Antibiotics, Sedatives, and Catecholamines Further Compromise Sepsis-Induced Immune Suppression in Peripheral Blood Mononuclear Cells.

OBJECTIVES: We hypothesized that the immunosuppressive effects associated with antibiotics, sedatives, and catecholamines amplify sepsis-associated immune suppression through mitochondrial dysfunction, and there is a cumulative effect when used in combination. We thus sought to determine the impact of the exemplar drugs ciprofloxacin, propofol, and norepinephrine, used alone and in combination, at clinically relevant concentrations, on the ex vivo functionality of peripheral blood mononuclear cells (PBMCs) drawn from healthy, infected, and septic individuals. DESIGN: In vitro/ex vivo investigation. SETTING: University laboratory. SUBJECTS: Healthy volunteers, infected (nonseptic) patients in the emergency department, and septic ICU patients. INTERVENTIONS: PBMCs were isolated from these subjects and treated with ciprofloxacin (100 µg/mL), propofol (50 µg/mL), norepinephrine (10 µg/mL), or all three drugs combined, with and without lipopolysaccharide (100 ng/mL) for 6 or 24 hours. Comparison was made between study groups and against untreated cells. Measurements were made of cell viability, cytokine production, phagocytosis, human leukocyte antigen-DR (HLA-DR) status, mitochondrial membrane potential, mitochondrial reactive oxygen species production, and oxygen consumption. Gene expression in immune and metabolic pathways was investigated in PBMCs sampled from healthy volunteers coincubated with septic serum. MEASUREMENTS AND RESULTS: Coincubation with each of the drugs reduced cytokine production and phagocytosis in PBMCs isolated from septic patients, and healthy volunteers coincubated with septic serum. No effect was seen on HLA-DR surface expression. No cumulative effects were seen with the drug combination. Sepsis-induced changes in gene expression and mitochondrial functionality were not further affected by addition of any of the drugs. CONCLUSION: Drugs commonly used in critical care lead to significant immune dysfunction ex vivo and enhance sepsis-associated immunosuppression. Further studies are required to identify underlying mechanisms and potential impact on patient outcomes.

Clinical and molecular evidence of accelerated ageing following very preterm birth.

BACKGROUND: The mechanisms responsible for the associations between very preterm birth and a higher risk of poor cardiovascular and metabolic health in adult life are unknown. METHODS: Here, we compare the clinical and molecular phenotypes of healthy, normal-weight young adults (18-27 years), born very preterm (<33 weeks gestational age (GA)) and at full-term (37-42 weeks GA). Outcomes included whole-body MRI, hepatic and muscle 1H MRS, blood pressure measurements and telomere length. RESULTS: We recruited 156 volunteers, 69 preterm (45 women; 24 men) and 87 born at full-term (45 women; 42 men). Preterm individuals had a significantly altered blood pressure profile, including higher systolic blood pressure (SBP mmHg: preterm men 133.4 ± 10.1, term men 23.0 ± 6.9; preterm women 124.3 ± 7.1, term women 118.4 ± 8.0, p < 0.01 for all). Furthermore, preterm men had fewer long telomeres (145-48.5 kb: preterm men 14.1 ± 0.9%, term men 17.8 ± 1.1%, p < 0.05; 48.5-8.6 kb: preterm men 28.2 ± 2.6, term men 37.0 ± 2.4%, p < 0.001) and a higher proportion of shorter telomeres (4.2-1.3 kb: preterm men 40.4 ± 3.5%, term men 29.9 ± 3.2%, p < 0.01). CONCLUSION: Our data indicate that healthy young adults born very preterm manifest clinical and molecular evidence of accelerated ageing.

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients.

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

RNA-Seq of Huntington's disease patient myeloid cells reveals innate transcriptional dysregulation associated with proinflammatory pathway activation.

Innate immune activation beyond the central nervous system is emerging as a vital component of the pathogenesis of neurodegeneration. Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The systemic innate immune system is thought to act as a modifier of disease progression; however, the molecular mechanisms remain only partially understood. Here we use RNA-sequencing to perform whole transcriptome analysis of primary monocytes from thirty manifest HD patients and thirty-three control subjects, cultured with and without a proinflammatory stimulus. In contrast with previous studies that have required stimulation to elicit phenotypic abnormalities, we demonstrate significant transcriptional differences in HD monocytes in their basal, unstimulated state. This includes previously undetected increased resting expression of genes encoding numerous proinflammatory cytokines, such as IL6 Further pathway analysis revealed widespread resting enrichment of proinflammatory functional gene sets, while upstream regulator analysis coupled with Western blotting suggests that abnormal basal activation of the NFĸB pathway plays a key role in mediating these transcriptional changes. That HD myeloid cells have a proinflammatory phenotype in the absence of stimulation is consistent with a priming effect of mutant huntingtin, whereby basal dysfunction leads to an exaggerated inflammatory response once a stimulus is encountered. These data advance our understanding of mutant huntingtin pathogenesis, establish resting myeloid cells as a key source of HD immune dysfunction, and further demonstrate the importance of systemic immunity in the potential treatment of HD and the wider study of neurodegeneration.

Characterisation of immune cell function in fragment and full-length Huntington's disease mouse models.

Inflammation is a growing area of research in neurodegeneration. In Huntington's disease (HD), a fatal inherited neurodegenerative disease caused by a CAG-repeat expansion in the gene encoding huntingtin, patients have increased plasma levels of inflammatory cytokines and circulating monocytes that are hyper-responsive to immune stimuli. Several mouse models of HD also show elevated plasma levels of inflammatory cytokines. To further determine the degree to which these models recapitulate observations in HD patients, we evaluated various myeloid cell populations from different HD mouse models to determine whether they are similarly hyper-responsive, as well as measuring other aspects of myeloid cell function. Myeloid cells from each of the three mouse models studied, R6/2, HdhQ150 knock-in and YAC128, showed increased cytokine production when stimulated. However, bone marrow CD11b(+) cells did not show the same hyper-responsive phenotype as spleen and blood cells. Furthermore, macrophages isolated from R6/2 mice show increased levels of phagocytosis, similar to findings in HD patients. Taken together, these results show significant promise for these mouse models to be used to study targeting innate immune pathways identified in human cells, thereby helping to understand the role the peripheral immune system plays in HD progression.

Short-term anesthesia inhibits formalin-induced extracellular signal-regulated kinase (ERK) activation in the rostral anterior cingulate cortex but not in the spinal cord.

BACKGROUND: The rostral anterior cingulate cortex (rACC) has been implicated in the negative affective response to injury, and importantly, it has been shown that activation of extracellular signal-regulated kinase (ERK) signaling in the rACC contributes to the full expression of the affective component of pain in rodents. In this study, we investigated whether administration of anesthesia at the time of injury could reduce phosphorylated-ERK (PERK) expression in the rACC, which might eliminate the negative affective component of noxious stimulation. Intraplantar hindpaw formalin stimulation, an aversive event in the awake animal, was given with or without general isoflurane anesthesia, and PERK expression was subsequently quantified in the rACC using immunohistochemistry. Furthermore, as numerous studies have demonstrated the importance of spinal ERK signaling in the regulation of nociceptive behaviour, we also examined PERK in the superficial dorsal horn of the spinal cord. FINDINGS: Formalin injection with and without short-term (<10 min) general isoflurane anesthesia induced the same level of PERK expression in spinal cord laminae I-II. However, PERK expression was significantly inhibited across all laminae of the rACC in animals anesthetized during formalin injection. The effect of anesthesia was such that levels of PERK were the same in formalin and sham treated anesthesized animals. CONCLUSIONS: This study is the first to demonstrate that isoflurane anesthesia can inhibit formalin-induced PERK in the rACC and therefore might eliminate the unpleasantness of restraint associated with awake hindpaw injection.

Ribosomal DNA copy number is associated with body mass in humans and other mammals.

Body mass results from a complex interplay between genetics and environment. Previous studies of the genetic contribution to body mass have excluded repetitive regions due to the technical limitations of platforms used for population scale studies. Here we apply genome-wide approaches, identifying an association between adult body mass and the copy number (CN) of 47S-ribosomal DNA (rDNA). rDNA codes for the 18 S, 5.8 S and 28 S ribosomal RNA (rRNA) components of the ribosome. In mammals, there are hundreds of copies of these genes. Inter-individual variation in the rDNA CN has not previously been associated with a mammalian phenotype. Here, we show that rDNA CN variation associates with post-pubertal growth rate in rats and body mass index in adult humans. rDNA CN is not associated with rRNA transcription rates in adult tissues, suggesting the mechanistic link occurs earlier in development. This aligns with the observation that the association emerges by early adulthood.

Perspectives on the Home Monitoring of Macular Disease.

Recent advancements in imaging technology have led to increasing interest in home monitoring of macular disease. The prevalence of macular disease is projected to rise considerably over time, leading to a significant burden on hospital services for age-related macular degeneration and diabetic macular edema. Home monitoring has the potential to augment conventional hospital assessment and so enable improved access to clinical care for low- and moderate-risk patients, while also allowing sensitive detection of early signs of disease that may require prompt intervention. Despite this, there are significant considerations before large-scale implementation could be possible. These are related to both the current availability of home monitoring technology and the logistical barriers to its widespread introduction. Access to home monitoring is also likely to be more challenging in lower-income communities and countries, with subsequent implications for health inequality that will need to be considered and addressed appropriately.

Differential Associations Between Two Markers of Probable Sarcopenia and Continuous Orthostatic Hemodynamics in The Irish Longitudinal Study on Ageing.

BACKGROUND: Sarcopenia and orthostatic hypotension are growing age-related health burdens associated with adverse outcomes, including falls. Despite a possible pathophysiological link, the association between the 2 disorders is not well elucidated. We sought to investigate this relationship in The Irish Longitudinal Study on Ageing (TILDA). METHODS: Data from 2 858 participants at wave 3 of TILDA were analyzed. Probable sarcopenia was defined as per the European Working Group on Sarcopenia in Older People revised definition cutoffs (hand grip strength [HGS] <27 kg in men, <16 kg in women, and/or 5-chair stand test [5CST] time >15 seconds). Participants underwent an active stand orthostatic test with continuous blood pressure (BP) monitoring. Multilevel mixed-effects models, controlling for possible confounders, were used to assess the effect of probable sarcopenia by HGS and 5CST criteria on the change in BP after standing. RESULTS: HGS- and 5CST-defined probable sarcopenia were independently associated with an attenuated BP recovery at 10-20 seconds poststand (systolic BP: ß -0.54, p < .001; ß -0.25, p < .001). On average, those meeting HGS probable sarcopenia criteria had a significantly lower BP at 20, 30, and 40 seconds (differences in systolic BP: -5.01 mmHg, -3.68 mmHg, -2.32 mmHg, p < .05 for all). Those meeting 5CST probable sarcopenia criteria had a significant difference in systolic BP at 20 seconds (-1.94 mmHg, p = .002) but not at 30 or 40 seconds. CONCLUSION: Probable sarcopenia had a significant association with delayed orthostatic BP recovery, with HGS-defined probable sarcopenia having a stronger association than 5CST-defined probable sarcopenia. Results support a modest but significant pathophysiological link between probable sarcopenia and orthostatic hypotension.

Structural brain signatures of frailty, defined as accumulation of self-reported health deficits in older adults.

Background: Frailty in older adults has been associated with reduced brain health. However, structural brain signatures of frailty remain understudied. Our aims were: (1) Explore associations between a frailty index (FI) and brain structure on magnetic resonance imaging (MRI). (2) Identify the most important FI features driving the associations. Methods: We designed a cross-sectional observational study from a population-based study (The Irish Longitudinal Study on Aging: TILDA). Participants aged ≥50 years who underwent the wave 3 MRI sub-study were included. We measured cortex, basal ganglia, and each of the Desikan-Killiany regional volumes. Age-and sex-adjusted correlations were performed with a 32-item self-reported FI that included conditions commonly tested for frailty in research and clinical settings. A graph theory analysis of the network composed by each FI item and cortex volume was performed. White matter fiber integrity was quantified using diffusion tensor imaging (DTI). Results: In 523 participants (mean age 69, 49% men), lower cortex and thalamic volumes were independently associated with higher FI. Sensory and functional difficulties, diabetes, polypharmacy, knee pain, and self-reported health were the main FI associations with cortex volume. In the network analysis, cortex volume had a modest influence within the frailty network. Regionally, higher FI was significantly associated with lower volumes in both orbitofrontal and temporal cortices. DTI analyses revealed inverse associations between the FI and the integrity of some association bundles. Conclusion: The FI used had a recognizable but subtle structural brain signature in this sample. Only some FI deficits were directly associated with cortex volume, suggesting scope for developing FIs that include metrics more specifically related with brain health in future aging neuroscience studies.

Brain connectivity in frailty: Insights from The Irish Longitudinal Study on Ageing (TILDA).

Frailty in older adults is associated with greater risk of cognitive decline. Brain connectivity insights could help understand the association, but studies are lacking. We applied connectome-based predictive modeling to a 32-item self-reported Frailty Index (FI) using resting state functional MRI data from The Irish Longitudinal Study on Ageing. A total of 347 participants were included (48.9% male, mean age 68.2 years). From connectome-based predictive modeling, we obtained 204 edges that positively correlated with the FI and composed the "frailty network" characterised by connectivity of the visual network (right); and 188 edges that negatively correlated with the FI and formed the "robustness network" characterized by connectivity in the basal ganglia. Both networks' highest degree node was the caudate but with different patterns: from caudate to visual network in the frailty network; and to default mode network in the robustness network. The FI was correlated with walking speed but not with metrics of global cognition, reinforcing the matching between the FI and the brain connectivity pattern found (main predicted connectivity in basal ganglia).

Rare anatomical variant of the spinal accessory nerve: case report and comprehensive review.

Historical studies of the anatomy of the spinal accessory nerve (SAN) have reported conflicting results regarding its relationship with the internal jugular vein (IJV). A literature review was undertaken to establish the prevalence of anatomical variations of the SAN encountered during routine neck dissection surgery, in order to increase awareness and reduce morbidity associated with iatrogenic SAN injury. The published literature was analysed by qualitative synthesis and nine articles were yielded following application of the inclusion and exclusion criteria. Incidences of the SAN lateral to the IJV and medial to the IJV ranged from 39.8% to 96.6%, and 2.6% to 57.4%, respectively. Five of the studies reported incidences of the SAN traversing the IJV, which occurred in 0.9% to 2.8% of cases. One study reported an isolated variant of the SAN dividing around the IJV, with a prevalence of 0.5%. We present a case report demonstrating the rare variant of the SAN traversing the IJV. Preoperative identification of rare anatomical association of the SAN and IJV may reduce perioperative injury to vital structures during neck dissection.

Longitudinal Study on Sustained Attention to Response Task (SART): Clustering Approach for Mobility and Cognitive Decline.

The Sustained Attention to Response Task (SART) is a computer-based go/no-go task to measure neurocognitive function in older adults. However, simplified average features of this complex dataset lead to loss of primary information and fail to express associations between test performance and clinically meaningful outcomes. Here, we combine a novel method to visualise individual trial (raw) information obtained from the SART test in a large population-based study of ageing in Ireland and an automatic clustering technique. We employed a thresholding method, based on the individual trial number of mistakes, to identify poorer SART performances and a fuzzy clusters algorithm to partition the dataset into 3 subgroups, based on the evolution of SART performance after 4 years. Raw SART data were available for 3468 participants aged 50 years and over at baseline. The previously reported SART visualisation-derived feature 'bad performance', indicating the number of SART trials with at least 4 mistakes, and its evolution over time, combined with the fuzzy c-mean (FCM) algorithm, individuated 3 clusters corresponding to 3 degrees of physiological dysregulation. The biggest cluster (94% of the cohort) was constituted by healthy participants, a smaller cluster (5% of the cohort) by participants who showed improvement in cognitive and psychological status, and the smallest cluster (1% of the cohort) by participants whose mobility and cognitive functions dramatically declined after 4 years. We were able to identify in a cohort of relatively high-functioning community-dwelling adults a very small group of participants who showed clinically significant decline. The selected smallest subset manifested not only mobility deterioration, but also cognitive decline, the latter being usually hard to detect in population-based studies. The employed techniques could identify at-risk participants with more specificity than current methods, and help clinicians better identify and manage the small proportion of community-dwelling older adults who are at significant risk of functional decline and loss of independence.

Targeting of the Arf-like GTPase Arl3p to the Golgi requires N-terminal acetylation and the membrane protein Sys1p.

The GTPase Arl3p is required to recruit a second GTPase, Arl1p, to the Golgi in Saccharomyces cerevisiae. Arl1p binds to the GRIP domain, which is present in a number of long coiled-coil proteins or 'golgins'. Here we show that Arl3p is not myristoylated like most members of the Arf family, but is instead amino-terminally acetylated by the NatC complex. Targeting of Arl3p also requires a Golgi membrane protein Sys1p. The human homologues of Arl3p (Arf-related protein 1 (ARFRP1)) and Sys1p (hSys1) can be isolated in a complex after chemical cross-linking. This suggests that the targeting of ARFRP1/Arl3p to the Golgi is mediated by a direct interaction between its acetylated N terminus and Sys1p/hSys1.

The Importance of Age in the Prediction of Mortality by a Frailty Index: A Machine Learning Approach in the Irish Longitudinal Study on Ageing.

The quantification of biological age in humans is an important scientific endeavor in the face of ageing populations. The frailty index (FI) methodology is based on the accumulation of health deficits and captures variations in health status within individuals of the same age. The aims of this study were to assess whether the addition of age to an FI improves its mortality prediction and whether the associations of the individual FI items differ in strength. We utilized data from The Irish Longitudinal Study on Ageing to conduct, by sex, machine learning analyses of the ability of a 32-item FI to predict 8-year mortality in 8174 wave 1 participants aged 50 or more years. By wave 5, 559 men and 492 women had died. In the absence of age, the FI was an acceptable predictor of mortality with AUCs of 0.7. When age was included, AUCs improved to 0.8 in men and 0.9 in women. After age, deficits related to physical function and self-rated health tended to have higher importance scores. Not all FI variables seemed equally relevant to predict mortality, and age was by far the most relevant feature. Chronological age should remain an important consideration when interpreting the prognostic significance of an FI.

SART and Individual Trial Mistake Thresholds: Predictive Model for Mobility Decline.

The Sustained Attention to Response Task (SART) has been used to measure neurocognitive functions in older adults. However, simplified average features of this complex dataset may result in loss of primary information and fail to express associations between test performance and clinically meaningful outcomes. Here, we describe a new method to visualise individual trial (raw) information obtained from the SART test, vis-à-vis age, and groups based on mobility status in a large population-based study of ageing in Ireland. A thresholding method, based on the individual trial number of mistakes, was employed to better visualise poorer SART performances, and was statistically validated with binary logistic regression models to predict mobility and cognitive decline after 4 years. Raw SART data were available for 4864 participants aged 50 years and over at baseline. The novel visualisation-derived feature bad performance, indicating the number of SART trials with at least 4 mistakes, was the most significant predictor of mobility decline expressed by the transition from Timed Up-and-Go (TUG) < 12 to TUG ≥ 12 s (OR = 1.29; 95% CI 1.14-1.46; p < 0.001), and the only significant predictor of new falls (OR = 1.11; 95% CI 1.03-1.21; p = 0.011), in models adjusted for multiple covariates. However, no SART-related variables resulted significant for the risk of cognitive decline, expressed by a decrease of ≥2 points in the Mini-Mental State Examination (MMSE) score. This novel multimodal visualisation could help clinicians easily develop clinical hypotheses. A threshold approach to the evaluation of SART performance in older adults may better identify subjects at higher risk of future mobility decline.

Comparison of Gait Speed Reserve, Usual Gait Speed, and Maximum Gait Speed of Adults Aged 50+ in Ireland Using Explainable Machine Learning.

Gait speed is a measure of general fitness. Changing from usual (UGS) to maximum (MGS) gait speed requires coordinated action of many body systems. Gait speed reserve (GSR) is defined as MGS-UGS. From a shortlist of 88 features across five categories including sociodemographic, cognitive, and physiological, we aimed to find and compare the sets of predictors that best describe UGS, MGS, and GSR. For this, we leveraged data from 3,925 adults aged 50+ from Wave 3 of The Irish Longitudinal Study on Ageing (TILDA). Features were selected by a histogram gradient boosting regression-based stepwise feature selection pipeline. Each model's feature importance and input-output relationships were explored using TreeExplainer from the Shapely Additive Explanations explainable machine learning package. The mean R a d j 2 (SD) from fivefold cross-validation on training data and the R a d j 2   score on test data were 0.38 (0.04) and 0.41 for UGS, 0.45 (0.04) and 0.46 for MGS, and 0.19 (0.02) and 0.21 for GSR. Each model selected features across all categories. Features common to all models were age, grip strength, chair stands time, mean motor reaction time, and height. Exclusive to UGS and MGS were educational attainment, fear of falling, Montreal cognitive assessment errors, and orthostatic intolerance. Exclusive to MGS and GSR were body mass index (BMI), and number of medications. No features were selected exclusively for UGS and GSR. Features unique to UGS were resting-state pulse interval, Center for Epidemiologic Studies Depression Scale (CESD) depression, sit-to-stand difference in diastolic blood pressure, and left visual acuity. Unique to MGS were standard deviation in sustained attention to response task times, resting-state heart rate, smoking status, total heartbeat power during paced breathing, and visual acuity. Unique to GSR were accuracy proportion in a sound-induced flash illusion test, Mini-mental State Examination errors, and number of cardiovascular conditions. No interactions were present in the GSR model. The four features that overall gave the most impactful interactions in the UGS and MGS models were age, chair stands time, grip strength, and BMI. These findings may help provide new insights into the multisystem predictors of gait speed and gait speed reserve in older adults and support a network physiology approach to their study.

Cannabidiol Modulates Mitochondrial Redox and Dynamics in MCF7 Cancer Cells: A Study Using Fluorescence Lifetime Imaging Microscopy of NAD(P)H.

The cannabinoid, cannabidiol (CBD), is part of the plant's natural defense system that when given to animals has many useful medicinal properties, including activity against cancer cells, modulation of the immune system, and efficacy in epilepsy. Although there is no consensus on its precise mode of action as it affects many cellular targets, CBD does appear to influence mitochondrial function. This would suggest that there is a cross-kingdom ability to modulate stress resistance systems that enhance homeostasis. As NAD(P)H autofluorescence can be used as both a metabolic sensor and mitochondrial imaging modality, we assessed the potential of this technique to study the in vitro effects of CBD using 2-photon excitation and fluorescence lifetime imaging microscopy (2P-FLIM) of NAD(P)H against more traditional markers of mitochondrial morphology and cellular stress in MCF7 breast cancer cells. 2P-FLIM analysis revealed that the addition of CBD induced a dose-dependent decrease in bound NAD(P)H, with 20 µM treatments significantly decreased the contribution of bound NAD(P)H by 14.6% relative to the control (p < 0.001). CBD also increased mitochondrial concentrations of reactive oxygen species (ROS) (160 ± 53 vs. 97.6 ± 4.8%, 20 µM CBD vs. control, respectively, p < 0.001) and Ca2+ (187 ± 78 vs. 105 ± 10%, 20 µM CBD vs. the control, respectively, p < 0.001); this was associated with a significantly decreased mitochondrial branch length and increased fission. These are all suggestive of mitochondrial stress. Our results support the use of NAD(P)H autofluorescence as an investigative tool and provide further evidence that CBD can modulate mitochondrial function and morphology in a dose-dependent manner, with clear evidence of it inducing oxidative stress at higher concentrations. This continues to support emerging data in the literature and may provide further insight into its overall mode of action, not only in cancer, but potentially its function in the plant and why it can act as a medicine.

A kisspeptin-10 analog with greater in vivo bioactivity than kisspeptin-10.

The kisspeptins are neuropeptides that stimulate the hypothalamo-pituitary-gonadal (HPG) axis. The smallest endogenous kisspeptin, kisspeptin-10 (KP-10), binds to the receptor KISS1R with a similar affinity to the full-length peptide, kisspeptin-54 (KP-54), but is less effective in vivo, possibly because of increased enzymatic breakdown or clearance. The kisspeptin system may have therapeutic potential in the treatment of reproductive disorders and endocrine cancers. We have rationally modified the structure of KP-10 and tested the binding affinity of these analogs for the KISS1R. Those analogs that bound with relatively high affinity to KISS1R were tested for ability to stimulate ERK1/2 phosphorylation in vitro and for their ability to stimulate the HPG axis in vivo. One analog, [dY](1)KP-10, bound to KISS1R with lower affinity to KP-10 and exhibited similar bioactivity in vitro. However, in vivo peripheral administration of [dY](1)KP-10 increased plasma LH and testosterone more potently than KP-10 itself at 20 min postinjection in mice. In addition, 60 min postinjection, 0.15 nmol [dY](1)KP-10 significantly increased total testosterone levels in mice whereas the same dose of KP-10 had no significant effect. Should manipulation of the kisspeptin/KISS1R signaling system prove therapeutically useful, long-lasting analogs such as [dY](1)KP-10 may have greater therapeutic potential than endogenous forms of kisspeptin.

Manganese enhancement in non-CNS organs.

Manganese-enhanced magnetic resonance imaging (MEMRI) is a novel imaging technique capable of monitoring calcium influx, in vivo. Manganese (Mn2+) ions, similar to calcium ions (Ca2+), are taken up by activated cells where their paramagnetic properties afford signal enhancement in T(1)-weighted MRI methodologies. In this study we have assessed Mn2+ distribution in mice using magnetization-prepared rapid gradient echo (MP-RAGE) based MRI, by measuring changes in T(1)-effective relaxation times (T(1)-eff), effective R(1)-relaxation rates (R(1)-eff) and signal intensity (SI) profiles over time. The manganese concentration in the tissue was also determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). Our results show a strong positive correlation between infused dose of MnCl2 and the tissue manganese concentration. Furthermore, we demonstrate a linear relationship between R(1)-eff and tissue manganese concentration and tissue-specific Mn2+ distribution in murine tissues following dose-dependent Mn2+ administration. This data provides an optimized MnCl2 dose regimen for an MP-RAGE based sequence protocol for specific target organs and presents a potential 3D MRI technique for in vivo imaging of Ca2+ entry during Ca2+-dependent processes in a wide range of tissues.