls.

NMR, IR/Raman, and structural properties in HNO and RNO (R = alkyl and aryl) metalloporphyrins with implication for the HNO-myoglobin complex.

Structural and functional details of heme protein complexes with HNO and the isoelectronic RNO (R = alkyl and aryl) molecules (metabolic intermediates) are largely unknown. We report a quantum chemical investigation of three characteristic spectroscopic properties, (1)H and (15)N NMR chemical shifts and NO vibrational frequencies in synthetic HNO and RNO heme complexes, with theory-versus-experiment correlation coefficients R(2) = 0.990-0.998. A new density functional theory (DFT) method was found to yield excellent predictions of experimental structures of HNO, RNO, and NO heme systems. Interestingly, this method also helps the identification of an excellent linear quantitative structure observable relationship between NO vibrational frequencies and bond lengths in all of these NO-containing systems. This suggests that NO vibrations are largely local effects of the NO bonds in these complexes and may help deduce the NO bond lengths from using experimental vibrational data in these systems. The NO vibrational frequencies in HNO, RNO, and NO metalloporphyrins were found to follow a general trend of NO > RNO > HNO complexes, as a result of the electron populations in the antibonding NO orbitals of NO < RNO < HNO complexes. Investigations of the NMR and IR/Raman spectroscopic data in HNO metal complexes show that HNO is a strong pi-acid. In addition, we performed the first quantum chemical investigation of the hydrogen-bond effect on HNO in MbHNO (Mb = myoglobin) models. On the basis of comparisons with experimental (1)H and (15)N NMR results and NO vibrational frequency in MbHNO, a dual hydrogen-bond mode for HNO in MbHNO was proposed. The enhanced stability from this dual hydrogen bonding may provide a basis for the unusual stability of MbHNO observed experimentally. These results should facilitate spectroscopic characterizations and structural investigations of HNO and RNO heme proteins and models.