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Aims The ageing heart naturally incurs a progressive decline in function and perfusion that available treatments cannot halt. However, some exceptional individuals maintain good health until the very late stage of their life due to favourable gene–environment interaction. We have previously shown that carriers of a longevity-associated variant (LAV) of the BPIFB4 gene enjoy prolonged health spans and lesser cardiovascular complications. Moreover, supplementation of LAV-BPIFB4 via an adeno-associated viral vector improves cardiovascular performance in limb ischaemia, atherosclerosis, and diabetes models. Here, we asked whether the LAV-BPIFB4 gene could address the unmet therapeutic need to delay the heart’s spontaneous ageing. Methods and results Immunohistological studies showed a remarkable reduction in vessel coverage by pericytes in failing hearts explanted from elderly patients. This defect was attenuated in patients carrying the homozygous LAV-BPIFB4 genotype. Moreover, pericytes isolated from older hearts showed low levels of BPIFB4, depressed pro-angiogenic activity, and loss of ribosome biogenesis. LAV-BPIFB4 supplementation restored pericyte function and pericyte-endothelial cell interactions through a mechanism involving the nucleolar protein nucleolin. Conversely, BPIFB4 silencing in normal pericytes mimed the heart failure pericytes. Finally, gene therapy with LAV-BPIFB4 prevented cardiac deterioration in middle-aged mice and rescued cardiac function and myocardial perfusion in older mice by improving microvasculature density and pericyte coverage. Conclusions We report the success of the LAV-BPIFB4 gene/protein in improving homeostatic processes in the heart’s ageing. These findings open to using LAV-BPIFB4 to reverse the decline of heart performance in older people.

The RAS family of GTPases (HRAS, KRAS, NRAS) continue to present formidable challenges to the design of compounds that successfully and specifically target oncogenic RAS mutant populations in cancer. Although recent advances have led to the successful development of inhibitors specific to KRAS G12C (such as Sotorasib [Amgen] and Adagrasib [Mirati Therapeutics]), ongoing efforts seek to develop additional targeted inhibitors for oncogenic mutants of KRAS. Development of new KRAS targeted inhibitors requires the understanding of how a mutation impacts the accessibility of key residues within the protein, along with how each candidate compound interacts with populations of mutant and wild-type proteins. Proteomic analyses, typically comprising peptide-based methods to confirm compound localization or quantify binding kinetics, represent a key validation step for each phase of the inhibitor development process. However, these methods may not always provide a clear picture of how effectively the compound binds to the protein, how specific the compound is to the target residue, and how concentration or incubation time may impact these factors. To address this, we have developed a novel assay to evaluate target engagement in vitro employing top-down proteomics, which entails the analysis of intact protein molecules and their modified forms (proteoforms). By analyzing intact labeled or compound-treated recombinant KRAS4B proteins, we can directly visualize and precisely characterize each species within a sample. This method allows us to easily detect nonspecific binding, identify targeting of incorrect residues, and characterize unanticipated compound reaction products within a short LC-MS run, all facilitated by targeted MS2 fragmentation. Moreover, we have developed complementary methods for RAS proteoform quantitation, allowing for the evaluation of compound engagement to all proteoforms with greater accuracy than traditional peptide-based methods. We first performed a proof-of-concept experiment using a panel of KRAS4B G12D_C118S proteins with selected residues modified to cysteine to evaluate reactivity to maleimide-biotin over time. We then applied our optimized top-down assay to determine the engagement of targeted covalent inhibitors Sotorasib and Adagrasib with KRAS WT, G12C, or G13C in both the active (GppNHp) and inactive (GDPβS) states. Finally, we selected three compounds from a large-scale screen against a UCSF Small Molecule Discovery Center compound library and evaluated each for specificity to KRAS4B G13C over KRAS4B WT and KRAS4B G12C. Our results show the concentration dependence of target engagement and directly map compound binding location(s) without disrupting the protein’s primary structure. Moreover, our ability to target discrete compound additions for top-down MS2 fragmentation provides context and order of preferential labeling of KRAS4B. Our hope is that this methodology may help accelerate the identification of new successful targeted inhibitors for KRAS and other RAS isoforms by the greater RAS community. Citation Format: Robert A. D'Ippolito, Caroline J. DeHart, Dana Rabara, Maria Abreu Blanco, Emily Alberico, Nitya Ramakrishnan, Stephanie Widmeyer, Simon Messing, David Turner, Michelle Arkin, Anna Maciag, Andrew Stephen, Dominic Esposito, Frank McCormick, Dwight V. Nissley. A top-down proteomic assay to evaluate KRAS4B-compound engagement [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A018.

KRAS mutations occur in roughly 15% of cancer patients, with KRAS G12D, KRAS G12V, and KRAS G12C substitutions constituting the majority of these cases. Cancer types also display puzzling preferences for certain RAS mutations; for instance, 49% of pancreatic ductal carcinomas (PDAC) are driven by KRAS G12D substitutions compared to 35% of colon adenocarcinomas (COAD) and 17% of lung adenocarcinomas (LUAD)1. In contrast, KRAS G12C mutations drive only 3% of PDAC and 10% of COAD, but 41% of LUAD cases1. Some of this discrepancy can be explained by mutagen exposure, as there is evidence that tobacco smoke carcinogens drive the predominance of KRAS G12C mutations in lung cancer. However, it is possible that inherent functional differences between the mutant proteins also contribute to such preferences across cancer types. These oncogenic substitutions do endow subtle and unique differences in biochemical properties, but it is unknown whether this translates to differences in cellular signaling potential. KRAS activates the MAPK and PI3K signaling pathways by binding directly to RAF1 or the p110 catalytic subunit of PI3K. These interactions are important signaling nodes that result in cellular proliferation, survival, movement, differentiation, and immune evasion, and therefore drive cancer when constitutively activated by mutant KRAS. Biochemical assessments of relative effector affinity between KRAS mutants lack crucial elements present in a cellular environment, such as GDP/GTP ratios, membrane interactions, subcellular localization, and engagement with other proteins. Here, we optimize a cell-based Bioluminescent Resonance Energy Transfer (BRET) system in HEK293T cells using NanoLuc- and mVenus-tagged proteins to quantify relative effector affinities between KRAS mutants. We assess the affinities of KRAS4b G12C, KRAS4b G12D, and KRAS4b WT for effectors p110α and RAF1(52-188), along with RAS binding deficient control mutants p110α T208D/K227A and RAF1(52-188)R89L, and show statistical differences in effector engagement between the wildtype and mutant proteins. We also demonstrate the efficacy of this optimized system as a drug screening tool by assessing compounds in clinical development for their ability to disrupt mutant KRAS-effector interactions within a cellular environment. 1Prior IA, Hood FE, Hartley JL. The frequency of Ras mutations in cancer. Cancer Research 2020. Citation Format: Megan Rigby, John Columbus, Vanessa Wall, Dominic Esposito, Thomas Turbyville, Dhirendra Simanshu, Dwight Nissley, Frank McCormick, Anna Maciag. Bioluminescence resonance energy transfer (BRET) as a tool for assessing mutant KRAS-effector affinity and drug efficacy [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B027.

Aims: Homozygosity for a 4-missense single-nucleotide-polymorphism-haplotype of the human BPIFB4 gene is enriched in long-living individuals. Delivery of this Longevity Associated Variant (LAV) improved revascularisation and reduced endothelial dysfunction and atherosclerosis in mice through a mechanism involving the stromal cell-derived factor-1 (SDF-1). Here, we investigated if delivery of the LAV-BPIFB4 gene may attenuate the progression of diabetic cardiomyopathy. Methods and Results: Compared with age-matched lean controls, diabetic db/db mice showed altered echocardiographic indices of diastolic and systolic function and histological evidence of microvascular rarefaction, lipid accumulation, and fibrosis in the myocardium. All these alterations, as well as endothelial dysfunction, were prevented by systemic LAV-BPIFB4 gene therapy using an adeno-associated viral vector serotype 9 (AAV9). In contrast, AAV9-wild type (WT)-BPIFB4 exerted no benefit. Interestingly, the LAV-BPIFB4-treated mice showed increased SDF-1 levels in peripheral blood and myocardium and upregulation of cardiac myosin heavy chain isoform alpha, a contractile protein that was reduced in diabetic hearts. SDF-1 upregulation was instrumental to LAV-BPIFB4-induced benefit as both haemodynamic and structural improvements were inhibited by an orally active antagonist of the SDF-1 CXCR4 receptor. Conclusions: In mice with type-2 diabetes, LAV-BPIFB4 gene therapy promotes an advantageous remodelling of the heart, allowing it to better withstand diabetes-induced stress. These results support the viability of transferring healthy characteristics of longevity to attenuate diabetic cardiac disease.

In the ongoing global COVID-19 pandemic, male sex is a risk factor for severe disease and death, and the reasons for these clinical discrepancies are largely unknown. The aim of this work is to study the influence of sex on the course of infection and the differences in prognostic markers between genders in COVID-19 patients. Our cohort consisted of 64 adult patients (n = 34 men and n = 30 women) with PCR-proven SARS-CoV-2 infection. Further, a group of patients was characterized by a different severity degree (n = 8 high- and n = 8 low-grade individuals for both male and female patients). As expected, the serum concentrations of LDH, fibrinogen, CRP, and leucocyte count in men were significantly higher than in females. When serum concentrations of the inflammatory cytokines, including IL-6, IL-2, IP-10 and IL-4 and chemokines like MCP-1, were measured with multiplex ELISA, no significant differences between male and female patients were found. In COVID-19 patients, we recently attributed a new prognostic value to BPIFB4, a natural defensin against dysregulation of the immune responses. Here, we clarify that BPIFB4 is inversely related to the disease degree in men but not in women. Indeed, higher levels of BPIFB4 characterized low-grade male patients compared to high-grade ones. On the contrary, no significant difference was reported between low-grade female patients and high-grade ones. In conclusion, the identification of BPIFB4 as a biomarker of mild/moderate disease and its sex-specific activity would open an interesting field for research to underpin gender-related susceptibility to the disease.

Lay abstract Aging is the major risk factor for disease development. Long-living individuals (LLIs) are subjects older than 90 years that represent an invaluable model to study mechanisms underpinning longevity and healthy aging. Circulating levels of soluble receptor for advanced glycation end products (sRAGE) change with aging and can forecast the cardiovascular risk, which is reduced in centenarians. sRAGE is composed of two isoforms, the cleaved RAGE (cRAGE) and the secretory RAGE (esRAGE), that are known to inhibit the oxidative stress and inflammatory activities of their ligands such the advanced glycation end products (AGEs). In this study, we measured the plasmatic levels of both sRAGE isoforms and AGEs in LLIs (90–105 years) and control subjects (11–89 years). We found that cRAGE decreases with age in controls and LLIs. esRAGE increases in LLIs and AGEs increase in controls with age but decrease in LLIs. AGEs/esRAGE ratio and cRAGE were able to discriminate controls from LLIs. Hence, LLIs are characterized by a lower AGEs/sRAGE ratio, due to esRAGE increase and AGEs reduction that may explain their reduced cardiovascular and metabolic risk. Besides, circulating cRAGE could be considered a reliable marker of chronological age, while esRAGE a protective factor associated with longevity.

BACKGROUND:There is an increasing concern about age-related frailty because of the growing number of elderly people in the general population. The Longevity-Associated Variant (LAV) of the human BPIFB4 gene was found to correct endothelial dysfunction, one of the mechanisms underlying frailty, in aging mice whereas the RV-BPIFB4 variant induced opposite effects. Thus, we newly hypothesize that, besides being associated with life expectancy, BPIFB4 polymorphisms can predict frailty.Aim and Results: Here we investigated if the BPIFB4 haplotypes, LAV, wild-type (WT) and RV, differentially associate with frailty in a cohort of 237 elderly subjects from Calabria region in Southern Italy. Moreover, we studied the effect of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer on the progression of frailty in aging mice. We found an inverse correlation of the homozygous LAV-BPIFB4 haplotype with frailty in elderly subjects. Conversely, carriers of the RV-BPIFB4 haplotype showed an increase in the frailty status and risk of death. Moreover, in old mice, LAV-BPIFB4 gene transfer delayed frailty progression.CONCLUSIONS:These data indicate that specific BPIFB4 haplotypes could represent useful genetic markers of frailty. In addition, horizontal transfer of a healthy gene variant can attenuate frailty in aging organisms.

Understanding the role that a researcher recording oral history biographical testimonies performs during a conversation (who they become to the narrator, and what boundaries and ethics they need), are a condition of a well-done recording. Due to specifics of the XX century history, the oralists often deal with people whose lives have been marked by some kind of trauma A social order to remember the past means an order to bear witness to the tragic or traumatic experiences. This is a difficult task not only for the narrator but also for the researcher, who are usually not psychologists but sociologists, historians or anthropologists. In the article, the author confronts the theory regulating the ethics of oral histor recordings with empirical research.

Background Healthy longevity is the result of the interaction between favourable environment and unique genetic makeup. We showed that horizontal transfer of a longevity-associated gene variant (LAV-BPIFB4) improves endothelial function and accelerates the recovery from ischemia. Purpose To determine if the benefit of LAV-BPIFB4 gene therapy can be extended to diabetic cardiomyopathy. Methods and results We confirmed that human diabetic patients with heart failure (n=13) show a decreased cardiac expression of BPIFB4 compared with healthy subjects (n=10). Obese db/db mice received a systemic injection of adeno-associated viral vector (AAV9)-LAV-BPIFB4, AAV9-wild type (WT)-BPIFB4 (both 100 μL at 1×1012 GC/mL) or vehicle before the onset of cardiomyopathy, and were euthanised four weeks later for histological, metabolic and transcriptional analyses. Echocardiographic evaluation (n=8/group), performed at baseline and after gene therapy, showed that LAV-BPIFB4 treatment, despite not resolving hyperglycaemia, improved left ventricular function compared with the other groups. Histological analyses of the hearts (n=5 to 10/group) revealed that LAV-BPIFB4 reduced myocardial fibrosis and increased angiogenesis compared with vehicle and WT-hearts; moreover, LAV increased the expression of the alpha-isoform of the cardiac myosin heavy chain, which is associated with a superior cardiomyocyte contractility. Interestingly, LAV-BPIFB4 treatment induced an increase in cardiac SDF1 expression compared with WT and vehicle, despite the mechanism linking the two events is still unknown. The oral administration of the CXCR4 antagonist AMD-070, given at 2 mg/kg/day for four weeks, abolished several of the beneficial effects exerted by the LAV-BPIFB4 therapy in the obese diabetic mice, as assessed by echocardiography and histology (n=7/group). At the molecular level, next-generation RNA sequencing (n=3 to 4 /group) showed 8 genes were differentially expressed by LAV-BPIFB4-hearts compared with vehicle-hearts. These genes are associated with mitochondrial and metabolic functions. Among them, changes in the UCP3, HMGCS2, CS, ATPB and TOMM20 expression were also validated at the protein level by western blotting. Lipidomics using ultrahigh-performance liquid chromatography-mass spectrometry (n=6 or 7/group) showed 63 metabolites differentially expressed by LAV-BPIFB4- compared with vehicle-hearts, with only 3 (two cardiolipins and one glycerophospholipid) returning close to the non-diabetic phenotype following LAV-BPIFB4 treatment. Conclusions This study newly shows the possibility of transferring the benefit of salutary polymorphic gene variants to protect the cardiovascular system from metabolic pressure. Rather than combating pathogenic mechanisms, the strategy activates alternative pathways overriding disease risk factors. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): British Heart Foundation project grant “Longevity-associated BPIFB4 gene therapy for treatment of ischemic disease”

Bitter taste receptors (TAS2R) are involved in a variety of non-tasting physiological processes, including immune-inflammatory ones. Therefore, their genetic variations might influence various traits. In particular, in different populations of South Italy (Calabria, Cilento, and Sardinia), polymorphisms of TAS2R16 and TAS238 have been analysed in association with longevity with inconsistent results. A meta-analytic approach to quantitatively synthesize the possible effect of the previous variants and, possibly, to reconcile the inconsistencies has been used in the present paper. TAS2R38 variants in the Cilento population were also analysed for their possible association with longevity and the obtained data have been included in the relative meta-analysis. In population from Cilento no association was found between TAS2R38 and longevity, and no association was observed as well, performing the meta-analysis with data of the other studies. Concerning TAS2R16 gene, instead, the genotype associated with longevity in the Calabria population maintained its significance in the meta-analysis with data from Cilento population, that, alone, were not significant in the previously published study. In conclusion, our results suggest that TAS2R16 genotype variant is associated with longevity in South Italy.

The longevity-associated variant (LAV) of the bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) has been found significantly enriched in long-living individuals. Neuroinflammation is a key player in Huntington’s disease (HD), a neurodegenerative disorder caused by neural death due to expanded CAG repeats encoding a long polyglutamine tract in the huntingtin protein (Htt). Herein, we showed that striatal-derived cell lines with expanded Htt (STHdh Q111/111) expressed and secreted lower levels of BPIFB4, when compared with Htt expressing cells (STHdh Q7/7), which correlated with a defective stress response to proteasome inhibition. Overexpression of LAV-BPIFB4 in STHdh Q111/111 cells was able to rescue both the BPIFB4 secretory profile and the proliferative/survival response. According to a well-established immunomodulatory role of LAV-BPIFB4, conditioned media from LAV-BPIFB4-overexpressing STHdh Q111/111 cells were able to educate Immortalized Human Microglia—SV40 microglial cells. While STHdh Q111/111 dying cells were ineffective to induce a CD163 + IL-10high pro-resolving microglia compared to normal STHdh Q7/7, LAV-BPIFB4 transduction promptly restored the central immune control through a mechanism involving the stromal cell-derived factor-1. In line with the in vitro results, adeno-associated viral-mediated administration of LAV-BPIFB4 exerted a CXCR4-dependent neuroprotective action in vivo in the R6/2 HD mouse model by preventing important hallmarks of the disease including motor dysfunction, body weight loss, and mutant huntingtin protein aggregation. In this view, LAV-BPIFB4, due to its pleiotropic ability in both immune compartment and cellular homeostasis, may represent a candidate for developing new treatment for HD.

Background Innate immunity utilizes components of sensory signal transduction such as bitter and sweet taste receptors. In fact, empirical evidence has shown bitter and sweet taste receptors to be an integral component of antimicrobial immune response in upper respiratory tract infections. Since an efficient immune response plays a key role in the attainment of longevity, it is not surprising that the rs978739 polymorphism of the bitter taste receptor TAS2R16 gene has been shown to be associated with longevity in a population of 941 individuals ranging in age from 20 to 106 years from Calabria (Italy). There are many possible candidate genes for human longevity, however of the many genes tested, only APOE and FOXO3 survived to association in replication studies. So, it is necessary to validate in other studies genes proposed to be associated with longevity. Thus, we analysed the association of the quoted polymorphism in a population of long lived individuals (LLIs) and controls from another Italian population from Cilento. Methods The analysis has been performed on data previously obtained with genome-wide association study on a population of LLIs (age range 90–109 years) and young controls (age range 18–45 years) from Cilento (Italy). Results Statistical power calculations showed that the analysed cohort represented by 410 LLIs and 553 young controls was sufficiently powered to replicate the association between rs978739 and the longevity phenotype according to the effect size and frequencies described in the previous paper, under a dominant and additive genetic model. However, no evidence of association between rs978739 and the longevity phenotype was observed according to the additive or dominant model. Conclusion There are several reasons for the failure of the confirmation of a previous study. However, the differences between the two studies in terms of environment of the population adopted and of the criteria of inclusion have made difficult the replication of the findings.

One of the basis of exceptional longevity is the maintaining of the balance between inflammatory and anti-inflammatory networks. The monocyte-macrophages activation plays a major role in tuning the immune responses, by oscillating between patrolling-protective to inflammatory status. Longevity-associated variant (LAV) of bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) activates calcium, PKC-alpha, and eNOS, rescuing endothelial dysfunction in aged mice and inducing revascularization. The BPIFB4’s increment in serum of healthy long-living individuals (LLIs) compared to nonhealthy ones, its therapeutic potential in improving vascular homeostasis, which depends on immune system, together with its expression in bone marrow myeloid cells, suggests that LAV-BPIFB4 may improve immune regulation. Here we show that human monocytes exposed to LAV-BPIFB4 protein increased co-stimulatory molecules in resting state and reduced pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) after activating stimuli. Accordingly, a low percentage of CD69+ activated lymphocytes are found among LAV-BPIFB4-treated peripheral blood mononuclear cells (PBMCs). Moreover, human monocyte-derived dendritic cells (DCs) generated in presence of LAV-BPIFB4 secreted higher anti-(IL-10 and TGF-β) and lower pro-inflammatory (TNF-α and IL-1β) cytokines. Accordingly, LLIs’ plasma showed higher levels of circulating IL-10 and of neutralizing IL-1 receptor antagonist (IL-1RA) compared to controls. Thus, LAV-BPIFB4 effects on myeloid compartment could represent one example of a genetic predisposition carried by LLIs to protect from immunological dysfunctions.

Background The aim of this study was to analyse the role of GM allotypes, i.e. the hereditary antigenic determinants expressed on immunoglobulin polypeptide chains, in the attainment of longevity. The role played by immunoglobulin allotypes in the control of immune responses is well known as well as the role of an efficient immune response in longevity achievement. So, it is conceivable that particular GM allotypes may contribute to the generation of an efficient immune response that supports successful ageing, hence longevity. Methods In order to show if GM allotypes play a role in the achievement of longevity, we typed the DNA of 95 Long-living individuals (LLIs) and 96 young control individuals (YCs) from South Italy for GM3/17 and GM23+/− alleles. Results To demonstrate the role of GM allotypes in the attainment of longevity we compared genotype and allele frequencies of GM allotypes between LLIs and YCs. A global chi-square test (3 × 2) shows that the distribution of genotypes at the GM 3/17 locus is highly significantly different in LLIs from that observed in YCs (p

BPIFB4 is associated with exceptional longevity: four single-nucleotide polymorphisms distinguish the wild-type form from a longevity-associated variant conferring positive effects on blood pressure. The effect of a rare variant (RV; allele frequency, 4%) on blood pressure is unknown. Here, we show that overexpression of RV-BPIFB4 in ex-vivo mouse vessels impairs phosphorylation of endothelial nitric oxide synthase (eNOS), blunting acetylcholine-evoked vasorelaxation; in vivo, virally mediated overexpression of RV-BPIFB4 increases blood pressure, an action absent in eNOS-deficient mice. In humans, we found RV carriers to have increased diastolic blood pressure, a finding that was more marked in subjects on anti-hypertensive medication; moreover, recombinant RV-BPIFB4 protein impaired eNOS function in ex-vivo human vessels. Thus, RV-BPIFB4 acts directly on blood pressure homeostasis and may represent a novel biomarker of vascular dysfunction and hypertension.

Progranulin (GRN) gene mutations have been genetically associated with FTD and are present in about 23% of patients with familial FTD. However, the neurobiology of this secreted glycoprotein remains unclear. Here, we report the identification of three pedigrees of Southern Italian extraction in whom FTD segregates with autosomal dominant inheritance patterns. We present evidence that all the available patients in these three familial cases are carrying the rare GRN gene exon six deletion g10325_10331delCTGCTGT (relative to nt 1 in NG_007886.1), alias Cys157LysfsX97. This mutation was previously described in two sporadic cases but was never associated with familial cases. Our patients demonstrate heterogeneous clinical phenotypes, such as the behavioral variant (bv-FTD) in the affected men and the nonfluent/agrammatic variant of primary progressive aphasia (nfvPPA) in the affected woman. Haploinsufficiency was revealed by both quantitative RT-PCR of the gene and protein analyses. These findings provide further support for a previously proposed role for the GRN gene in the genetic etiology of FTD and its phenotypic variability.

Progranulin (GRN) gene mutations have been genetically associated with frontotemporal dementia (FTD) and are present in about 23% of patients with familial FTD. However, the neurobiology of this secreted glycoprotein remains unclear. Here, we report the identification of 3 pedigrees of Southern Italian extraction in whom FTD segregates with autosomal dominant inheritance patterns. We present evidence that all the available patients in these 3 familial cases are carrying the rare GRN gene exon 6 deletion g10325_10331delCTGCTGT (relative to nt 1 in NG_007886.1), alias Cys157LysfsX97. This mutation was previously described in 2 sporadic cases but was never associated with familial cases. Our patients demonstrate heterogeneous clinical phenotypes, such as the behavioral variant (bvFTD) in the affected men and the nonfluent/agrammatic variant of primary progressive aphasia (nfvPPA) in the affected woman. Haploinsufficiency was revealed by both quantitative real-time PCR of the gene and protein analyses. These findings provide further support for a previously proposed role for the GRN gene in the genetic etiology of FTD and its phenotypic variability.

Two new pyrazolate-based ligands, N'-[1-(3-acetyl-4-methyl-1H-pyrazol-5-yl)ethylidene]-2-(hydroxyimino)propanehydrazide (L1) and 5-[(E)-1-(2-{(E)-2-(hydroxyimino}propanoyl}hydrazono)ethyl]-4-methyl-1H-pyrazole-3-carboxylic acid (L2), were synthesized and studied for zinc(II) complexation. A set of pH-dependent UV/Vis measurements has been performed to determine the complex formation properties of L2. According to the calculations, in solution, L2 forms variously protonated mononuclear (ZnII/L2 = 1:1) and dinuclear (ZnII/L2 = 2:1) complexes. The reaction of the deprotonated ligands with hydrated ZnII salts and slow diffusion of ammonia into the reaction mixtures gave mononuclear [Zn(L1-2H)(NH3)2]center dot DMF (1) and trinuclear mu-pyrazolato-bridged [Zn3(L2-3H)2(NH3)5]center dot 4H2O (3). In both complexes, the zinc ions are in the same distorted trigonal-bipyramidal environment, coordinated to two nitrogen atoms of the ammonia and one oxygen and two nitrogen atoms of the pyrazolate and hydrazide groups. The molecular structures of all of the ligands and complexes have been elucidated by X-ray crystallography. (Less)

A series of dizinc(II) complexes based on the pyrazolate ligands 3-[(1E)-N-hydroxyethanimidoyl]-4-methyl-1H-pyrazole-5-carboxylic acid (H(3)L(1)), (1E,1'E)-1,1'-(4-methyl-1H-pyrazole-3,5-diyl)diethanone dihydrazone (HL(2)), (E,E)-(4-methyl-1H-pyrazole-3,5-diyl)bis(methylmethanone) dioxime (H(3)L(3)), (E,E)-(4-phenyl-1H-pyrazole-3,5-diyl)bis(phenylmethanone) dioxime (H(3)L(4)), and 1H-pyrazole-3,5-dicarboxylic acid (H(3)L(5)) have been synthesized and investigated as functional models of phosphoesterases, focusing on correlations between the hydrolytic activity and molecular parameters of the bimetallic core. Speciation of the various dizinc complexes in solution has been determined potentiometrically, and the structures in the solid state have been established by X-ray crystallography. The hydrolysis of two phosphoesters, an RNA model 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) and the pesticide paraoxon-ethyl (POE), promoted by the dinuclear phosphoesterase model complexes has been investigated in DMSO/buffered water (1:1) at 50 degrees C as a function of complex concentration, substrate concentration, and pH. Drastic differences in the hydrolytic activities of [Zn(2)(HL(1))(2)](0), [Zn(2)(L(2))(2)](2+), [Zn(2)(H(2)L(3))(2)](2+), and [Zn(2)(HL(5))(2)](2-) are observed and can be attributed to molecular peculiarities. Pyrazolate-bridged dinuclear zinc(II) complexes seem to provide a sufficient number of coordination sites for both activating the substrate and generating the nucleophile, where the phosphate esters are preferentially bound in a bidentate bridging fashion (in the case of HPNP) and in a monodentate fashion (in the case of POE).

The proteins belonging to the Fur family are global regulators of gene expression involved in the response to several environmental stresses and to the maintenance of divalent cation homeostasis. The Mycobacterium tuberculosis genome encodes two Fur-like proteins, FurA and a protein formerly annotated FurB. Since in this paper we show that it represents a zinc uptake regulator, we refer to it as Zur. The gene encoding Zur is found in an operon together with the gene encoding a second transcriptional regulator (Rv2358). In a previous work we demonstrated that Rv2358 is responsible for the zinc-dependent repression of the Rv2358- zur operon, favoring the hypothesis that these genes represent key regulators of zinc homeostasis. In this study we generated a zur mutant in M. tuberculosis , examined its phenotype, and characterized the Zur regulon by DNA microarray analysis. Thirty-two genes, presumably organized in 16 operons, were found to be upregulated in the zur mutant. Twenty-four of them belonged to eight putative transcriptional units preceded by a conserved 26-bp palindrome. Electrophoretic mobility shift experiments demonstrated that Zur binds to this palindrome in a zinc-dependent manner, suggesting its direct regulation of these genes. The proteins encoded by Zur-regulated genes include a group of ribosomal proteins, three putative metal transporters, the proteins belonging to early secretory antigen target 6 (ESAT-6) cluster 3, and three additional proteins belonging to the ESAT-6/culture filtrate protein 10 (CFP-10) family known to contain immunodominant epitopes in the T-cell response to M. tuberculosis infection.

We performed a whole-genome association analysis of lung tumor susceptibility using dense SNP maps ( approximately 1 SNP per 20 kb) in inbred mice. We reproduced the pulmonary adenoma susceptibility 1 (Pas1) locus identified in previous linkage studies and further narrowed this quantitative trait locus (QTL) to a region of less than 0.5 Mb in which at least two genes, Kras2 (Kirsten rat sarcoma oncogene 2) and Casc1 (cancer susceptibility candidate 1; also known as Las1), are strong candidates. Casc1 knockout mouse tumor bioassays showed that Casc1-deficient mice were susceptible to chemical induction of lung tumors. We also found three more genetic loci for lung adenoma development. Analysis of one of these candidate loci identified a previously uncharacterized gene Lasc1, bearing a nonsynonymous substitution (D102E). We found that the Lasc1 Glu102 allele preferentially promotes lung tumor cell growth. Our findings demonstrate the prospects for using dense SNP maps in laboratory mice to refine previous QTL regions and identify genetic determinants of complex traits.

Objectives: To determine the influence of the essential element status on blood concentrations of lead and other toxic metals. Design and methods: A group of 157 children from Katowice, an industrial area in Poland, was investigated for concentrations of lead and cadmium in whole blood, and mercury, selenium, zinc, copper, and magnesium in whole blood and serum. Relations between these elements, serum ferritin, hematological parameters, as well as serum selenoprotein P and glutathione peroxidase (GSH-px) were examined. Conversion factors for element concentrations (μmol to μg): lead 207.19, cadmium 112.41, mercury 200.59, selenium 78.96, magnesium 24.31, copper 63.55, and zinc 65. Results: Blood lead was negatively associated with concentrations of selenium in whole blood and serum as well as selenoprotein P and glutathione peroxidase in serum. The association was mainly apparent at low blood lead concentrations, which may indicate an influence of selenium on the kinetics of lead, rather than an effect of lead on the selenium status. Children with low serum ferritin levels had statistically higher blood cadmium levels and a tendency for higher blood lead levels, indicating increased gastrointestinal absorption of these metals at reduced iron stores. Blood lead was negatively correlated with mean corpuscular hemoglobin concentration, which may reflect the effect of lead on hemoglobin synthesis. There was an association between blood mercury and selenium, indicating a common source of intake through fish consumption. Conclusions: The results indicate that selenium and iron status may influence the kinetics of lead.

Resveratrol-a natural polyphenolic compound-was first discovered in the 1940s. Although initially used for cancer therapy, it has shown beneficial effects against most cardiovascular and cerebrovascular diseases. A large part of these effects are related to its antioxidant properties. Here we review: (a) the sources, the metabolism, and the bioavailability of resveratrol; (b) the ability of resveratrol to modulate redox signalling and to interact with multiple molecular targets of diverse intracellular pathways; (c) its protective effects against oxidative damage in cardio-cerebro-vascular districts and metabolic disorders such as diabetes; and (d) the evidence for its efficacy and toxicity in humans. The overall aim of this review is to discuss the frontiers in the field of resveratrol's mechanisms, bioactivity, biology, and health-related use.

A rexinoid, targretin, and two retinoids, 9-cis retinoic acid (9cRA) and 4-hydroxyphenylretinamide (4HPR), were examined for their effects on gene expression in rat mammary gland, liver, and lung tissues. The chemopreventive effects of these agents have largely been attributed to their ability to interact with retinoic acid receptors (RAR) and/or retinoid X receptors (RXR). Targretin interacts with the RXR receptors. 9cRA interacts with both the RAR and RXR receptors, whereas 4HPR has a moderate affinity primarily for RAR γ. Based on previous studies on mammary chemoprevention, targretin (150 mg/kg diet), 9cRA (100 mg/kg diet), and 4HPR (782 mg/kg diet), were administered to rats continually in their diet for 7 days. Tissue- and agent-specific expression differences were determined by comparing tissues from treated rats with those from rats given a control diet. There were significantly more changes associated with targretin than 9cRA or 4HPR. Only a limited number of expression changes were found with 4HPR treatment. For each organ, targretin- and 9cRA-treated tissues clustered closely together, whereas 4HPR-treated tissues clustered with the tissues from the control diet group. In contrast to 9cRA treatment, targretin treatment altered genes that involved fatty acid metabolism and modulation of various cytochromes P450 in the liver, clearly demonstrating the very disparate nature of these two retinoids. These expression signatures could provide useful pharmacodynamic biomarkers for retinoid treatment and chemoprevention. [Mol Cancer Ther 2006;5(4):1060–72]

Transplacental exposure to environmental tobacco smoke (ETS) is a possible cancer risk factor in offspring. The authors exposed pregnant Sprague-Dawley rats to a relevant dose of ETS (1 mg/m3) from gestation day 4 to days 16 or 21. They then assayed tissues for levels of 8-oxo-2'-deoxyguanosine (8-oxo-dG), a marker of oxidative deoxyribonucleic acid damage. ETS exposure ending on gestation day 16 resulted in statistically significant increases in 8-oxo-dG in maternal liver and kidney and in fetal kidney. On gestation day 21, there were significant 8-oxo-dG increases in fetal liver and brain. These gestational stage- and tissue-specific increases of 1.2- to 1.4-fold are similar to the putative relative increases in risk of human cancers related to ETS.