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In age-related neurodegenerative diseases, like Alzheimer's and Parkinson's, disease-specific proteins become aggregation-prone and form amyloid-like deposits. Depletion of SERF proteins ameliorates this toxic process in worm and human cell models for diseases. Whether SERF modifies amyloid pathology in mammalian brain, however, has remained unknown. Here, we generated conditional Serf2 knockout mice and found that full-body deletion of Serf2 delayed embryonic development, causing premature birth and perinatal lethality. Brain-specific Serf2 knockout mice, on the other hand, were viable, and showed no major behavioral or cognitive abnormalities. In a mouse model for amyloid-β aggregation, brain depletion of Serf2 altered the binding of structure-specific amyloid dyes, previously used to distinguish amyloid polymorphisms in the human brain. These results suggest that Serf2 depletion changed the structure of amyloid deposits, which was further supported by scanning transmission electron microscopy, but further study will be required to confirm this observation. Altogether, our data reveal the pleiotropic functions of SERF2 in embryonic development and in the brain and support the existence of modifying factors of amyloid deposition in mammalian brain, which offer possibilities for polymorphism-based interventions., (© 2023 Stroo et al.)

Background: The population of people living with HIV is aging, and with aging come emergent comorbidities, including osteoporosis, for which screening and treatment are becoming increasingly important. Osteoporosis prevalence among those living with HIV is 3 times greater than among HIV-uninfected controls., Objective: To assess and describe osteoporosis risk factors, screening, diagnosis, and treatment for people 50 years of age or older living with HIV and receiving care at a multidisciplinary HIV primary care clinic., Methods: A retrospective chart review of people 50 years of age or older living with HIV was conducted at the John Ruedy Clinic in Vancouver, British Columbia, between June 1, 2016, and June 1, 2019. Patients who had had fewer than 2 yearly follow-up appointments were excluded., Results: A total of 146 patients were included in the analysis; most were male ( n = 134, 92%), and the median age was 55 years. Patients had a median of 3 osteoporosis risk factors (in addition to age and HIV infection), and 145 patients had at least 1 risk factor. All screening for osteoporosis was conducted by dual-energy X-ray absorptiometry (DXA). Thirty-nine (27%) of the patients were screened with DXA, 92 (63%) were not screened, and 15 (10%) already had a diagnosis of osteoporosis. The DXA screening identified osteoporosis in an additional 10 patients and osteopenia in 22 patients. Treatments for patients with osteoporosis included bisphosphonates ( n = 15, 60%) and vitamin D or calcium (or both), without any other medications ( n = 4, 16%). In the overall study population, 32 (22%) of the patients were taking calcium and 46 (32%) were taking vitamin D., Conclusions: Many patients aged 50 years or older and receiving HIV care at the John Ruedy Clinic had or were at risk for osteoporosis. An opportunity exists to increase screening and treatment of these individuals. A multidisciplinary team may be crucial in achieving this goal., Competing Interests: Competing interests: None declared., (2022 Canadian Society of Hospital Pharmacists. All content in the Canadian Journal of Hospital Pharmacy is copyrighted by the Canadian Society of Hospital Pharmacy. In submitting their manuscripts, the authors transfer, assign, and otherwise convey all copyright ownership to CSHP.)

Chemical modifications of RNA molecules can affect translation in multiple ways. Therefore, it is critical to understand how their absence changes cellular translation dynamics and in particular codon-specific translation. In this chapter, we discuss the application of ribosome profiling to analyze changes in codon-specific translation and differential translation in Saccharomyces cerevisiae and human cells., (Copyright © 2021 Elsevier Inc. All rights reserved.)

C. elegans is widely used to investigate biological processes related to health and disease. To study protein localization, fluorescently-tagged proteins can be used in vivo or immunohistochemistry can be performed in whole worms. Here, we describe a technique to localize a protein of interest at a subcellular level in C. elegans lysates, which can give insight into the location, function and/or toxicity of proteins., Competing Interests: Competing interests The authors declare no conflicts of interest or competing interests.

Protein aggregation is a hallmark of several neurodegenerative diseases and is associated with impaired protein homeostasis. This imbalance is caused by the loss of the protein's native conformation, which ultimately results in its aggregation or abnormal localization within the cell. Using a C. elegans model of polyglutamine diseases, we describe in detail the filter retardation assay, a method that captures protein aggregates in a cellulose acetate membrane and allows its detection and quantification by immunoblotting., Competing Interests: Competing interests The authors declare no conflicts of interest or competing interests.

Dendrite pruning of Drosophila sensory neurons during metamorphosis is induced by the steroid hormone ecdysone through a transcriptional program. In addition, ecdysone activates the eukaryotic initiation factor 4E-binding protein (4E-BP) to inhibit cap-dependent translation initiation. To uncover how efficient translation of ecdysone targets is achieved under these conditions, we assessed the requirements for translation initiation factors during dendrite pruning. We found that the canonical cap-binding complex eIF4F is dispensable for dendrite pruning, but the eIF3 complex and the helicase eIF4A are required, indicating that differential translation initiation mechanisms are operating during dendrite pruning. eIF4A and eIF3 are stringently required for translation of the ecdysone target Mical, and this depends on the 5' UTR of Mical mRNA. Functional analyses indicate that eIF4A regulates eIF3-mRNA interactions in a helicase-dependent manner. We propose that an eIF3-eIF4A-dependent alternative initiation pathway bypasses 4E-BP to ensure adequate translation of ecdysone-induced genes., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation 2,3 . Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U 34 ) tRNA (U 34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U 34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U 34 enzymes. Mechanistically, U 34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U 34 enzymes and HIF1α. Together, these results demonstrate that U 34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

Protein aggregation is associated with age-related neurodegenerative disorders, such as Alzheimer's and polyglutamine diseases. As a causal relationship between protein aggregation and neurodegeneration remains elusive, understanding the cellular mechanisms regulating protein aggregation will help develop future treatments. To identify such mechanisms, we conducted a forward genetic screen in a C. elegans model of polyglutamine aggregation and identified the protein MOAG-2/LIR-3 as a driver of protein aggregation. In the absence of polyglutamine, MOAG-2/LIR-3 regulates the RNA polymerase III-associated transcription of small non-coding RNAs. This regulation is lost in the presence of polyglutamine, which mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol. We then show biochemically that MOAG-2/LIR-3 can also catalyze the aggregation of polyglutamine-expanded huntingtin. These results suggest that polyglutamine can induce an aggregation-promoting activity of MOAG-2/LIR-3 in the cytosol. The concept that certain aggregation-prone proteins can convert other endogenous proteins into drivers of aggregation and toxicity adds to the understanding of how cellular homeostasis can be deteriorated in protein misfolding diseases., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein homeostasis is fundamental for cell function and survival, because proteins are involved in all aspects of cellular function, ranging from cell metabolism and cell division to the cell's response to environmental challenges. Protein homeostasis is tightly regulated by the synthesis, folding, trafficking and clearance of proteins, all of which act in an orchestrated manner to ensure proteome stability. The protein quality control system is enhanced by stress response pathways, which take action whenever the proteome is challenged by environmental or physiological stress. Aging, however, damages the proteome, and such proteome damage is thought to be associated with aging-related diseases. In this review, we discuss the different cellular processes that define the protein quality control system and focus on their role in protein conformational diseases. We highlight the power of using small organisms to model neurodegenerative diseases and how these models can be exploited to discover genetic modulators of protein aggregation and toxicity. We also link findings from small model organisms to the situation in higher organisms and describe how some of the genetic modifiers discovered in organisms such as worms are functionally conserved throughout evolution. Finally, we demonstrate that the non-coding genome also plays a role in maintaining protein homeostasis. In all, this review highlights the importance of protein and RNA homeostasis in neurodegenerative diseases.

Artificial microRNA (miRNA) expression vectors have been developed and used for RNA interference. The secondary structure of artificial miRNA is important for RNA interference efficacy. We designed two groups of six artificial splicing miRNA 155-based miRNAs (SM155-based miRNAs) with the same target in the coding region or 3' UTR of a target gene and studied their RNA silencing efficiency and interferon β (IFN-β) induction effects. SM155-based miRNA with a mismatch at the +1 position and a bulge at the +11, +12 positions in a miRNA precursor stem-loop structure showed the highest gene silencing efficiency and lowest IFN-β induction effect (increased IFN-β mRNA level by 10% in both target cases), regardless of the specificity of the target sequence, suggesting that pSM155-based miRNA with this design could be a valuable miRNA expression vector.

Background: In earlier work, it was shown that patients with septic shock who also have adrenal insufficiency experience a benefit in terms of lower mortality rates with hydrocortisone supplementation. As such, the adrenocorticotropic hormone (ACTH) stimulation test has been used frequently to identify these patients. However, recent evidence has suggested that the identification and treatment of adrenal insufficiency in patients with septic shock does not reduce mortality. These results call into question the utility of the ACTH stimulation test in this patient population., Objectives: To determine the indications for ordering the ACTH stimulation test for critically ill patients at a tertiary care hospital and to classify the indications as either appropriate (e.g., primary adrenal insufficiency or medication-induced suppression of the hypothalamus-pituitary-adrenal axis) or inappropriate (e.g., patients with septic shock, prior etomidate exposure, or absence of steroid use)., Methods: A retrospective analysis of health care records was conducted for all patients who had been admitted to the intensive care unit and who had undergone an ACTH stimulation test during 2007. For each patient, the indication for the test was identified and classified as appropriate or inappropriate., Results: A total of 35 ACTH stimulation tests were performed during the study period, of which 8 (23%) were classified as having an appropriate indication and 27 (77%) as having an inappropriate indication. Of the tests with an inappropriate indication, 15 (56%) were ordered for patients with septic shock. However, the number of ACTH tests ordered for this indication declined as the year progressed., Conclusions: The ACTH stimulation test was often used inappropriately for patients with septic shock. Over time, there appeared to be a trend away from use of this test in this patient population, perhaps reflecting increasing awareness of the lack of benefit.

Recently, artificial microRNA (amiRNA) has become a promising RNA interference (RNAi) technology. Here, we describe a flexible and reliable method for constructing both single- and multi-amiRNA expression vectors. Two universal primers, together with two specific primers carrying the encoding sequence of amiRNA were designed and utilized to synthesize the functional amiRNA cassette through a one-step PCR. With appropriate restriction sites, the synthesized amiRNA cassettes can be cloned into any site of different destination vectors. Using the method, we constructed both single- and multi-amiRNA expression vectors to target three reporter genes, which code firefly luciferase (Fluc), enhanced green fluorescent protein (EGFP) and beta-galactosidase (LacZ), respectively. The expressions of three genes were all specifically inhibited by either the corresponding single- or the multi-amiRNA expression vector in 293T cells. And the RNAi efficiency of each amiRNA produced by both single- and multi-amiRNA expression vectors was comparable.

This pilot study examines the smoking behaviors of patients treated with either risperidone alone or in combination with clozapine. Smoking behavior was evaluated using expired carbon monoxide (CO) measurements, the Fagerstrom Test for Nicotine Dependence (FTND), and a semi-structured interview. Our results indicate that patients co-prescribed clozapine with risperidone smoke significantly less than patients treated with risperidone alone (19.1+/-9.3 vs. 37.8+/-19.1 ppm CO, respectively, P=0.03). These data are consistent with previous studies showing that clozapine treatment is associated with significantly reduced smoking behavior relative to other antipsychotic agents.