Diet, Pace of Biological Aging, and Risk of Dementia in the Framingham Heart Study.

OBJECTIVE: People who eat healthier diets are less likely to develop dementia, but the biological mechanism of this protection is not well understood. We tested the hypothesis that healthy diet protects against dementia because it slows the pace of biological aging. METHODS: We analyzed Framingham Offspring Cohort data. We included participants ≥60 years-old, free of dementia and having dietary, epigenetic, and follow-up data. We assessed healthy diet as long-term adherence to the Mediterranean-Dash Intervention for Neurodegenerative Delay diet (MIND, over 4 visits spanning 1991-2008). We measured the pace of aging from blood DNA methylation data collected in 2005-2008 using the DunedinPACE epigenetic clock. Incident dementia and mortality were defined using study records compiled from 2005 to 2008 visit through 2018. RESULTS: Of n = 1,644 included participants (mean age 69.6, 54% female), n = 140 developed dementia and n = 471 died over 14 years of follow-up. Greater MIND score was associated with slower DunedinPACE and reduced risks for dementia and mortality. Slower DunedinPACE was associated with reduced risks for dementia and mortality. In mediation analysis, slower DunedinPACE accounted for 27% of the diet-dementia association and 57% of the diet-mortality association. INTERPRETATION: Findings suggest that slower pace of aging mediates part of the relationship of healthy diet with reduced dementia risk. Monitoring pace of aging may inform dementia prevention. However, a large fraction of the diet-dementia association remains unexplained and may reflect direct connections between diet and brain aging that do not overlap other organ systems. Investigation of brain-specific mechanisms in well-designed mediation studies is warranted. ANN NEUROL 2024;95:1069-1079.

Ancient DNA gives new insights into a Norman Neolithic monumental cemetery dedicated to male elites.

The Middle Neolithic in western Europe is characterized by monumental funerary structures, known as megaliths, along the Atlantic façade. The first manifestations of this phenomenon occurred in modern-day France with the long mounds of the Cerny culture. Here, we present genome-wide data from the fifth-millennium BCE site of Fleury-sur-Orne in Normandy (France), famous for its impressively long monuments built for selected individuals. The site encompasses 32 monuments of variable sizes, containing the burials of 19 individuals from the Neolithic period. To address who was buried at the site, we generated genome-wide data for 14 individuals, of whom 13 are males, completing previously published data [M. Rivollat et al., Sci. Adv. 6, eaaz5344 (2020)]. Population genetic and Y chromosome analyses show that the Fleury-sur-Orne group fits within western European Neolithic genetic diversity and that the arrival of a new group is detected after 4,000 calibrated BCE. The results of analyzing uniparentally inherited markers and an overall low number of long runs of homozygosity suggest a patrilineal group practicing female exogamy. We find two pairs of individuals to be father and son, buried together in the same monument/grave. No other biological relationship can link monuments together, suggesting that each monument was dedicated to a genetically independent lineage. The combined data and documented father­son line of descent suggest a male-mediated transmission of sociopolitical authority. However, a single female buried with an arrowhead, otherwise considered a symbol of power of the male elite of the Cerny culture, questions a strictly biological sex bias in the burial rites of this otherwise "masculine" monumental cemetery.

Dairy products and brain structure in French older adults.

Among food groups with putative benefits for brain structures, dairy products (DP) have been poorly studied. The sample included participants without dementia from the ancillary brain imaging study of the Three-City cohort who were aged 65+ years, had their DP intake assessed with a FFQ at baseline and underwent an anatomical scan 3 years (n 343) or 9 years (n 195) after completing the dietary survey. The frequencies of consumption of total DP, milk and cheese were not associated with brain structure. Compared with the lowest frequency, the highest frequency of fresh DP (F-DP) consumption (< 0·5 v. > 1·5 times/d) was significantly associated with a lower medial temporal lobe volume (MTLV) (ß = -1·09 cm3, 95 % CI - 1·83, -0·36) 9 years later. In this population-based study of older adults, the consumption of F-DP more than 1·5 times/d was associated with a lower MTLV, which is considered an early biomarker of Alzheimer's disease, 9 years later. This original study should be replicated in different settings before conclusions are drawn.

Impact of volemia at admission on the effect of collateral status on functional outcomes in patients undergoing endovascular thrombectomy.

BACKGROUND AND PURPOSE: Having good collaterals is associated with better clinical outcomes in patients undergoing endovascular thrombectomy. This study aims to evaluate whether the effect of collateral status on functional outcomes is modified by volemia at admission. METHODS: This is a single-center, retrospective analysis of patients who had acute proximal anterior circulation occlusion and underwent endovascular thrombectomy between January 2019 and June 2022. Volemia at admission, evaluated by blood urea nitrogen-to-creatinine ratio, was used to dichotomize patients into dehydrated and hydrated groups. The primary outcome was functional independence (90-day modified Rankin Scale score = 0-2). Secondary outcomes were the rates of successful reperfusion, 24-h symptomatic intracranial hemorrhage, and 90-day all-cause mortality. Multivariable logistic regression analysis was used to assess the interaction between collateral status and volemia at admission on outcomes. RESULTS: A total of 290 patients were enrolled, among whom having good collaterals was associated with functional independence (adjusted odds ratio [OR] = 2.71, 95% confidence interval [CI] = 1.41-5.22, p = 0.003). Having good collaterals benefited dehydrated patients (adjusted OR = 3.33, 95% CI = 1.45-7.63, p = 0.004) but not hydrated patients (adjusted OR = 2.21, 95% CI = 0.73-6.68, p = 0.161). However, an interaction between collaterals and volemia at admission on functional independence was not observed (p = 0.319). The rates of successful reperfusion, symptomatic intracerebral hemorrhage, and all-cause mortality were similar between those with good and poor collaterals in both dehydrated and hydrated patients. CONCLUSIONS: The effect of collateral status on the functional independence of patients undergoing thrombectomy is not modified by volemia at admission.

Postoperative blood glucose increase is associated with futile recanalization in patients with successful thrombectomy: a retrospective study.

BACKGROUND: Timely recognition of futile recanalization might enable a prompt response and an improved outcome in post-thrombectomy patients. This study aims to evaluate whether postoperative blood glucose increase (BGI) could act as an indicator of futile recanalization in patients receiving a successful thrombectomy. METHODS: This is a single-center, retrospective analysis of patients with anterior circulation large-vessel occlusion and successful thrombectomy between February 2019 and June 2022. BGI was defined as a higher level of blood glucose at the first postoperative morning than at admission. Futile recanalization was defined as patients with a modified Rankin Scale score of 3-6 at 90 days after onset. Multivariable binary logistic regression was used to assess the association of BGI with futile recanalization. RESULTS: A total of 276 patients were enrolled, amongst which 120 patients (43.5%) had BGI. Futile recanalization was more prevalent among patients with BGI compared to those without (70.0 vs. 49.4%, P = 0.001). After adjusting for potential confounders, BGI was associated with a higher likelihood of futile recanalization (adjusted OR: 2.97, 95%CI: 1.50-5.86, P = 0.002). This association was consistently observed regardless of diabetes history, occlusion site, time from symptom onset to groin puncture, or reperfusion status. CONCLUSION: Our findings support BGI serving as an indicator of futile recanalization in patients with anterior circulation large-vessel occlusion and successful thrombectomy.

Dietary factors and brain health.

PURPOSE OF REVIEW: Nutrition is a complex exposure (i.e., the food exposome) that influences brain function and health through multiple pathways. We review recent epidemiological studies that have improved the characterization of the food exposome and brain health in humans and have revealed promising nutrition-based strategies to prevent cognitive aging. RECENT FINDINGS: A selection of epidemiological research from the past 18 months of both observational and clinical studies is presented, with a focus on novel findings, including novel nutrient and diet patterns, diet-related approaches to rescue brain energetics defects in aging, and biomarker-based studies to decipher specific neurobiological pathways of nutrition and brain health. SUMMARY: Although healthy diets such as the Mediterranean diet promote brain health throughout life, specific diets, such as the Mediterranean-Dietary Approaches to Stop Hypertension Intervention for Neurodegenerative Delay diet, or specific nutrients (LC n-3 polyunsaturated fatty acids, carotenoids, vitamin D, B vitamins, polyphenols) alone or in combination, may prevent cognitive aging. Diet management approaches to rescue brain energetics defects such as the Modified Mediterranean-ketogenic diet may be promising to prevent neurodegenerative diseases. Expanding research also suggests that promotion of a healthy gut microbiome through prebiotic foods may preserve the diet-gut-brain axis with aging. Future studies should explore more individualized preventive approaches through a 'precision nutrition' framework.

Opportunities for Molecular Imaging in Multiple Sclerosis Management: Linking Probe to Treatment.

Imaging has been a critical component of multiple sclerosis (MS) management for nearly 40 years. The visual information derived from structural MRI, that is, signs of blood-brain barrier disruption, inflammation and demyelination, and brain and spinal cord atrophy, are the primary metrics used to evaluate therapeutic efficacy in MS. The development of targeted imaging probes has expanded our ability to evaluate and monitor MS and its therapies at the molecular level. Most molecular imaging probes evaluated for MS applications are small molecules initially developed for PET, nearly half of which are derived from U.S. Food and Drug Administration-approved drugs and those currently undergoing clinical trials. Superparamagnetic and fluorinated particles have been used for tracking circulating immune cells (in situ labeling) and immunosuppressive or remyelinating therapeutic stem cells (ex vivo labeling) clinically using proton (hydrogen 1 [1H]) and preclinically using fluorine 19 (19F) MRI. Translocator protein PET and 1H MR spectroscopy have been demonstrated to complement imaging metrics from structural (gadolinium-enhanced) MRI in nine and six trials for MS disease-modifying therapies, respectively. Still, despite multiple demonstrations of the utility of molecular imaging probes to evaluate the target location and to elucidate the mechanisms of disease-modifying therapies for MS applications, their use has been sparse in both preclinical and clinical settings.

CEST MRI and MALDI imaging reveal metabolic alterations in the cervical lymph nodes of EAE mice.

BACKGROUND: Multiple sclerosis (MS) is a neurodegenerative disease, wherein aberrant immune cells target myelin-ensheathed nerves. Conventional magnetic resonance imaging (MRI) can be performed to monitor damage to the central nervous system that results from previous inflammation; however, these imaging biomarkers are not necessarily indicative of active, progressive stages of the disease. The immune cells responsible for MS are first activated and sensitized to myelin in lymph nodes (LNs). Here, we present a new strategy for monitoring active disease activity in MS, chemical exchange saturation transfer (CEST) MRI of LNs. METHODS AND RESULTS: We studied the potential utility of conventional (T2-weighted) and CEST MRI to monitor changes in these LNs during disease progression in an experimental autoimmune encephalomyelitis (EAE) model. We found CEST signal changes corresponded temporally with disease activity. CEST signals at the 3.2 ppm frequency during the active stage of EAE correlated significantly with the cellular (flow cytometry) and metabolic (mass spectrometry imaging) composition of the LNs, as well as immune cell infiltration into brain and spinal cord tissue. Correlating primary metabolites as identified by matrix-assisted laser desorption/ionization (MALDI) imaging included alanine, lactate, leucine, malate, and phenylalanine. CONCLUSIONS: Taken together, we demonstrate the utility of CEST MRI signal changes in superficial cervical LNs as a complementary imaging biomarker for monitoring disease activity in MS. CEST MRI biomarkers corresponded to disease activity, correlated with immune activation (surface markers, antigen-stimulated proliferation), and correlated with LN metabolite levels.

Interest of novel N-alkylpyridinium-indolizine hybrids in the field of Alzheimer's disease: Synthesis, characterization and evaluation of antioxidant activity, cholinesterase inhibition, and amyloid fibrillation interference.

A small library of molecules combining indolizine and N-alkyl pyridinium was synthesized and evaluated in a multi-target-directed-ligand strategy for Alzheimer's disease (AD) treatment. The new compounds were classified in three series depending on the number of methylene residues linking the two heterocycles (Ind-PyCx with x = 0, 2 or 3). The molecules were synthesized from the corresponding bis-pyridines by two-step formation of the indolizine core including mono-alkylation of pyridine and 1,3-dipolar cycloaddition with an alkylpropiolate. Their activities against AD's key-targets were evaluated in vitro: acetyl- and butyrylcholinesterase (AChE and BChE) inhibition, antioxidant properties and inhibition of amyloid fibril formation. None of the three series showed significant activities against all the targets. The Ind-PyC2 and Ind-PyC3 series are active on eeAChE and hAChE (µM IC50 values). Most of the positively charged molecules from these two series also appeared active against eqBChE, however they lost their activity on hBChE. Comparative molecular modeling of 13 and 15 docked in hAChE and hBChE highlighted the importance of the substituent (p-methoxybenzoyl or methyloxycarbonyl, respectively) located on the indolizine C-3 for the binding. The larger molecule 13 fits more tightly at the active site of the two enzymes than 15 that shows a larger degree of freedom. The Ind-PyC2 and Ind-PyC3 hybrids displayed some antioxidant activity when tested at 750 µg/mL (up to 95% inhibition of DPPH radical scavenging for 10). In both series, most hybrids were also able to interact with amyloid fibers, even if the inhibitory effect was observed at a high 100 µM concentration. The Ind-PyC0 molecules stand out completely due to their spectroscopic properties which prevent their evaluation by Ellman's and ThT assays. However, these molecules showed interesting features in the presence of preformed fibers. In particular, the strong increase in fluorescence of 3 in the presence of amyloid fibers is very promising for its use as a fibrillation fluorescent reporter dye.

FUT7 promotes the malignant transformation of follicular thyroid carcinoma through α1,3-fucosylation of EGF receptor.

Aberrant protein glycosylation is involved in many diseases including cancer. This study investigated the role of fucosyltransferase VII (FUT7) in the progression of follicular thyroid carcinoma (FTC). FUT7 expression was found to be upregulated in FTC compared to paracancerous thyroid tissue, and in FTC with T2 stage of TMN classification compared to FTC with T1 stage. FUT7 overexpression promoted cell proliferation, epithelial-mesenchymal transition (EMT), and the migration and invasion of primary FTC cell line FTC-133. Consistently, FUT7 knock-down inhibited cell proliferation, EMT, as well as the migration and invasion of the metastatic FTC cell line FTC-238. Mechanistic investigation revealed that FUT7 catalyzed the α1,3-fucosylation of epidermal growth factor receptor (EGFR) in FTC cells. The extent of glycan α1,3-fucosylation on EGFR was positively correlated with the activation of EGFR in the presence/absence of epidermal growth factor (EGF) treatment. Furthermore, FUT7 was shown to enhance EGF-induced progression of FTC cells through mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways. These findings provide a new perspective on FUT7 that may be a novel diagnostic and therapeutic target of FTC.

Brief exposure to hyperglycemia activates dendritic cells in vitro and in vivo.

Dendritic cells are key players in regulating immunity. These cells both activate and inhibit the immune response depending on their cellular environment. Their response to hyperglycemia, a condition common amongst diabetics wherein glucose is abnormally elevated, remains to be elucidated. In this study, the phenotype and immune response of dendritic cells exposed to hyperglycemia were characterized in vitro and in vivo using the streptozotocin-induced diabetes model. Dendritic cells were shown to be sensitive to hyperglycemia both during and after differentiation from bone marrow precursor cells. Dendritic cell behavior under hyperglycemic conditions was found to vary by phenotype, among which, tolerogenic dendritic cells were particularly sensitive. Expression of the costimulatory molecule CD86 was found to reliably increase when dendritic cells were exposed to hyperglycemia. Additionally, hydrogel-based delivery of the anti-inflammatory molecule interleukin-10 was shown to partially inhibit these effects in vivo.

Monitoring diffuse injury during disease progression in experimental autoimmune encephalomyelitis with on resonance variable delay multiple pulse (onVDMP) CEST MRI.

Multiple sclerosis (MS) is an autoimmune disorder that targets myelin proteins and results in extensive damage in the central nervous system in the form of focal lesions as well as diffuse molecular changes. Lesions are currently detected using T1-weighted, T2-weighted, and gadolinium-enhanced magnetic resonance imaging (MRI); however, monitoring such lesions has been shown to be a poor predictor of disease progression. Chemical exchange saturation transfer (CEST) MRI is sensitive to many of the biomolecules in the central nervous system altered in MS that cannot be detected using conventional MRI. We monitored disease progression in an experimental autoimmune encephalomyelitis (EAE) model of MS using on resonance variable delay multiple pulse (onVDMP) CEST MRI. Alterations in onVDMP signal were observed in regions responsible for hindlimb function throughout the central nervous system. Histological analysis revealed glial activation in areas highlighted in onVDMP CEST MRI. onVDMP signal changes in the 3rd ventricle preceded paralysis onset that could not be observed with conventional MRI techniques. Hence, the onVDMP CEST MRI signal has potential as a novel imaging biomarker and predictor of disease progression in MS.

Cranial trepanation and healing process in modern patients-Bioarchaeological and anthropological implications.

The diagnosis of cranial trepanation in the archaeological record has always been a big challenge for archaeologists. The identification is first and foremost based on the shape and edges of the opening, and in cases where the individual survived, the shape and edges resulted from both the surgical procedure and the healing process. Because of the lack of reliable data on the osseous remodelling process of the skull, it is difficult to distinguish true trepanations from some pathological or physiological defects called pseudotrepanations. Furthermore, it is challenging to investigate the post-operative survival time. The purpose of this paper was to summarize observations made on a total of 90 archaeological samples of cranial lesions interpreted as 'partially' or 'completely' healed trepanations, and compare them with 14 modern case studies. Observations made on monitoring post-operative scans of modern patients, at varying times after craniotomy, provided a preliminary timetable for the successive post-surgery bony changes in the skull, and confirmed that the process of osseous remodelling does lead to smooth and rounded edges of the profile of the opening. However, contrary to what has been observed in several archaeological case studies, none of the cases shows a complete closure of the cranial vault. The sharp vertical edges of the opening become bevelled at late stages of healing, which has to be taken into consideration when interpreting the procedures and methods in past population. By bridging bioarchaeology and medical sciences, this study adds to previous discussions on the diagnosis of healed trepanation in the archaeological record, by providing detailed descriptions of morphological changes at various healing stages, that may help archaeologists to identify ancient trepanations more accurately.

Blood polyunsaturated omega-3 fatty acids, brain atrophy, cognitive decline, and dementia risk.

INTRODUCTION: We searched for consistent associations of an omega-3 index in plasma (sum of eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) with several dementia-related outcomes in a large cohort of older adults. METHODS: We included 1279 participants from the Three-City study, non-demented at the time of blood measurements at baseline, with face-to-face neuropsychological assessment and systematic detection of incident dementia over a 17-year follow-up. An ancillary study included 467 participants with up to three repeated brain imaging exams over 10 years. RESULTS: In multivariable models, higher levels of plasma EPA+DHA were consistently associated with a lower risk of dementia (hazard ratio for 1 standard deviation = 0.87 [95% confidence interval, 0.76-0.98]), and a lower decline in global cognition (P = .04 for change over time), memory (P = .06), and medial temporal lobe volume (P = .02). DISCUSSION: This prospective study provides compelling evidence for a relationship between long-chain omega-3 fatty acids levels and lower risks for dementia and related outcomes.

CEST MRI with distribution-based analysis for assessment of early stage disease activity in a mouse model of multiple sclerosis: An initial study.

Imaging biomarkers that can detect pathological changes at an early stage of multiple sclerosis (MS) may allow earlier therapeutic intervention with an improved outcome. Using a mouse model of MS, termed as experimental autoimmune encephalomyelitis (EAE), we performed chemical exchange saturation transfer (CEST) MRI at a very early stage before symptom onset (6 days post-induction) for assessment of changes in tissues that appear "normal" with conventional MRI. The collected CEST Z-spectra signals (Ssat /S0 ) were analyzed using a histogram-guided method to determine the contributions from various offset frequencies. Histogram analysis showed that EAE mice exhibit a more heterogeneous distribution with lower peak heights in the hindbrain compared with naïve mice at saturation offsets of 1 and 2 ppm. At these two offsets, both the mean Ssat /S0 and the mean MTRasym values in the cerebellum and brain stem are significantly different between EAE and naïve mice (P < 0.05). Immunofluorescent staining validated the presence of neuroinflammation, with IBA1-positive cells detected throughout the hindbrain including the cerebellum and brain stem. Follow-up MRI at the symptom onset (score = 1.5-2.5, 13 days post-induction) confirmed gadolinium-enhanced periventricular lesions. CEST Z-spectra signals also changed by this time. The proposed three-level histogram-oriented analysis is simple to execute and robust for detecting subtle changes in Z-spectra signals, which does not require a priori knowledge of damage locations or contributing offset components. CEST MRI signals at 1 and 2 ppm were sensitive to the subtle pathological changes at an early stage in EAE mice, and have potential as novel imaging biomarkers complementary to functional and physiological MRI measures.

NCAM Mimetic Peptides: Potential Therapeutic Target for Neurological Disorders.

The neural cell adhesion molecule (NCAM) plays a pivotal role in the development and maintenance of the nervous system via homophilic (NCAM-NCAM) and heterophilic (NCAM-other molecules) interactions. Many synthetic peptides have been engineered to mimic these interactions and induce NCAM-downstream signaling pathways. Such NCAM mimetics have displayed neuritogenic and neuroprotective properties, as well as synaptic modulation in vitro and in vivo. Furthermore, they have been used successfully in preclinical studies to treat neurological disorders including stroke, traumatic brain injury and Alzheimer's disease. This review focuses on recent progress in the development of NCAM mimetic peptides, in particular, on establishing C3, plannexin, and FGL as therapeutic candidates for neurological disorders.

Structural insights and membrane binding properties of MGD1, the major galactolipid synthase in plants.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipid components of photosynthetic membranes, and hence the most abundant lipids in the biosphere. They are essential for assembly and function of the photosynthetic apparatus. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by MGDG synthase 1 (MGD1), which transfers a galactosyl residue from UDP-galactose to diacylglycerol (DAG). MGD1 is a monotopic protein that is embedded in the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids. Here we present two crystal structures of MGD1: one unliganded and one complexed with UDP. MGD1 has a long and flexible region (approximately 50 amino acids) that is required for DAG binding. The structures reveal critical features of the MGD1 catalytic mechanism and its membrane binding mode, tested on biomimetic Langmuir monolayers, giving insights into chloroplast membrane biogenesis. The structural plasticity of MGD1, ensuring very rapid capture and utilization of DAG, and its interaction with anionic lipids, possibly driving the construction of lipoproteic clusters, are consistent with the role of this enzyme, not only in expansion of the inner envelope membrane, but also in supplying MGDG to the outer envelope and nascent thylakoid membranes.

Probing the acceptor active site organization of the human recombinant ß1,4-galactosyltransferase 7 and design of xyloside-based inhibitors.

Among glycosaminoglycan (GAG) biosynthetic enzymes, the human ß1,4-galactosyltransferase 7 (hß4GalT7) is characterized by its unique capacity to take over xyloside derivatives linked to a hydrophobic aglycone as substrates and/or inhibitors. This glycosyltransferase is thus a prime target for the development of regulators of GAG synthesis in therapeutics. Here, we report the structure-guided design of hß4GalT7 inhibitors. By combining molecular modeling, in vitro mutagenesis, and kinetic measurements, and in cellulo analysis of GAG anabolism and decorin glycosylation, we mapped the organization of the acceptor binding pocket, in complex with 4-methylumbelliferone-xylopyranoside as prototype substrate. We show that its organization is governed, on one side, by three tyrosine residues, Tyr(194), Tyr(196), and Tyr(199), which create a hydrophobic environment and provide stacking interactions with both xylopyranoside and aglycone rings. On the opposite side, a hydrogen-bond network is established between the charged amino acids Asp(228), Asp(229), and Arg(226), and the hydroxyl groups of xylose. We identified two key structural features, i.e. the strategic position of Tyr(194) forming stacking interactions with the aglycone, and the hydrogen bond between the His(195) nitrogen backbone and the carbonyl group of the coumarinyl molecule to develop a tight binder of hß4GalT7. This led to the synthesis of 4-deoxy-4-fluoroxylose linked to 4-methylumbelliferone that inhibited hß4GalT7 activity in vitro with a Ki 10 times lower than the Km value and efficiently impaired GAG synthesis in a cell assay. This study provides a valuable probe for the investigation of GAG biology and opens avenues toward the development of bioactive compounds to correct GAG synthesis disorders implicated in different types of malignancies.