Knockdown of CALM2 increases the sensitivity to afatinib in HER2-amplified gastric cancer cells by regulating the Akt/FoxO3a/Puma axis.

Gastric cancer (GC) is a global health issue that lacks effective treatment options. Afatinib is a tyrosine kinase inhibitor (TKI) that has shown promising results in the treatment of GC. However, resistance to afatinib is inevitable and hampers its clinical application. To date, there is limited knowledge regarding the mechanisms underlying the resistance of GC cells to afatinib. This study aimed to identify novel factors that may contribute to the resistance of GC cells to afatinib. We found that upregulation of calmodulin 2 (CALM2), a member of the CALM family, confers resistance to afatinib in GC cells. Knockdown of CALM2 can overcome the resistance to afatinib by promoting mitochondrial apoptosis in a caspase-dependent manner. Mechanistically, it was found that the downregulation of CALM2 led to the upregulation of the FoxO3a/Puma axis. Inhibition of either FoxO3a or Puma abrogated the effects of CALM2 downregulation in GC cells. In addition, we revealed that CALM2 knockdown inhibited Akt signaling, which is responsible for blocking the FoxO3a/Puma axis. Altogether, our results indicated that CALM2 could be considered a potential target to overcome the resistance of GC cells to afatinib.

Reduction of Chronic Wasting Disease Prion Seeding Activity following Digestion by Mountain Lions.

Chronic wasting disease (CWD) is a transmissible prion disease first observed in the 1960s in North America. This invariably fatal disease affects multiple cervid species in the wild and in captivity. In addition to the several known transmission pathways involving cervid host species, prions have been detected in the feces of crows and coyotes after consumption of experimentally spiked tissues. This raises questions about the role of cervid consumers in the perpetuation of CWD. Mountain lions have been shown to preferentially select CWD-infected prey and are also apparently resistant to infection. In this study, two captive mountain lions were fed ground mule deer muscle tissue spiked with brain-derived CWD prions, and lion feces were collected for 1 week afterward. The input brain and resulting fecal materials were analyzed using the highly sensitive real-time quaking-induced conversion (RT-QuIC) assay to quantify prion seeding activity. We recovered only 2.8 to 3.9% of input CWD prions after passage through the mountain lions' gastrointestinal tracts. Interestingly, CWD prions were shed only in the first defecation following consumption. Our data support the possibility that mountain lions feeding upon infected carcasses could excrete CWD prions in their feces over a short period of time but also suggest that most of the ingested prions are eliminated or sequestered by this large predator. IMPORTANCE CWD prions appear to spread naturally among susceptible cervid species in captivity and in the wild. A better understanding of all the ways these prions move, persist, and subsequently infect target species through the environment is critical to developing comprehensive disease control strategies. In our study, we show limited, transient pass-through of CWD prions in an apex predator, the mountain lion, using the highly sensitive RT-QuIC assay on feces collected after lions were fed prion-spiked muscle tissue. Prions were detected in feces only in the first defecation after exposure. Moreover, the amount of CWD prions recovered in feces was reduced by >96% after passing through the lion digestive system. This indicates that mountain lions may have some potential to distribute CWD prions within their home ranges but that they also effectively eliminate most of the CWD prions they consume.

Marine fog inputs appear to increase methylmercury bioaccumulation in a coastal terrestrial food web.

Coastal marine atmospheric fog has recently been implicated as a potential source of ocean-derived monomethylmercury (MMHg) to coastal terrestrial ecosystems through the process of sea-to-land advection of foggy air masses followed by wet deposition. This study examined whether pumas (Puma concolor) in coastal central California, USA, and their associated food web, have elevated concentrations of MMHg, which could be indicative of their habitat being in a region that is regularly inundated with marine fog. We found that adult puma fur and fur-normalized whiskers in our marine fog-influenced study region had a mean (±SE) total Hg (THg) (a convenient surrogate for MMHg) concentration of 1544 ± 151 ng g-1 (N = 94), which was three times higher (P < 0.01) than mean THg in comparable samples from inland areas of California (492 ± 119 ng g-1, N = 18). Pumas in California eat primarily black-tailed and/or mule deer (Odocoileus hemionus), and THg in deer fur from the two regions was also significantly different (coastal 28.1 ± 2.9, N = 55, vs. inland 15.5 ± 1.5 ng g-1, N = 40). We suggest that atmospheric deposition of MMHg through fog may be contributing to this pattern, as we also observed significantly higher MMHg concentrations in lace lichen (Ramalina menziesii), a deer food and a bioindicator of atmospheric deposition, at sites with the highest fog frequencies. At these ocean-facing sites, deer samples had significantly higher THg concentrations compared to those from more inland bay-facing sites. Our results suggest that fog-borne MMHg, while likely a small fraction of Hg in all atmospheric deposition, may contribute, disproportionately, to the bioaccumulation of Hg to levels that approach toxicological thresholds in at least one apex predator. As global mercury levels increase, coastal food webs may be at risk to the toxicological effects of increased methylmercury burdens.

The pro-apoptotic protein Bmf co-operates with Bim and Puma in neuron death induced by ß-amyloid or NGF deprivation.

The pro-apoptotic Bcl-2 homology 3 domain only (BH3-only) proteins are central regulators of cell death in various physiological and pathological conditions, including Alzheimer's disease (AD). Bcl-2 modifying factor (Bmf) is one such BH3-only protein that is implicated in various death paradigms such as anoikis, seizures, cancer and autoimmunity. It also co-operates with other BH3-only proteins such as Bim in various death paradigms. However, its role in neurodegeneration is under-investigated. Here, we report for the first time the essential role of Bmf and its co-operativity with direct activator BH3-only proteins Bim and Puma in neuron death induced by beta-amyloid (Aß) toxicity or NGF deprivation. Oligomeric Aß is main pathologic species in AD and NGF deprivation is relevant for both developmental as well as pathologic neuron death. We find that Bmf over-expression causes cell death and Bmf knockdown protects neurons against death evoked by Aß or NGF deprivation. We also find that Bmf co-operates with other important BH3-only proteins such as Bim and Puma in neuron death induced by Aß or NGF deprivation. Simultaneous knocking down of these molecules by their respective shRNAs provide enhanced protection against Aß. Taken together, our results elucidate the essential role of Bmf and its co-operative effects with already known neuron death inducers, Bim and Puma, in neuron death evoked by Aß treatment or NGF deprivation.

ZNF509S1 downregulates PUMA by inhibiting p53K382 acetylation and p53-DNA binding.

Expression of the POK family protein ZNF509L, and -its S1 isoform, is induced by p53 upon exposure to genotoxic stress. Due to alternative splicing of the ZNF509 primary transcript, ZNF509S1 lacks the 6 zinc-fingers and C-terminus of ZNF509L, resulting in only one zinc-finger. ZNF509L and -S1 inhibit cell proliferation by activating p21/CDKN1A and RB transcription, respectively. When cells are exposed to severe DNA damage, p53 activates PUMA (p53-upregulated modulator of apoptosis) transcription. Interestingly, apoptosis due to transcriptional activation of PUMA by p53 is attenuated by ZNF509S1. Thus we investigated the molecular mechanism(s) underlying the transcriptional attenuation and anti-apoptotic effects of ZNF509S1. We show that ZNF509S1 modulation of p53 activity is important in PUMA gene transcription by modulating post-translational modification of p53 by p300. ZNF509S1 directly interacts with p53 and inhibits p300-mediated acetylation of p53 lysine K382, with deacetylation of p53 K382 leading to decreased DNA binding at the p53 response element 1 of the PUMA promoter. ZNF509S1 may play a role not only in cell cycle arrest, by activating RB expression, but also in rescuing cells from apoptotic death by repressing PUMA expression in cells exposed to severe DNA damage.

Evaluation of cardiopulmonary parameters and recovery from anesthesia in cougars (Puma concolor) anesthetized with detomidine/ketamine and isoflurane or sevoflurane / Avaliação de parâmetros cardiorrespiratórios e recuperação da anestesia em pumas (Puma concolor) anestesiados com detomidina/cetamina e isoflurano ou sevoflurano

The aim of this study was to assess the cardiopulmonary effects, the onset time after the administration of a detomidine/ketamine combination, and the recovery from anesthesia of cougars (Puma concolor) anesthetized with detomidine/ketamine and isoflurane or sevoflurane for abdominal ultrasound imaging. Fourteen animals were randomly allocated into two experimental groups: GISO (n=7) and GSEVO (n=7). Chemical restraint was performed using 0.15mg/kg detomidine combined with 5mg/kg ketamine intramuscularly; anesthesia induction was achieved using 2mg/kg propofol intravenously and maintenance with isoflurane (GISO) or sevoflurane (GSEVO). The following parameters were assessed: heart rate, respiratory rate, systolic and diastolic arterial blood pressure, mean arterial blood pressure, oxyhemoglobin saturation, rectal temperature, central venous pressure, and end-tidal carbon dioxide. The time to sternal recumbency (TSR) and time to standing position (TSP) were also determined. There was not statistically significant difference for the cardiopulmonary variables or TSP whereas TSR was significantly shorter in GSEVO. The time to onset of anesthesia was 11.1±1.2 minutes and 11.3±1.8 minutes for GISO and GSEVO, respectively. The anesthesia of cougars with detomidine/ketamine and isoflurane or sevoflurane was conducted with safety, cardiopulmonary stability, and increased time to sternal recumbency in the GISO group.

O objetivo do presente estudo foi avaliar os efeitos cardiorrespiratórios e a recuperação anestésica de onças-pardas (Puma concolor) submetidas à anestesia com detomidina/cetamina e isofluorano ou sevofluorano para avaliação ultrassonográfica abdominal. Para isso, foram utilizados 14 animais divididos aleatoriamente em dois grupos experimentais GISO (n=7) e GSEVO (n=7). Foram submetidos à contenção química com detomidina 0,15mg/kg associada à cetamina 5mg/kg pela via intramuscular, induzidos com propofol 2mg/kg pela via intravenosa e mantidos com isofluorano (GISO) ou sevofluorano. Foram avaliados os parâmetros: frequência cardíaca e respiratória, pressão arterial sistólica, média e diastólica saturação de oxihemoglobina, temperatura retal, pressão venosa central e fração expirada de dióxido de carbono. O tempo para adoção de decúbito esternal e posição quadrupedal também foram avaliados. Não houve diferença estatística para as variáveis cardiorrespiratórias e no tempo para adoção da posição quadrupedal. O tempo para adoção de decúbito esternal foi significativamente menor no GSEVO em relação ao GISO. Concluiu-se que a anestesia de onças pardas com detomidina/cetamina e isoflurano ou sevoflurano foi realizada de maneira segura, com estabilidade cardiorrespiratória e com aumento no tempo para adoção de decúbito esternal no GISO.

Mammalian energetics. Instantaneous energetics of puma kills reveal advantage of felid sneak attacks.

Pumas (Puma concolor) live in diverse, often rugged, complex habitats. The energy they expend for hunting must account for this complexity but is difficult to measure for this and other large, cryptic carnivores. We developed and deployed a physiological SMART (species movement, acceleration, and radio tracking) collar that used accelerometry to continuously monitor energetics, movements, and behavior of free-ranging pumas. This felid species displayed marked individuality in predatory activities, ranging from low-cost sit-and-wait behaviors to constant movements with energetic costs averaging 2.3 times those predicted for running mammals. Pumas reduce these costs by remaining cryptic and precisely matching maximum pouncing force (overall dynamic body acceleration = 5.3 to 16.1g) to prey size. Such instantaneous energetics help to explain why most felids stalk and pounce, and their analysis represents a powerful approach for accurately forecasting resource demands required for survival by large, mobile predators.

High proportion of male faeces in jaguar populations.

Faeces provide relevant biological information which includes, with the application of genetic techniques, the sex and identity of individuals that defecated, thus providing potentially useful data on the behaviour and ecology of individuals, as well as the dynamics and structure of populations. This paper presents estimates of the sex ratio of different felid species (jaguar, Panthera onca; puma, Puma concolor; and ocelot/margay, Leopardus pardalis/Leopardus wiedi) as observed in field collected faeces, and proposes several hypotheses that could explain the strikingly high proportion of faeces from male jaguars. The proportion of male and female faeces was estimated using a non-invasive faecal sampling method in 14 study areas in Mexico and Brazil. Faecal samples were genetically analysed to identify the species, the sex and the individual (the latter only for samples identified as belonging to jaguars). Considering the three species, 72.6% of faeces (n = 493) were from males; however, there were significant differences among them, with the proportion from males being higher for jaguars than for pumas and ocelots/margays. A male-bias was consistently observed in all study areas for jaguar faeces, but not for the other species. For jaguars the trend was the same when considering the number of individuals identified (n = 68), with an average of 4.2±0.56 faeces per male and 2.0±0.36 per female. The observed faecal marking patterns might be related to the behaviour of female jaguars directed toward protecting litters from males, and in both male and female pumas, to prevent interspecific aggressions from male jaguars. The hypothesis that there are effectively more males than females in jaguar populations cannot be discarded, which could be due to the fact that females are territorial and males are not, or a tendency for males to disperse into suboptimal areas for the species.

Suspected secondary thiafentanil intoxication in a captive mountain lion (Puma concolor).

Inadvertent ingestion of thiafentanil oxalate by a captive adult female mountain lion (Puma concolor) caused a prolonged clinical syndrome that included sedation and depression, muscle tension, and myopathy that was incompletely antagonized by naltrexone HCl. A serum chemistry profile revealed markedly elevated creatinine phosphokinase (CK; 490,450 IU/l), alanine aminotransferase (ALT; 1,896 IU/l), and aspartate aminotransferase (AST; 4,321 IU/l) 2 days after onset. The affected animal's condition gradually improved over the next 15 days in response to supportive therapy that included diazepam (5 mg as needed), Normasol R (3 l/day), dexamethasone (tapering dose starting at 1 mg/kg), and ketoprofen (1 mg/kg). She eventually recovered completely. Based on these observations, carcasses of animals immobilized with thiafentanil should be marked and disposed of properly to preclude opportunities for secondary exposure and potential intoxication in scavenging species. In addition, caution is advised when using thiafentanil in animals that could be preyed upon before full metabolism of the drug.