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Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by severe deficits in social communication and interaction, repetitive movements, abnormal focusing on objects, or activity that can significantly affect the quality of life of the afflicted. Neuronal and glial cells have been implicated. It has a genetic component but can also be triggered by environmental factors or drugs. For example, prenatal exposure to valproic acid or acetaminophen, or ingestion of propionic acid, can increase the risk of ASD. Recently, epigenetic influences on ASD have come to the forefront of investigations on the etiology, prevention, and treatment of this disorder. Epigenetics refers to DNA modifications that alter gene expression without making any changes to the DNA sequence. Although an increasing number of pharmaceuticals and environmental chemicals are being implicated in the etiology of ASD, here, we specifically focus on the molecular influences of the abovementioned chemicals on epigenetic alterations in neuronal and glial cells and their potential connection to ASD. We conclude that a better understanding of these phenomena can lead to more effective interventions in ASD.

Background: Sexual dysfunction is a common side effect of Serotonergic antidepressants (SA) treatment, and persists in some patients despite drug discontinuation, a condition termed post-SSRI sexual dysfunction (PSSD). The risk for PSSD is unknown but is thought to be rare and difficult to assess. This study aims to estimate the risk of erectile dysfunction (ED) and PSSD in males treated with SAs., Methods: A 19-year retrospective cohort analysis was conducted using a computerized database of the largest HMO in Israel. ED was defined by phosphodiesterase-5 inhibitors prescriptions. 12,302 males aged 21-49 met the following criteria: non-smokers, no medical or psychiatric comorbidities or medications associated with ED, no alcohol or substance use. Logistic regression was used for estimation of ED risk in SA-treated subjects compared to non-SA-treated controls, assessed with and without the effects of age, body mass index (BMI), socioeconomic status (SES), depression and anxiety, yielding crude and adjusted odds ratios (cOR and aOR, respectively)., Results: SAs were associated with an increased risk for ED (cOR = 3.6, p < 0.000001, 95% CI  2.8-4.8), which remained significant after adjusting for age, SES, BMI, depression and anxiety (aOR = 3.2, p < 0.000001, 95% CI  2.3-4.4). The risk for PSSD was 1 in 216 patients (0.46%) treated with SAs. The prevalence of PSSD was 4.3 per 100,000., Conclusions: This work offers a first assessment of the small but significant risk of irreversible ED associated with the most commonly prescribed class of antidepressants which should enhance the process of receiving adequate informed consent for therapy., (© 2023. The Author(s).)

Parkinson's disease (PD) is a progressive neurodegenerative disease associated with loss of dopaminergic neurons in the substantia nigra pars compacta. Although aging is the primary cause, environmental and genetic factors have also been implicated in its etiology. In fact, the sporadic nature of PD (i.e., unknown etiology) renders the uncovering of the exact pathogenic mechanism(s) or development of effective pharmacotherapies challenging. In search of novel neuroprotectants, we showed that butyrate (BUT), a short-chain fatty acid, protects against salsolinol (SALS)-induced toxicity in human neuroblastoma-derived SH-SY5Y cells, which are considered an in-vitro model of PD. Dihydromyricetin (DHM), a flavonoid derived from Asian medicinal plant, has also shown effectiveness against oxidative damage and neuroinflammation, hallmarks of neurodegenerative diseases. Here we show that pretreatment of SH-SY5Y cells with DHM concentration-dependently prevented SALS-induced toxicity and that a combination of DHM and BUT resulted in a synergistic protection. The effects of both DHM and BUT in turn could be completely blocked by flumazenil (FLU), a GABA A antagonist acting at benzodiazepine receptor site, and by bicuculline (BIC), a GABA A antagonist acting at orthosteric site. Beta-hydroxybutyrate (BHB), a free fatty acid 3 (FA3) receptor antagonist, also fully blocked the protective effect of DHM. BHB was shown previously to only partially block the protective effect of BUT. Thus, there are some overlaps and some distinct differences in protective mechanisms of DHM and BUT against SALS-induced toxicity. It is suggested that a combination of DHM and BUT may have therapeutic potential in PD. However, further in-vivo verifications are necessary., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Throughout the animal kingdom, our two chemical senses, olfaction and gustation, are defined by two primary factors: genomic architecture of the organisms and their living environment. During the past three years of the global COVID-19 pandemic, these two sensory modalities have drawn much attention at the basic science and clinical levels because of the strong association of olfactory and gustatory dysfunction with viral infection. Loss of our sense of smell alone, or together with a loss of taste, has emerged as a reliable indicator of COVID-19 infection. Previously, similar dysfunctions have been detected in a large cohort of patients with chronic conditions. The research focus remains on understanding the persistence of olfactory and gustatory disturbances in the post-infection phase, especially in cases with long-term effect of infection (long COVID). Also, both sensory modalities show consistent age-related decline in studies aimed to understand the pathology of neurodegenerative conditions. Some studies using classical model organisms show an impact on neural structure and behavior in offspring as an outcome of parental olfactory experience. The methylation status of specific odorant receptors, activated in parents, is passed on to the offspring. Furthermore, experimental evidence indicates an inverse correlation of gustatory and olfactory abilities with obesity. Such diverse lines of evidence emerging from basic and clinical research studies indicate a complex interplay of genetic factors, evolutionary forces, and epigenetic alterations. Environmental factors that regulate gustation and olfaction could induce epigenetic modulation. However, in turn, such modulation leads to variable effects depending on genetic makeup and physiological status. Therefore, a layered regulatory hierarchy remains active and is passed on to multiple generations. In the present review, we attempt to understand the experimental evidence that indicates variable regulatory mechanisms through multilayered and cross-reacting pathways. Our analytical approach will add to enhancement of prevailing therapeutic interventions and bring to the forefront the significance of chemosensory modalities for the evaluation and maintenance of long-term health.

Epigenetic changes in pancreatic beta cells caused by sustained high blood glucose levels, as seen in prediabetic conditions, may contribute to the etiology of diabetes. To delineate a direct cause and effect relationship between high glucose and epigenetic changes, we cultured human pancreatic beta cells derived from induced pluripotent stem cells and treated them with either high or low glucose, for 14 days. We then used the Arraystar 4x180K HG19 RefSeq Promoter Array to perform whole-genome DNA methylation analysis. A total of 478 gene promoters, out of a total of 23,148 present on the array (2.06%), showed substantial differences in methylation ( p < 0.01). Out of these, 285 were hypomethylated, and 193 were hypermethylated in experimental vs. control. Ingenuity Pathway Analysis revealed that the main pathways and networks that were differentially methylated include those involved in many systems, including those related to development, cellular growth, and proliferation. Genes implicated in the etiology of diabetes, including networks involving glucose metabolism, insulin secretion and regulation, and cell cycle regulation, were notably altered. Influence of upstream regulators such as MRTFA, AREG, and NOTCH3 was predicted based on the altered methylation of their downstream targets. The study validated that high glucose levels can directly cause many epigenetic changes in pancreatic beta cells, suggesting that this indeed may be a mechanism involved in the etiology of diabetes., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this manuscript., (Copyright © 2023 Rasha A. Alhazzaa et al.)

Toxicity induced by binge alcohol drinking, particularly in adolescent and young adults, is of major medical and social consequence. Recently, we reported that butyrate, a short chain fatty acid, can protect against ethanol (ETOH)-induced toxicity in an in vitro model. In this study, we sought to evaluate the potential effectiveness of dihydromyricetin (DHM), a natural bioactive flavonoid, alone or in combination with butyrate in the same model. Exposure of SH-SY5Y cells for 24 h to 500 mM ETOH resulted in approximately 40% reduction in cell viability, which was completely prevented by 0.1 μM DHM. Combinations of DHM and butyrate provided synergistic protection against alcohol toxicity. Whereas butyrate effect was shown to be mediated primarily through fatty acid receptor 3 activation, DHM protection appears to be mediated primarily via benzodiazepine receptor site of GABA A receptor. This is based on the finding that DHM's effect could be completely prevented by pretreatment with flumazenil, a selective antagonist at this site, but not by bicuculline, a selective antagonist at the actual GABA A receptor binding site. These findings suggest potential utility of DHM alone or in combination with butyrate against ETOH-induced toxicity., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: A set of enduring conditions have been reported in the literature involving persistent sexual dysfunction after discontinuation of serotonin reuptake inhibiting antidepressants, 5 alpha-reductase inhibitors and isotretinoin., Objective: To develop diagnostic criteria for post-SSRI sexual dysfunction (PSSD), persistent genital arousal disorder (PGAD) following serotonin reuptake inhibitors, post-finasteride syndrome (PFS) and post-retinoid sexual dysfunction (PRSD)., Methods: The original draft was designed using data from two published case series (Hogan et al., 2014 and Healy et al., 2018), which represent the largest public collections of data on these enduring conditions. It was further developed with the involvement of a multidisciplinary panel of experts., Results: A set of criteria were agreed upon for each of the above conditions. Features of PSSD, PFS and PRSD commonly include decreased genital and orgasmic sensation, decreased sexual desire and erectile dysfunction. Ancillary non-sexual symptoms vary depending on the specific condition but can include emotional blunting and cognitive impairment. PGAD presents with an almost mirror image of unwanted sensations of genital arousal or irritability in the absence of sexual desire. A new term, post-SSRI asexuality, is introduced to describe a dampening of sexual interest and pleasure resulting from a pre-natal or pre-teen exposure to a serotonin reuptake inhibitor., Conclusions: These criteria will help in both clinical and research settings. As with all criteria, they will likely need modification in the light of developments.

Alcohol use disorder (AUD), brought about by excessive alcohol use, is associated with damages to several organs including the brain. Chronic excessive use of alcohol can compromise intestinal integrity, leading to changes in gut microbiota (GM) composition known as dysbiosis. Dysbiosis, by disruption of the gut-brain axis (GBA), further exacerbates the deleterious effects of alcohol. One of the fermentation by-products of GM is butyrate (BUT), a short-chain fatty acid (SCFA) that plays an important role in maintaining homeostasis of the GBA. Alcohol metabolism results in formation of acetaldehyde, a highly reactive compound that reacts with dopamine in the brain to form toxic adducts such as salsolinol. Recent studies indicate potential neuro-protective effects of BUT against various toxicants including salsolinol. Here, we sought to investigate whether BUT can also protect against alcohol toxicity. Pretreatment of neuroblastoma-derived SH-SY5Y cells with 500 mM ethanol (ETOH) for 24 h resulted in approximately 40% reduction in cell viability, which was totally blocked by 10 µM of either BUT or AR 420,626 (AR), a selective fatty acid 3 receptor (FA3R) agonist. The neuro-protective effects of both BUT and AR were significantly (80%) attenuated by beta-hydroxy butyrate (BHB), a selective FA3R antagonist. Interestingly, combination of BUT and AR resulted in synergistic protection against ETOH, which was totally blocked by BHB. These findings suggest potential utility of butyrate and/or FA3R agonists against ETOH-induced toxicity., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Parkinson's disease (PD), a progressive neurodegenerative disorder, is associated with the destruction of dopamine neurons in the substantia nigra (SN) and the formation of Lewy bodies in basal ganglia. Risk factors for PD include aging, as well as environmental and genetic factors. Recent converging reports suggest a role for the gut microbiome and epigenetic factors in the onset and/or progression of PD. Of particular relevance and potential therapeutic targets in this regard are histone deacetylases (HDACs), enzymes that are involved in chromatin remodeling. Butyrate, a short-chain fatty acid (FA) produced in the gut and presumably acting via several G protein-coupled receptors (GPCRs) including FA3 receptors (FA3Rs), is a well-known HDAC inhibitor that plays an important role in maintaining homeostasis of the gut-brain axis. Recently, its significance in regulation of some critical brain functions and usefulness in neurodegenerative diseases such as PD has been suggested. In this study we sought to determine whether butyrate may have protective effects against salsolionl (SALS)-induced toxicity in SH-SY5Y cells. SALS, an endogenous product of aldehyde and dopamine condensation, may be selectively toxic to dopaminergic neurons. SH-SY5Y cells, derived from human neuroblastoma cells, are used as a model of these neurons. Exposure of SH-SY5Y cells for 24 h to 400 μM SALS resulted in approximately 60% cell death, which was concentration-dependently prevented by butyrate. The effects of butyrate in turn were significantly attenuated by beta-hydroxy butyrate (BHB), a selective FA3R antagonist. Moreover, a selective FA3R agonist (AR 420626) also provided protective effects against SALS, which was totally blocked by BHB. These findings provide further support that butyrate or an agonist of FA3R may be of therapeutic potential in PD.

The C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) may be of therapeutic potential in motor impairments associated with Parkinson disease (PD). Since depression is a common co-morbid condition with PD, we undertook this study to determine whether Hc-TeTx might also show antidepressant-like properties and whether central brain-derived neurotrophic factor (BDNF) and/or tumor necrosis factor (TNF)-alpha are also affected by it. Adult male Wistar-Kyoto rats, a putative animal model of depression, were treated with various doses of Hc-TeTx (0, 20, 40 and 60 μg/kg, IM) and their performance in the open field locomotor activity (OFLA) as well as in the forced swim test (FST) was evaluated at 24 h, one week and two weeks after the single injection. A separate group of rats were injected with 60 μg/kg Hc-TeTx and sacrificed 24 h later for neurochemical evaluations. Hc-TeTx resulted in a dose-dependent decrease in immobility score after 24 h, whereas OFLA was not affected. Concomitant with the 24 h behavioral effects, the levels of hippocampal and frontal cortical BDNF were significantly increased, whereas the levels of TNF-alpha in both these areas were significantly decreased. The decrease in immobility scores following higher doses of Hc-TeTx were still evident after one week, but not 2 weeks of rest. These results indicate long lasting antidepressant effects of a single Hc-TeTx dose and suggest potential utility of Hc-TeTx in PD-depression co-morbidity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Manganese (Mn) and iron (Fe) are trace elements that are essential for proper growth and physiological functions as both play critical role in a variety of enzymatic reactions. At high concentrations, however, they can be toxic and cause neurodegenerative disorders, particularly Parkinson-like syndromes. Nicotine, on the other hand, has been shown to have neuroprotective effects against various endogenous or exogenous toxins that selectively damage the dopaminergic cells. These cells include neuroblastoma-derived SH-SY5Y cells which express significant dopaminergic activity. However, practically no information on possible neuroprotective effects of nicotine against toxicity induced by trace elements is available. Therefore, in this study we investigated the effects of nicotine on toxicity induced by manganese or iron in these cells. Exposure of SH-SY5Y cells for 24 h to manganese (20 μM) or iron (20 μM) resulted in approximately 30% and 35% toxicity, respectively. Pretreatment with nicotine (1 μM) completely blocked the toxicities of Mn and Fe. The effects of nicotine, in turn, were blocked by selective nicotinic receptor antagonists. Thus, dihydro-beta erythroidine (DHBE), a selective alpha 4-beta 2 subtype antagonist and methyllycaconitine (MLA), a selective alpha7 antagonist, as well as mecamylamine, a non-selective nicotinic antagonist all dose-dependently blocked the protective effects of nicotine against both Mn and Fe. These findings provide further support for the potential utility of nicotine or nicotinic agonists in Parkinson's disease-like neurodegenerative disorders, including those that might be precipitated by trace elements, such as Fe and Mn. Moreover, both alpha4-beta2 and alpha7 nicotinic receptor subtypes appear to mediate the neuroprotective effects of nicotine against toxicity induced by these two trace metals., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

With the aging population growing and the incidence of neurodegenerative diseases on the rise, the researchers in the field are yet more urgently challenged to slow and/or reverse the devastating consequences of such progression. The challenge is further enforced by psychiatric co-morbid conditions, particularly the feeling of despair in these population. Fortunately, as our understanding of the neurobiological substrates of maladies affecting the central nervous system increases, more therapeutic options are also presented. In this short review while providing evidence of shared biological substrates between Parkinson's disease and depression, novel therapeutic targets and drugs are suggested. The emphasis will be on neuroplasticity underscored by roles of neurotrophic and inflammatory factors. Examples of few therapeutic drugs as well as future directions are also touched upon., (© 2019 Elsevier Inc. All rights reserved.)

Background: Appreciable evidence suggest that dysbiosis in microbiota, reflected in gut microbial imbalance plays a key role in the pathogenesis of neuropsychiatric disorders including depression and inflammatory diseases. Recently, the antidepressant properties of ketamine have gained prominence due to its fast and long lasting effects. Additional uses for ketamine in inflammatory disorders such as irritable bowel syndrome have been suggested. However, ketamine's exact mechanism of action and potential effects on microbiome is not known. Here, we examined the effects of low dose ketamine, known to induce antidepressant effects, on stool microbiome profile in adult male Wistar rats. Animals (5/group) were injected intraperitoneally with ketamine (2.5 mg/kg) or saline, daily for 7 days and sacrificed on day 8 when intestinal stools were collected and stored at - 80 °C. DNA was extracted from the samples and the 16 S rRNA gene-based microbiota analysis was performed using 16S Metagenomics application., Results: At genus-level, ketamine strikingly amplified Lactobacillus, Turicibacter and Sarcina by 3.3, 26 and 42 fold, respectively. Conversely, opportunistic pathogens Mucispirillum and Ruminococcus were reduced by approximately 2.6 and 26 fold, respectively, in ketamine group. Low levels of Lactobacillus and Turicibacter are associated with various disorders including depression and administration of certain species of Lactobacillus ameliorates depressive-like behavior in animal models. Hence, some of the antidepressant effects of ketamine might be mediated through its interaction with these gut bacteria. Additionally, high level of Ruminococcus is positively associated with the severity of irritable bowel syndrome (IBS), and some species of Mucispirillum have been associated with intestinal inflammation. Indirect evidence of anti-inflammatory role of Sarcina has been documented. Hence, some of the anti-inflammatory effects of ketamine and its usefulness in specific inflammatory diseases including IBS may be mediated through its interaction with these latter bacteria., Conclusion: Our data suggest that at least some of the antidepressant and anti-inflammatory effects of daily ketamine treatment for 7 days may be mediated via its interaction with specific gut bacteria. These findings further validate the usefulness of microbiome as a target for therapeutic intervention and call for more detailed investigation of microbiome interaction with central mediators of mood and/or inflammatory disorders.

The dose-dependent effects of alcohol, where the initial euphoric and stimulant effects initiated by the exposure to low ethanol levels can quickly lead to a deadly consequence are well established. Thus, high blood alcohol concentration (BAC), as seen in alcoholics, can cause significant damage to various organs. At low concentrations (e.g., 10 mg% or lower), however, beneficial effects of alcohol, particularly on cardiovascular function have been reported. Although, the latter assertion has been challenged by recent epidemiological studies, protective effects of low alcohol concentrations in vitro and in vivo relevant to the central nervous system (CNS) is well documented. In this review, the mechanism(s) leading to the detrimental effects of high BAC, as well as the beneficial effects of low BAC will be discussed. In addition, gender consideration is touched upon. Although further investigation is clearly warranted, it may be concluded that at least some of the beneficial outcomes of low BAC, including possible neuroprotection and antidepressant-like effects, may be due to elevation of the neurotropic factors and reduction of inflammatory mediators, whereas detrimental outcomes associated with high BAC, including neurotoxicity and depressive-like behavior may be due to reduction in neurotropic factors and elevation of inflammatory mediators. Furthermore, new research strategies are suggested.

Ethanol (EtOH) is one of the most frequently abused drugs with heavy health, economic, and societal burdens. Although moderate to low EtOH may have some neuroprotective effects, heavy EtOH consumption associated with high blood alcohol level (BAL) can be quite detrimental. The brain is particularly vulnerable to the damaging effects of high BAL, leading to neuronal loss, cognitive, and behavioral deficits. Although the exact causes of these detriments are not fully elucidated, it is believed that damage to the cholinergic system is at least partially responsible for the cognitive impairment. Thus, high BAL may result in selective apoptotic damage to the cholinergic neurons. Donepezil (DON), a centrally acting, reversible and non-competitive acetylcholinesterase (AChE) inhibitor, approved for use in Alzheimer's disease (AD), may also attenuate EtOH-induced cognitive impairment. Cognitive effects of DON might be due to an anti-apoptotic activity as some AChE inhibitors have been shown to have this property. The aim of this study was to determine whether DON might protect against EtOH-induced toxicity and whether such protection might be apoptotically mediated. We exposed the human neuroblastoma-derived, SH-SY5Y cells to a relatively high concentration of EtOH (500 mM) for 24 h and evaluated the effects of two concentrations of DON (0.1 and 1.0 μM) on alcohol-induced toxicity and caspase-3, an apoptotic marker. We found a dose-dependent protection of DON against EtOH-induced toxicity as well as dose-dependent attenuation of EtOH-induced increases in caspase-3 levels. Thus, DON may inhibit apoptosis as well as alcohol-induced toxicity.

Drug addiction affects a large extent of young people and disadvantaged populations. Drugs of abuse impede brain circuits or affect the functionality of brain circuits and interfere with bodily functions. Cannabinoids (Δ9-tetrahydrocannabinol) form key constituents of marijuana derived from the cannabis plant. Marijuana is a frequently used illegal drug in the USA. Here, we review the effects of cannabinoids at the epigenetic level and the potential role of these epigenetic effects in health and disease. Epigenetics is the study of alterations in gene expression that are transmitted across generations and take place without an alteration in DNA sequence, but are due to modulation of chromatin associated factors by environmental effects. Epigenetics is now known to offer an extra mechanism of control over transcription and how genes are expressed. Insights from research at the genetic and epigenetic level potentially provide venues that allow the translation of the biology of abused drugs to new means of how to treat marijuana substance use disorder or other addictions using pharmacotherapeutic tools.

Commonly used pharmaceutical drugs might alter the epigenetic state of cells, leading to varying degrees of long-term repercussions to human health. To test this hypothesis, we cultured HEK-293 cells in the presence of 50  μ M citalopram, a common antidepressant, for 30 days and performed whole-genome DNA methylation analysis using the NimbleGen Human DNA Methylation 3x720K Promoter Plus CpG Island Array. A total of 626 gene promoters, out of a total of 25,437 queried genes on the array (2.46%), showed significant differential methylation ( p < 0.01); among these, 272 were hypomethylated and 354 were hypermethylated in treated versus control. Using Ingenuity Pathway Analysis, we found that the chief gene networks and signaling pathways that are differentially regulated include those involved in nervous system development and function and cellular growth and proliferation. Genes implicated in depression, as well as genetic networks involving nucleic acid metabolism, small molecule biochemistry, and cell cycle regulation were significantly modified. Involvement of upstream regulators such as BDNF, FSH, and NF κ B was predicted based on differential methylation of their downstream targets. The study validates our hypothesis that pharmaceutical drugs can have off-target epigenetic effects and reveals affected networks and pathways. We view this study as a first step towards understanding the long-term epigenetic consequences of prescription drugs on human health.

The detrimental effects of heavy drinking and smoking are multiplied when the two are combined. Treatment modalities for each and especially for the combination are very limited. Although in low concentration, alcohol and nicotine, each may have beneficial effects including neuroprotection, their combination, instead of providing additive protection, may actually lead to toxicity in cell cultures. Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous 38 amino-acid peptide with demonstrated protection against neuronal injury, trauma as well as various endogenous and exogenous toxic agents. The aim of this study was to investigate whether PACAP may also protect against toxicity induced by high alcohol, high nicotine, or the combination of low alcohol and nicotine concentrations, and if so, whether this effect was mediated via PAC1 receptor. We used the neuroblastoma-derived SH-SY5Y cells and applied various colorimetric assays for determination of cell viability or toxicity. Results indicate that PACAP blocks toxicity induced by high alcohol and high nicotine as well as their combination at low concentrations. The effects of PACAP in turn were blocked by the PACAP antagonist (PACAP 6-38), indicating involvement of the PACAP receptor PAC1 and possibly vasoactive intestinal peptide (VIP) receptors in PACAP's protection. Moreover, no combined toxicity of low alcohol and low nicotine could be detected in calcium-free medium. These findings suggest possible beneficial effects of PACAP in preventing alcohol and nicotine toxicity and that calcium contributes to the damage induced by combination of low alcohol and nicotine in SH-SY5Y cells.

Introduction: Although a role for alpha-2 adrenoceptors (alpha-2 ARs) in alcohol use disorder (AUD) and depression is suggested, very little information on a direct interaction between alcohol and these receptors is available., Methods: In this study adult female Wistar and Wistar-Kyoto (WKY) rats, a putative animal model of depression, were exposed to alcohol vapor 3h daily for 10days (blood alcohol concentration ∼150mg%) followed by daily injection of 10mg/kg of imipramine (IMP, a selective norepinephrine NE/serotonin reuptake inhibitor) or nomifensine (NOMI, a selective NE/dopamine reuptake inhibitor). On day 11 animals were tested for open field locomotor activity (OFLA) and forced swim test (FST) and were sacrificed 2h later for measurement of alpha-2 ARs densities in the frontal cortex and hippocampus using [3H]RX 821002 as the specific ligand., Results: Chronic alcohol treatment increased the immobility in the FST, without affecting OFLA in both Wistar and WKY rats, suggesting induction of depressive-like behavior in Wistar rats and an exacerbation of this behavior in WKY rats. Alcohol treatment also resulted in an increase in cortical but not hippocampal alpha-2 ARs densities in both Wistar and WKY rats. The behavioral effects of alcohol were completely blocked by IMP and NOMI and the neurochemical effects (increases in alpha-2 ARs) were significantly attenuated by both drugs in both strains., Conclusions: The results suggest a role for cortical alpha-2 ARs in alcohol withdrawal-induced depression and that selective subtype antagonists of these receptors may be of adjunct therapeutic potential in AUD-depression co-morbidity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A subset of patients with oral squamous cell carcinoma (OSCC), the most common subtype of head and neck squamous cell carcinoma (HNSCC), harbor dysplastic lesions (often visually identified as leukoplakia) prior to cancer diagnosis. Although evidence suggest that leukoplakia represents an initial step in the progression to cancer, signaling networks driving this progression are poorly understood. Here, we applied in silico Pathway Activation Network Decomposition Analysis (iPANDA), a new bioinformatics software suite for qualitative analysis of intracellular signaling pathway activation using transcriptomic data, to assess a network of molecular signaling in OSCC and pre-neoplastic oral lesions. In tumor samples, our analysis detected major conserved mitogenic and survival signaling pathways strongly associated with HNSCC, suggesting that some of the pathways identified by our algorithm, but not yet validated as HNSCC related, may be attractive targets for future research. While pathways activation landscape in the majority of leukoplakias was different from that seen in OSCC, a subset of pre-neoplastic lesions has demonstrated some degree of similarity to the signaling profile seen in tumors, including dysregulation of the cancer-driving pathways related to survival and apoptosis. These results suggest that dysregulation of these signaling networks may be the driving force behind the early stages of OSCC tumorigenesis. While future studies with larger leukoplakia data sets are warranted to further estimate the values of this approach for capturing signaling features that characterize relevant lesions that actually progress to cancers, our platform proposes a promising new approach for detecting cancer-promoting pathways and tailoring the right therapy to prevent tumorigenesis., Competing Interests: EM, IO, KL, MT and AZ are affiliated with Insilico Medicine, Inc., a company engaged in aging research, which uses and provides both paid and free services using a variety of pathway activation scoring algorithms and hence may have competing financial interests. The remaining authors declare no conflict of interest.

Since time immemorial humans have utilized natural products and therapies for their healing properties. Even now, in the age of genomics and on the cusp of regenerative medicine, the use of complementary and alternative medicine (CAM) approaches represents a popular branch of health care. Furthermore, there is a trend towards a unified medical philosophy referred to as Integrative Medicine (IM) that represents the convergence of CAM and conventional medicine. The IM model not only considers the holistic perspective of the physiological components of the individual, but also includes psychological and mind-body aspects. Justification for and validation of such a whole-systems approach is in part dependent upon identification of the functional pathways governing healing, and new data is revealing relationships between therapies and biochemical effects that have long defied explanation. We review this data and propose a unifying theme: IM's ability to affect healing is due at least in part to epigenetic mechanisms. This hypothesis is based on a mounting body of evidence that demonstrates a correlation between the physical and mental effects of IM and modulation of gene expression and epigenetic state. Emphasis on mapping, deciphering, and optimizing these effects will facilitate therapeutic delivery and create further benefits., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

While primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide, it still does not have a clear mechanism that can explain all clinical cases of the disease. Elevated IOP is associated with increased accumulation of extracellular matrix (ECM) proteins in the trabecular meshwork (TM) that prevents normal outflow of aqueous humor (AH) and has damaging effects on the fine mesh-like lamina cribrosa (LC) through which the optic nerve fibers pass. Applying a pathway analysis algorithm, we discovered that an elevated level of TGFβ observed in glaucoma-affected tissues could lead to pro-fibrotic pathway activation in TM and in LC. In turn, activated pro-fibrotic pathways lead to ECM remodeling in TM and LC, making TM less efficient in AH drainage and making LC more susceptible to damage from elevated IOP via ECM transformation in LC. We propose pathway targets for potential therapeutic interventions to delay or avoid fibrosis initiation in TM and LC tissues.

In our current climate of rapid technological progress, it seems counterintuitive to think that modern science can learn anything of ethical value from the dark recesses of the nineteenth century or earlier. However, this happens to be quite true, with plenty of knowledge and wisdom to be gleaned by studying our scientific predecessors. Presently, our journals are flooded with original concepts and potential breakthroughs, a continuous stream of ideas pushing the frontiers of knowledge ever forward. Some ideas flourish while others flounder; but what sets the two apart? The distinguishing feature between success and failure within this context is the ability to discern the appropriate time to accept an innovation with open arms, versus when to take a more cautious approach. And the primary arbiters for whether an idea will catch on or not are the professional audience. I illustrate this concept by comparing the initial reception of two innovative ideas from Medicine's past: sterile technique, and prefrontal lobotomy. Sterile technique was first introduced by Dr. Ignaz Semmelweis and was initially ridiculed and rejected, with Semmelweis eventually dying in exile. Conversely, lobotomy was accepted and lauded and its inventor, Dr. Egas Moniz, won the Nobel Prize for his "discovery". This begs the question: why was a technique with the potential to save millions of lives initially rejected, whereas paradoxically, one that compromised and sometimes destroyed lives, accepted? Here I explore and analyze the potential reasons why, suggest how we can learn from these mistakes of the past and apply new insight to some current ethical dilemmas.

In this paper we present cellular senescence as the ultimate driver of the aging process, as a "causal nexus" that bridges microscopic subcellular damage with the phenotypic, macroscopic effect of aging. It is important to understand how the various types of subcellular damage correlated with the aging process lead to the larger, visible effects of anatomical aging. While it has always been assumed that subcellular damage (cause) results in macroscopic aging (effect), the bridging link between the two has been hard to define. Here, we propose that this bridge, which we term the "causal nexus", is in fact cellular senescence. The subcellular damage itself does not directly cause the visible signs of aging, but rather, as the damage accumulates and reaches a critical mass, cells cease to proliferate and acquire the deleterious "senescence-associated secretory phenotype" (SASP) which then leads to the macroscopic consequences of tissue breakdown to create the physiologically aged phenotype. Thus senescence is a precondition for anatomical aging, and this explains why aging is a gradual process that remains largely invisible during most of its progression. The subcellular damage includes shortening of telomeres, damage to mitochondria, aneuploidy, and DNA double-strand breaks triggered by various genetic, epigenetic, and environmental factors. Damage pathways acting in isolation or in concert converge at the causal nexus of cellular senescence. In each species some types of damage can be more causative than in others and operate at a variable pace; for example, telomere erosion appears to be a primary cause in human cells, whereas activation of tumor suppressor genes is more causative in rodents. Such species-specific mechanisms indicate that despite different initial causes, most of aging is traced to a single convergent causal nexus: senescence. The exception is in some invertebrate species that escape senescence, and in non-dividing cells such as neurons, where senescence still occurs, but results in the SASP rather than loss of proliferation plus SASP. Aging currently remains an inevitable endpoint for most biological organisms, but the field of cellular senescence is primed for a renaissance and as our understanding of aging is refined, strategies capable of decelerating the aging process will emerge.

Emerging evidence suggests that sexual dysfunction emerging during treatment with selective serotonin reuptake inhibitors (SSRIs) and/or serotonin-norepinephrine reuptake inhibitors (SNRIs) persists in some patients beyond drug discontinuation (post-SSRI sexual dysfunction [PSSD]). We sought to identify and characterize a series of such cases and explore possible explanatory factors and exposure-response relationship. Subjects who responded to an invitation in a forum dedicated to PSSD filled out a survey via online software. Case probability was defined according to the following 3 categories of increasing presumed likelihood of PSSD. Noncases did not meet the criteria for possible cases. Possible cases were subjects with normal pretreatment sexual function who first experienced sexual disturbances while using a single SSRI/SNRI, which did not resolve upon drug discontinuation for 1 month or longer as indicated by Arizona Sexual Experience Scale scores. High-probability cases were also younger than 50-year-olds; did not have confounding medical conditions, medications, or drug use; and had normal scores on the Hospital Anxiety and Depression Scale. Five hundred thirty-two (532) subjects completed the survey, among which 183 possible cases were identified, including 23 high-probability cases. Female sex, genital anesthesia, and depression predicted current sexual dysfunction severity, but dose/defined daily dose ratio and anxiety did not. Genital anesthesia did not correlate with depression or anxiety, but pleasureless orgasm was an independent predictor of both depression and case probability. Limitations of the study include retrospective design and selection and report biases that do not allow generalization or estimation of incidence. However, our findings add to previous reports and support the existence of PSSD, which may not be fully explained by alternative nonpharmacological factors related to sexual dysfunction, including depression and anxiety.

For the past several decades, research in understanding the molecular basis of human aging has progressed significantly with the analysis of premature aging syndromes. Progerin, an altered form of lamin A, has been identified as the cause of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), and may be a contributing causative factor in normal aging. However, the question of whether HGPS actually recapitulates the normal aging process at the cellular and organismal level, or simply mimics the aging phenotype is widely debated. In the present study we analyzed publicly available microarray datasets for fibroblasts undergoing cellular aging in culture, as well as fibroblasts derived from young, middle-age, and old-age individuals, and patients with HGPS. Using GeroScope pathway analysis and drug discovery platform we analyzed the activation states of 65 major cellular signaling pathways. Our analysis reveals that signaling pathway activation states in cells derived from chronologically young patients with HGPS strongly resemble cells taken from normal middle-aged and old individuals. This clearly indicates that HGPS may truly represent accelerated aging, rather than being just a simulacrum. Our data also points to potential pathways that could be targeted to develop drugs and drug combinations for both HGPS and normal aging.

Age-related macular degeneration (AMD) is a major cause of blindness in older people and is caused by loss of the central region of the retinal pigment epithelium (RPE). Conventional methods of gene expression analysis have yielded important insights into AMD pathogenesis, but the precise molecular pathway alterations are still poorly understood. Therefore we developed a new software program, "AMD Medicine", and discovered differential pathway activation profiles in samples of human RPE/choroid from AMD patients and controls. We identified 29 pathways in RPE-choroid AMD phenotypes: 27 pathways were activated in AMD compared to controls, and 2 pathways were activated in controls compared to AMD. In AMD, we identified a graded activation of pathways related to wound response, complement cascade, and cell survival. Also, there was downregulation of two pathways responsible for apoptosis. Furthermore, significant activation of pro-mitotic pathways is consistent with dedifferentiation and cell proliferation events, which occur early in the pathogenesis of AMD. Significantly, we discovered new global pathway activation signatures of AMD involved in the cell-based inflammatory response: IL-2, STAT3, and ERK. The ultimate aim of our research is to achieve a better understanding of signaling pathways involved in AMD pathology, which will eventually lead to better treatments.

In the last two decades we have witnessed a paradigm shift in our understanding of cells so radical that it has rewritten the rules of biology. The study of cellular reprogramming has gone from little more than a hypothesis, to applied bioengineering, with the creation of a variety of important cell types. By way of metaphor, we can compare the discovery of reprogramming with the archeological discovery of the Rosetta stone. This stone slab made possible the initial decipherment of Egyptian hieroglyphics because it allowed us to see this language in a way that was previously impossible. We propose that cellular reprogramming will have an equally profound impact on understanding and curing human disease, because it allows us to perceive and study molecular biological processes such as differentiation, epigenetics, and chromatin in ways that were likewise previously impossible. Stem cells could be called "cellular Rosetta stones" because they allow also us to perceive the connections between development, disease, cancer, aging, and regeneration in novel ways. Here we present a comprehensive historical review of stem cells and cellular reprogramming, and illustrate the developing synergy between many previously unconnected fields. We show how stem cells can be used to create in vitro models of human disease and provide examples of how reprogramming is being used to study and treat such diverse diseases as cancer, aging, and accelerated aging syndromes, infectious diseases such as AIDS, and epigenetic diseases such as polycystic ovary syndrome. While the technology of reprogramming is being developed and refined there have also been significant ongoing developments in other complementary technologies such as gene editing, progenitor cell production, and tissue engineering. These technologies are the foundations of what is becoming a fully-functional field of regenerative medicine and are converging to a point that will allow us to treat almost any disease.

Epigenetics has the potential to explain various biological phenomena that have heretofore defied complete explication. This review describes the various types of endogenous human developmental milestones such as birth, puberty, and menopause, as well as the diverse exogenous environmental factors that influence human health, in a chronological epigenetic context. We describe the entire course of human life from periconception to death and chronologically note all of the potential internal timepoints and external factors that influence the human epigenome. Ultimately, the environment presents these various factors to the individual that influence the epigenome, and the unique epigenetic and genetic profile of each individual also modulates the specific response to these factors. During the course of human life, we are exposed to an environment that abounds with a potent and dynamic milieu capable of triggering chemical changes that activate or silence genes. There is constant interaction between the external and internal environments that is required for normal development and health maintenance as well as for influencing disease load and resistance. For example, exposure to pharmaceutical and toxic chemicals, diet, stress, exercise, and other environmental factors are capable of eliciting positive or negative epigenetic modifications with lasting effects on development, metabolism and health. These can impact the body so profoundly as to permanently alter the epigenetic profile of an individual. We also present a comprehensive new hypothesis of how these diverse environmental factors cause both direct and indirect epigenetic changes and how this knowledge can ultimately be used to improve personalized medicine.

Hyaluronan is a high-molecular-weight glycosaminoglycan (GAG) prominent in the extracellular matrix. Emerging relatively late in evolution, it may have evolved to evade immune recognition. Chondroitin is a more ancient GAG and a possible hyaluronan precursor. Epimerization of a 4-hydroxyl in N-acetylgalactosamine in chondroitin to N-acetylglucosamine of hyaluronan is the only structural difference other than chain length between these two polymers. The axial 4-hydroxyl group extends out perpendicular from the equatorial plane of N-acetylgalactosamine in chondroitin. We suspect that this hydroxyl is a prime target for immune recognition. Conversion of a thumbs-up hydroxyl group into a thumbs-down position in the plane of the sugar endows hyaluronan with the ability to avoid immune recognition. Chitin is another potential precursor to hyaluronan. But regardless whether of chondroitin or of chitin origin, an ancient chondroitinase enzyme sequence seems to have been commandeered to catalyze the cleavage of the new hyaluronan substrate. The evolution of six hyaluronidase-like sequences in the human genome from a single chondroitinase as found in Caenorhabditis elegans can now be traced. Confirming our previous predictions, two duplication events occurred, with three hyaluronidase-like sequences occurring in the genome of Ciona intestinalis (sea squirt), the earliest known chordate. This was probably followed by en masse duplication, with six such genes present in the genome of zebra fish onwards. These events occurred, however, much earlier than predicted. It is also apparent on an evolutionary time scale that in several species, this gene family is continuing to evolve.

Peer review is a widely accepted instrument for raising the quality of science. Peer review limits the enormous unstructured influx of information and the sheer amount of dubious data, which in its absence would plunge science into chaos. In particular, peer review offers the benefit of eliminating papers that suffer from poor craftsmanship or methodological shortcomings, especially in the experimental sciences. However, we believe that peer review is not always appropriate for the evaluation of controversial hypothetical science. We argue that the process of peer review can be prone to bias towards ideas that affirm the prior convictions of reviewers and against innovation and radical new ideas. Innovative hypotheses are thus highly vulnerable to being "filtered out" or made to accord with conventional wisdom by the peer review process. Consequently, having introduced peer review, the Elsevier journal Medical Hypotheses may be unable to continue its tradition as a radical journal allowing discussion of improbable or unconventional ideas. Hence we conclude by asking the publisher to consider re-introducing the system of editorial review to Medical Hypotheses.

Impaired DSB repair has been implicated as a molecular mechanism contributing to the accelerating aging phenotype in Hutchinson-Gilford progeria syndrome (HGPS), but neither the extent nor the cause of the repair deficiency has been fully elucidated. Here we perform a quantitative analysis of the steady-state number of DSBs and the repair kinetics of ionizing radiation (IR)-induced DSBs in HGPS cells. We report an elevated steady-state number of DSBs and impaired repair of IR-induced DSBs, both of which correlated strongly with abnormal nuclear morphology. We recreated the HGPS cellular phenotype in human coronary artery endothelial cells for the first time by lentiviral transduction of GFP-progerin, which also resulted in impaired repair of IR-induced DSBs, and which correlated with abnormal nuclear morphology. Farnesyl transferase inhibitor (FTI) treatment improved the repair of IR-induced DSBs, but only in HGPS cells whose nuclear morphology was also normalized. Interestingly, FTI treatment did not result in a statistically significant reduction in the higher steady-state number of DSBs. We also report a delay in localization of phospho-NBS1 and MRE11, MRN complex repair factors necessary for homologous recombination (HR) repair, to DSBs in HGPS cells. Our results demonstrate a correlation between nuclear structural abnormalities and the DSB repair defect, suggesting a mechanistic link that may involve delayed repair factor localization to DNA damage. Further, our results show that similar to other HGPS phenotypes, FTI treatment has a beneficial effect on DSB repair., (Copyright © 2010. Published by Elsevier Inc.)

The term "Epigenetics" refers to DNA and chromatin modifications that persist from one cell division to the next, despite a lack of change in the underlying DNA sequence. The "epigenome" refers to the overall epigenetic state of a cell, and serves as an interface between the environment and the genome. The epigenome is dynamic and responsive to environmental signals not only during development, but also throughout life; and it is becoming increasingly apparent that chemicals can cause changes in gene expression that persist long after exposure has ceased. Here we present the hypothesis that commonly-used pharmaceutical drugs can cause such persistent epigenetic changes. Drugs may alter epigenetic homeostasis by direct or indirect mechanisms. Direct effects may be caused by drugs which affect chromatin architecture or DNA methylation. For example the antihypertensive hydralazine inhibits DNA methylation. An example of an indirectly acting drug is isotretinoin, which has transcription factor activity. A two-tier mechanism is postulated for indirect effects in which acute exposure to a drug influences signaling pathways that may lead to an alteration of transcription factor activity at gene promoters. This stimulation results in the altered expression of receptors, signaling molecules, and other proteins necessary to alter genetic regulatory circuits. With more chronic exposure, cells adapt by an unknown hypothetical process that results in more permanent modifications to DNA methylation and chromatin structure, leading to enduring alteration of a given epigenetic network. Therefore, any epigenetic side-effect caused by a drug may persist after the drug is discontinued. It is further proposed that some iatrogenic diseases such as tardive dyskinesia and drug-induced SLE are epigenetic in nature. If this hypothesis is correct the consequences for modern medicine are profound, since it would imply that our current understanding of pharmacology is an oversimplification. We propose that epigenetic side-effects of pharmaceuticals may be involved in the etiology of heart disease, cancer, neurological and cognitive disorders, obesity, diabetes, infertility, and sexual dysfunction. It is suggested that a systems biology approach employing microarray analyses of gene expression and methylation patterns can lead to a better understanding of long-term side-effects of drugs, and that in the future, epigenetic assays should be incorporated into the safety assessment of all pharmaceutical drugs. This new approach to pharmacology has been termed "phamacoepigenomics", the impact of which may be equal to or greater than that of pharmacogenetics. We provide here an overview of this potentially major new field in pharmacology and medicine.

Hyaluronidases are endoglycosidases that hydrolyze hyaluronan (HA), an abundant component of the extracellular matrix of vertebrate connective tissues. Six human hyaluronidase-related genes have been identified to date. Mutations in one of these genes cause a deficiency of hyaluronidase 1 (HYAL1) resulting in a lysosomal storage disorder, mucopolysaccharidosis (MPS) IX. We have characterized a mouse model of MPS IX and compared its phenotype with the human disease. The targeted Hyal1 allele in this model had a neomycin resistance cassette in exon 2 that replaced 753 bp of the coding region containing the predicted enzyme active site. As a result, Hyal1(-/-) animals had no detectable wild-type Hyal1 transcript, protein or serum activity. Hyal1 null animals were viable, fertile and showed no gross abnormalities at 1 year and 8 months of age. Histological studies of the knee joint showed a loss of proteoglycans occurring as early as 3 months that progressed with age. An increased number of chondrocytes displaying intense pericellular and/or cytoplasmic HA staining were detected in the epiphyseal and articular cartilage of null mice, demonstrating an accumulation of HA. Elevations of HA were not detected in the serum or non-skeletal tissues, indicating that osteoarthritis is the key disease feature in a Hyal1 deficiency. Hyal3 expression was elevated in Hyal1 null mice, suggesting that Hyal3 may compensate in HA degradation in non-skeletal tissues. Overall, the murine MPS IX model displays the key features of the human disease.

Introduction: Sexual dysfunctions such as low libido, anorgasmia, genital anesthesia, and erectile dysfunction are very common in patients taking selective serotonin reuptake inhibitors (SSRIs). It has been assumed that these side effects always resolve after discontinuing treatment, but recently, four cases were presented in which sexual function did not return to baseline. Here, we describe three more cases. Case #1: A 29-year-old with apparently permanent erectile dysfunction after taking fluoxetine 20 mg once daily for a 4-month period in 1996. Case #2: A 44-year-old male with persistent loss of libido, genital anesthesia, ejaculatory anhedonia, and erectile dysfunction after taking 20-mg once daily citalopram for 18 months. Case #3: A 28-year-old male with persistent loss of libido, genital anesthesia, and ejaculatory anhedonia since taking several different SSRIs over a 2-year period from 2003-2005., Results: No psychological issues related to sexuality were found in any of the three cases, and all common causes of sexual dysfunction such as decreased testosterone, increased prolactin or diabetes were ruled out. Erectile capacity is temporarily restored for Case #1 with injectable alprostadil, and for Case #2 with oral sildenafil, but their other symptoms remain. Case #3 has had some reversal of symptoms with extended-release methylphenidate, although it is not yet known if these prosexual effects will persist when the drug is discontinued., Conclusion: SSRIs can cause long-term effects on all aspects of the sexual response cycle that may persist after they are discontinued. Mechanistic hypotheses including persistent endocrine and epigenetic gene expression alterations were briefly discussed.

Nuclear lamins comprise the nuclear lamina, a scaffold-like structure that lines the inner nuclear membrane. B-type lamins are present in almost all cell types, but A-type lamins are expressed predominantly in differentiated cells, suggesting a role in maintenance of the differentiated state. Previous studies have shown that lamin A/C is not expressed during mouse development before day 9, nor in undifferentiated mouse embryonic carcinoma cells. To further investigate the role of lamins in cell phenotype maintenance and differentiation, we examined lamin expression in undifferentiated mouse and human embryonic stem (ES) cells. Wide-field and confocal immunofluorescence microscopy and semiquantitative reverse transcription-polymerase chain reaction analysis revealed that undifferentiated mouse and human ES cells express lamins B1 and B2 but not lamin A/C. Mouse ES cells display high levels of lamins B1 and B2 localized both at the nuclear periphery and throughout the nucleoplasm, but in human ES cells, B1 and B2 expression is dimmer and localized primarily at the nuclear periphery. Lamin A/C expression is activated during human ES cell differentiation before downregulation of the pluripotency marker Oct-3/4 but not before the downregulation of the pluripotency markers Tra-1-60, Tra-1-81, and SSEA-4. Our results identify the absence of A-type lamin expression as a novel marker for undifferentiated ES cells and further support a role for nuclear lamins in cell maintenance and differentiation.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease with widespread phenotypic features resembling premature aging. HGPS was recently shown to be caused by dominant mutations in the LMNA gene, resulting in the in-frame deletion of 50 amino acids near the carboxyl terminus of the encoded lamin A protein. Children with this disease typically succumb to myocardial infarction or stroke caused by severe atherosclerosis at an average age of 13 years. To elucidate further the molecular pathogenesis of this disease, we compared the gene expression patterns of three HGPS fibroblast cell strains heterozygous for the LMNA mutation with three normal, age-matched cell strains. We defined a set of 361 genes (1.1% of the approximately 33,000 genes analysed) that showed at least a 2-fold, statistically significant change. The most prominent categories encode transcription factors and extracellular matrix proteins, many of which are known to function in the tissues severely affected in HGPS. The most affected gene, MEOX2/GAX, is a homeobox transcription factor implicated as a negative regulator of mesodermal tissue proliferation. Thus, at the gene expression level, HGPS shows the hallmarks of a developmental disorder affecting mesodermal and mesenchymal cell lineages. The identification of a large number of genes implicated in atherosclerosis is especially valuable, because it provides clues to pathological processes that can now be investigated in HGPS patients or animal models.

Elevations in urinary hyaluronan have been used as the principal laboratory indicator for diagnosis of Hutchinson-Gilford Progeria Syndrome (HGPS). Previous reports have provided evidence suggesting that children with HGPS have altered hyaluronan metabolism as indicated by a mean 17-fold increase in urinary hyaluronan over normal values. In addition, adults with Werner's syndrome have elevated urinary hyaluronan and even more prominent elevations in serum hyaluronan over age-matched controls. It is not known whether serum hyaluronan is elevated or whether serum hyaluronan levels correlate with urinary hyaluronan levels in children with HGPS. In a large cohort of 19 HGPS patients, we sought to confirm elevations in urinary hyaluronan concentration, to establish whether serum hyaluronan is elevated, to measure the size of urinary hyaluronan, and to determine whether serum or urine hyaluronidase levels are altered. We have analyzed urinary and serum hyaluronan levels in patients with HGPS and control patients (1) by using an enzyme-linked immunosorbent assay (ELISA)-like method in which sample hyaluronan in solution and hyaluronan in solid phase compete for a solution of biotinylated hyaluronan-binding protein, and (2) by fluorophore-assisted carbohydrate electrophoresis. The size of urinary hyaluronan was measured by using Sepharose CL-6B size exclusion chromatography. Serum and urinary hyaluronidases were evaluated quantitatively, by using ELISA, and qualitatively, by using a gel detection method. HGPS patients did not show a significant elevation in either urinary or serum hyaluronan. We detected no difference in the size of urinary hyaluronan between HGPS children and age-matched controls. Serum and urinary hyaluronidase levels were not significantly different in normal and HGPS patients. These studies indicate that neither serum nor urinary hyaluronan concentration is a reliable diagnostic or prognostic marker for HGPS and underscore a difference between adult and childhood progerias.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by features reminiscent of marked premature ageing. Here, we present evidence of mutations in lamin A (LMNA) as the cause of this disorder. The HGPS gene was initially localized to chromosome 1q by observing two cases of uniparental isodisomy of 1q-the inheritance of both copies of this material from one parent-and one case with a 6-megabase paternal interstitial deletion. Sequencing of LMNA, located in this interval and previously implicated in several other heritable disorders, revealed that 18 out of 20 classical cases of HGPS harboured an identical de novo (that is, newly arisen and not inherited) single-base substitution, G608G(GGC > GGT), within exon 11. One additional case was identified with a different substitution within the same codon. Both of these mutations result in activation of a cryptic splice site within exon 11, resulting in production of a protein product that deletes 50 amino acids near the carboxy terminus. Immunofluorescence of HGPS fibroblasts with antibodies directed against lamin A revealed that many cells show visible abnormalities of the nuclear membrane. The discovery of the molecular basis of this disease may shed light on the general phenomenon of human ageing.

A hyaluronan-rich environment often correlate with tumor progression. and may be one mechanism for the invasive behavior of malignancies. Eradication of hyaluronan by hyaluronidase administration could reduce tumor aggressiveness and would provide, therefore, a new anti-cancer strategy. Hyaluronan interaction with its CD44 receptor and the resulting signal transduction events may be among the mechanisms for hyaluronan-associated cancer progression. We have shown previously that hyaluronidase treatment of breast cancer cells in vitro not only eradicates hyaluronan but also modifies expression of CD44 variant exons of tumor cells. We now determine if such effects occur in vivo and if it is accompanied by tumor regression. SCID mice bearing xenografts of human breast carcinomas were given intravenous hyaluronidase. Tumor volumes decreased 50% in 4 days. Tumor sections showed decreased hyaluronan. Intensity of staining for CD44s was not affected, whereas staining for specific CD44 variant exon isoforms was greatly reduced in residual tumors. Necrosis was not evident. Hyaluronidase, used previously as an adjunct in cancer treatment, presumably to enhance penetration of chemotherapeutic drugs, may itself have intrinsic anti-cancer activity. Removing peritumor hyaluronan appears to cause an irreversible change in tumor metabolism. Continuous hyaluronan binding to CD44 variant exon isoforms may also be required to stabilize inherently unstable isoforms that participate perhaps in tumor progression. Further investigation is required to confirm a cause and effect relationship between loss of hyaluronan, changes in CD44 variant exon expression and tumor reduction. If confirmed, hyaluronidase may provide a new class of anti-cancer therapeutics and one without toxic side effects., (Copyright 2002 Wiley-Liss, Inc.)

The human genome contains six hyaluronidase-like genes. Three genes (HYAL1, HYAL2 and HYAL3) are clustered on chromosome 3p21.3, and another two genes (HYAL4 and PH-20/SPAM1) and one expressed pseudogene (HYALP1) are similarly clustered on chromosome 7q31.3. The extensive homology between the different hyaluronidase genes suggests ancient gene duplication, followed by en masse block duplication, events that occurred before the emergence of modern mammals. Very recently we have found that the mouse genome also has six hyaluronidase-like genes that are also grouped into two clusters of three, in regions syntenic with the human genome. Surprisingly, the mouse ortholog of HYALP1 does not contain any mutations, and unlike its human counterpart may actually encode an active enzyme. Hyal-1 is the only hyaluronidase in mammalian plasma and urine, and is also found at high levels in major organs such as liver, kidney, spleen, and heart. A model is proposed suggesting that Hyal-2 and Hyal-1 are the major mammalian hyaluronidases in somatic tissues, and that they act in concert to degrade high molecular weight hyaluronan to the tetrasaccharide. Twenty-kDa hyaluronan fragments are generated at the cell surface in unique endocytic vesicles resulting from digestion by the glycosylphosphatidyl-inositol-anchored Hyal-2, transported intracellularly by an unknown process, and then further digested by Hyal-1. The two beta-exoglycosidases, beta-glucuronidase and beta-N-acetyl glucosaminidase, remove sugars from reducing termini of hyaluronan oligomers, and supplement the hyaluronidases in the catabolism of hyaluronan.