Antiproliferative Activity of King Cobra (Ophiophagus hannah) Venom l‐Amino Acid Oxidase

King cobra (Ophiophagus hannah) venom l ‐amino acid oxidase (LAAO), a heat‐stable enzyme, is an extremely potent antiproliferative agent against cancer cells when compared with LAAO isolated from other snake venoms. King cobra venom LAAO was shown to exhibit very strong antiproliferative activities against MCF‐7 (human breast adenocarcinoma) and A549 (human lung adenocarcinoma) cells, with an IC₅₀ value of 0.04 ± 0.00 and 0.05 ± 0.00 μg/mL, respectively, after 72‐hr treatment. In comparison, its cytotoxicity was about 3–4 times lower when tested against human non‐tumourigenic breast (184B5) and lung (NL 20) cells, suggesting selective antitumour activity. Furthermore, its potency in MCF‐7 and A549 cell lines was greater than the effects of doxorubicin, a clinically established cancer chemotherapeutic agent, which showed an IC₅₀ value of 0.18 ± 0.03 and 0.63 ± 0.21 μg/mL, respectively, against the two cell lines. The selective cytotoxic action of the LAAO was confirmed by phycoerythrin (PE) annexin V/7‐amino‐actinomycin (AAD) apoptotic assay, in which a significant increase in apoptotic cells was observed in LAAO‐treated tumour cells than in their non‐tumourigenic counterparts. The ability of LAAO to induce apoptosis in tumour cells was further demonstrated using caspase‐3/7 and DNA fragmentation assays. We also determined that this enzyme may target oxidative stress in its killing of tumour cells, as its cytotoxicity was significantly reduced in the presence of catalase (a H₂O₂ scavenger). In view of its heat stability and selective and potent cytotoxic action on cancer cells, king cobra venom LAAO can be potentially developed for treating solid tumours.     

How will new genetic technologies,such as gene editing,change reproductive decision-making? Views of high-risk couples

Couples at increased risk of having offspring with a specific genetic disorder who want to avoid having an affected child have several reproductive options including prenatal diagnosis (PND) and preimplantation genetic testing (PGT). In the future, non-invasive prenatal diagnosis (NIPD), germline gene editing (GGE) and somatic gene editing (SGE) might become available. This study explores if, and how, availability of new genetic technologies, including NIPD, GGE, SGE, would change reproductive decision-making of high-risk couples. In 2018, semi-structured interviews were conducted with 25 genetically at-risk couples. Couples previously had received genetic counselling for PND and PGT, and in most cases opted for (one of) these techniques, at one Dutch Clinical Genetics Center between 2013 and 2017. Considerations participants mentioned regarding the hypothetical use of NIPD, GGE and SGE, seem similar to considerations regarding PND and PGT and are reflected in underlying concepts. These include safety and burden for mother and child, and moral considerations. Couples generally favoured NIPD over PND as this would be safe and enables earlier diagnosis. Increased opportunities of having a ‘healthy’ embryo and less embryo disposal were considerations in favour of GGE. Some regarded GGE as unsafe and feared slippery slope scenarios. Couples were least favourable towards SGE compared to choosing for a genetic reproductive technology, because of the perceived burden for the affected offspring. With the possibly growing number of technological options, understanding high risk couples’ perspectives can assist in navigating the reproductive decision-making process. Counsellors should be prepared to counsel on more and complex reproductive options.Subject terms: Genetic testing, Genetic counselling, Genetic engineering     

Cryo-EM Structure of a Possum Enterovirus

Enteroviruses (EVs) represent a substantial concern to global health. Here, we present the cryo-EM structure of a non-human enterovirus, EV-F4, isolated from the Australian brushtail possum to assess the structural diversity of these picornaviruses. The capsid structure, determined to ~3 Å resolution by single particle analysis, exhibits a largely smooth surface, similar to EV-F3 (formerly BEV-2). Although the cellular receptor is not known, the absence of charged residues on the outer surface of the canyon suggest a different receptor type than for EV-F3. Density for the pocket factor is clear, with the entrance to the pocket being smaller than for other enteroviruses.     

Light exposure during sleep impairs cardiometabolic function

This study tested the hypothesis that acute exposure to light during nighttime sleep adversely affects next-morning glucose homeostasis and whether this effect occurs via reduced sleep quality, melatonin suppression, or sympathetic nervous system (SNS) activation during sleep. A total of 20 young adults participated in this parallel-group study design. The room light condition (n = 10) included one night of sleep in dim light (<3 lx) followed by one night of sleep with overhead room lighting (100 lx). The dim light condition (n = 10) included two consecutive nights of sleep in dim light. Measures of insulin resistance (morning homeostatic model assessment of insulin resistance, 30-min insulin area under the curve [AUC] from a 2-h oral glucose tolerance test) were higher in the room light versus dim light condition. Melatonin levels were similar in both conditions. In the room light condition, participants spent proportionately more time in stage N2 and less in slow wave and rapid eye movement sleep. Heart rate was higher and heart rate variability lower (higher sympathovagal balance) during sleep in the room light versus the dim light condition. Importantly, the higher sympathovagal balance during sleep was associated with higher 30-min insulin AUC, consistent with increased insulin resistance the following morning. These results demonstrate that a single night of exposure to room light during sleep can impair glucose homeostasis, potentially via increased SNS activation. Attention to avoiding exposure to light at night during sleep may be beneficial for cardiometabolic health.

Exposure to artificial light during the night is widespread globally, particularly in industrialized countries (1–3). Given that light and dark exposure patterns play a key role in the timing of many behaviors and physiological functions (4), exposure to light in the evening and night has been posited to be deleterious for human health and well-being (1, 5–10). Impacts of light exposure during sleep are not as well studied as other kinds of nighttime light exposure. However, a recent cross-sectional observation study noted that, compared to no light exposure during sleep, any self-reported artificial light exposure in the bedroom during sleep (small nightlight in room, light from outside room, or television/light in room) was associated with obesity in women (11). Furthermore, the incidence of obesity was highest in those who reported sleeping with a television or light on in the bedroom (11). These findings suggest that light in the bedroom during nighttime sleep may negatively influence metabolic regulation.Emerging evidence indicates that light exposure plays a role in human metabolic regulation, with evening light exposure having unfavorable effects on metabolic functions including decreased glucose tolerance and decreased insulin sensitivity (12, 13). In line with these data, we have previously shown that blue-enriched light exposure in the morning and evening alters glucose metabolism, with an increase in insulin resistance compared to dim light exposure (14). In addition, evidence indicates that nighttime indoor light exposure during the habitual sleep period while awake (15), and during sleep itself (16), likely has deleterious metabolic effects. A recent study prospectively measured light exposure in the bedroom during nighttime sleep and showed that higher levels of bedroom light exposure were associated with a higher incidence of type 2 diabetes in an elderly population (16). However, the exact mechanisms by which light exposure, particularly during nighttime sleep, impacts metabolic regulation are not well understood.A proposed pathway to explain the relationship between nighttime light exposure and altered metabolic function is via changes in sleep. Robust evidence from epidemiological and experimental studies indicates that nighttime light exposure, either from outdoor or indoor sources, has negative impacts on subjective and objective sleep quality as indicated by actigraphy or polysomnography (PSG) measures of reduced total sleep time (TST), sleep efficiency (SE), increased wake after sleep onset (WASO), reduced amount of slow wave sleep (SWS), or increased arousal index (AI) (17–20). Given the well-established contribution of sleep disruptions to metabolic dysfunction (21), it is plausible that nighttime light exposure alters glucose metabolism due to disturbances to sleep. However, nighttime light exposure also appears to have a direct effect on glucose regulation that is independent of sleep loss, as shown by a study that subjected healthy male individuals to sleep deprivation in the dark or to sleep deprivation with nighttime light exposure (22). This study showed that a full night of sleep deprivation with nighttime light exposure increased postprandial levels of insulin and glucagon-like peptide-1, increased insulin resistance, and reduced nighttime melatonin; these changes were not observed under conditions of sleep deprivation in darkness.A second proposed mechanism to explain the impairment of glucose metabolism from nighttime light exposure is via light-induced changes to the endogenous circadian system, including suppression and phase shifting of the melatonin rhythm (23). It is well established that light exposure suppresses melatonin secretion (24, 25), and several studies have implicated suppression of nighttime melatonin with incidence of diabetes (26) and insulin resistance (27). The association between altered melatonin levels and changes in glucose regulation may be explained by evidence that melatonin plays a role in the secretion and action of insulin (28–30). In particular, lower melatonin levels resulting from light exposure during the nighttime sleep period, in a fasting condition, have been suggested to alter melatonin’s facilitation of pancreatic β-cell recovery (31). Moreover, evidence shows that light exposure, even of moderate intensity, during the nighttime sleep period can produce a phase shift of the internal circadian system (32, 33). Given the established role of the circadian system in the control of glucose metabolism, light exposure during the nighttime sleep period could facilitate the misalignment between the central clock and peripheral clocks in metabolic tissues, with consequent negative impact on glucose homeostasis (34).A third potential mechanism is the effect of light exposure on autonomic nervous system (ANS) activity. Light exposure has an arousing effect on the sympathetic autonomic system as revealed by the increase in cortisol or heart rate (HR) associated with light exposure mainly during the morning and/or nighttime hours as compared to evening hours (35–37). Beyond the direct excitatory effect exerted by light exposure on sympathetic activity (35), alterations of the ANS characterized by a shift toward an increased sympathetic drive have also been suggested to mediate the negative effects of sleep disruption on many physiological systems such as glucose metabolism (38). Thus, it is plausible that light-induced autonomic activation, either directly and/or mediated by sleep disruption, significantly contributes to the observed relationship between nighttime light exposure and altered glucose metabolism. Notably, sympathetic overactivity has been shown to precede the development of insulin resistance and prediabetes and contribute to the development of obesity and metabolic syndrome (39–41).Prior studies have reported that light exposure during sleep increases HR and decreases HR variability (HRV), consistent with increased sympathetic activation (42–44). These studies either examined bright light (1,000 lx) over the entire sleep period (42) or lower light levels (50 lx or dawn simulation) early or late in the sleep period (43, 44). However, the effect of a single night of moderate room light exposure across the entire nighttime sleep period on autonomic activation and its impact on metabolic function has never been fully investigated.In the present study, we tested the hypothesis that room light exposure (100 lx) during habitual nighttime sleep is associated with increased insulin resistance as measured by the homeostatic model of insulin resistance (HOMA-IR), the Matsuda insulin sensitivity index, and impaired response to an oral glucose tolerance test (OGTT) the next morning. In addition, we hypothesized potential mechanisms of light-induced metabolic changes, such as reduced sleep quality, suppression of melatonin level, and elevated sympathetic activation (HR and HRV) during the sleep period.     

Phase I and pharmacological study of cytarabine and tanespimycin in relapsed and refractory acute leukemia

Background

In preclinical studies the heat shock protein 90 (Hsp90) inhibitor tanespimycin induced down-regulation of checkpoint kinase 1 (Chk1) and other client proteins as well as increased sensitivity of acute leukemia cells to cytarabine. We report here the results of a phase I and pharmacological study of the cytarabine + tanespimycin combination in adults with recurrent or refractory acute leukemia.

Design and Methods

Patients received cytarabine 400 mg/m²/day continuously for 5 days and tanespimycin infusions at escalating doses on days 3 and 6. Marrow mononuclear cells harvested before therapy, immediately prior to tanespimycin, and 24 hours later were examined by immunoblotting for Hsp70 and multiple Hsp90 clients.

Results

Twenty-six patients were treated at five dose levels. The maximum tolerated dose was cytarabine 400 mg/m²/day for 5 days along with tanespimycin 300 mg/m² on days 3 and 6. Treatment-related adverse events included disseminated intravascular coagulation (grades 3 and 5), acute respiratory distress syndrome (grade 4), and myocardial infarction associated with prolonged exposure to tanespimycin and its active metabolite 17-aminogeldanamycin. Among 21 evaluable patients, there were two complete and four partial remissions. Elevations of Hsp70, a marker used to assess Hsp90 inhibition in other studies, were observed in more than 80% of samples harvested 24 hours after tanespimycin, but down-regulation of Chk1 and other Hsp90 client proteins was modest.

Conclusions

Because exposure to potentially effective concentrations occurs only for a brief time in vivo, at clinically tolerable doses tanespimycin has little effect on resistance-mediating client proteins in relapsed leukemia and exhibits limited activity in combination with cytarabine.     

Progranulin expression correlates with dense‐core amyloid plaque burden in Alzheimer disease mouse models

Amyloid‐β (Aβ) plaques are pathological hallmarks of Alzheimer disease (AD). In addition, innate inflammatory responses, such as those mediated by microglia, are integral to the pathogenesis of AD. Interestingly, only dense‐core plaques and not diffuse plaques are associated with neuritic and inflammatory pathology in AD patients as well as in mouse AD models. However, the precise neuropathological changes that occur in the brain in response to amyloid deposition are largely unknown. To study the molecular mechanism(s) responsible for Aβ‐mediated neuropathology, we performed a gene expression analysis on laser‐microdissected brain tissue of Tg2576 and APPPS1 mice that are characterized by different types of amyloid plaques and genetic backgrounds. Data were validated by image and biochemical analyses on different ages of Tg2576, APPPS1, and Aβ42‐depositing BRI‐Aβ42 mice. Consistent with an important role of inflammatory responses in AD, we identified progranulin (mouse Grn; human GRN) as one of the top ten up‐regulated molecules in Tg2576 (≈8‐fold increased) and APPPS1 (≈2‐fold increased) mice compared to littermate controls, and among the eight significantly up‐regulated molecules common to both mouse models. In addition, Grn levels correlated significantly with amyloid load, especially the dense‐core plaque pathology (p < 0.001). We further showed that Grn is up‐regulated in microglia and neurons and neurites around dense‐core plaques, but not in astrocytes or oligodendrocytes, as has been shown in AD patients. Our data therefore support the ongoing use of these mouse models in drug trials, especially those with anti‐inflammatory compounds. Moreover, the correlation of Grn with increasing disease severity in AD mouse models prompts human studies exploring the viability of GRN as a disease biomarker. Because loss of GRN has recently been shown to cause frontotemporal dementia and serves as a risk factor for AD, the strong GRN reactivity around dense‐core plaques is consistent with an important role of this factor in AD pathogenesis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.