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Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein subcellular localization, consistent with important roles in infection.

Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMP[alpha] 2/IMP[Beta]1-dependent nuclear import by conferring direct binding to the IMP[alpha] 2/IMP[Beta]1 heterodimer, as well as to a truncated form of IMP[alpha] 2 lacking the IMP[Beta]-binding autoinhibitory domain ([delta]IBB-IMP[alpha] 2), and IMP[Beta]1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein subcellular localization, consistent with important roles in infection.

MCL-1 is an anti-apoptotic member of the BCL-2 protein family that ensures cell survival by blocking the intrinsic apoptotic cell death pathway1. MCL-1 is unique in being essential for early embryonic development and the survival of many cell types, including many cancer cells, which are not affected by the loss of the other anti-apoptotic BCL-2 family members1–4. Non-apoptotic functions of MCL-1 controlling mitochondrial ATP production and dynamics have been proposed to underlie this unique requirement for MCL-15–9. The relative contributions of the anti-apoptotic versus the non-apoptotic functions of MCL-1 in normal physiology have not been addressed. Here we replaced the coding sequence for MCL-1 with those for the anti-apoptotic proteins BCL-XL, BCL-2 or A1. We hypothesised that BCL-XL, BCL-2 and A1 may substitute for MCL-1 in the inhibition of apoptosis, but that they will not be able to replace MCL-1’s non-apoptotic function. Strikingly, Mcl-1Bcl-xL/Bcl-xL and Mcl-1Bcl-2/Bcl-2 embryos survived to embryonic day 14.5, greatly surpassing the pre-implantation lethality of Mcl-1−/− embryos at E3.5. This demonstrates that the non-apoptotic functions of MCL-1 are dispensable for early development. However, at later stages of development and life after birth many cell types, particularly ones with high energy demand, were found to require both the anti-apoptotic and the non-apoptotic functions of MCL-1. These findings reveal the relative importance of these distinct functions of MCL-1 in physiology, providing important information for basic biology and the advancement of MCL-1 inhibitors in cancer therapy.

Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum . The pathology of cerebral malaria is characterized by the accumulation of infected erythrocytes (IEs) in the microvasculature of the brain caused by parasite adhesins on the surface of IEs binding to human receptors on microvascular endothelial cells. The parasite and host molecules involved in this interaction are unknown. We selected three P. falciparum strains (HB3, 3D7, and IT/FCR3) for binding to a human brain endothelial cell line (HBEC-5i). The whole transcriptome of isogenic pairs of selected and unselected parasites was analyzed using a variant surface antigen-supplemented microarray chip. After selection, the most highly and consistently up-regulated genes were a subset of group A-like var genes ( HB3var3 , 3D7_PFD0020c , ITvar7 , and ITvar19 ) that showed 11- to >100-fold increased transcription levels. These var genes encode P. falciparum erythrocyte membrane protein (PfEMP)1 variants with distinct N-terminal domain types (domain cassette 8 or domain cassette 13). Antibodies to HB3var3 and PFD0020c recognized the surface of live IEs and blocked binding to HBEC-5i, thereby confirming the adhesive function of these variants. The clinical in vivo relevance of the HBEC-selected parasites was supported by significantly higher surface recognition of HBEC-selected parasites compared with unselected parasites by antibodies from young African children suffering cerebral malaria (Mann–Whitney test, P = 0.029) but not by antibodies from controls with uncomplicated malaria (Mann–Whitney test, P = 0.58). This work describes a binding phenotype for virulence-associated group A P. falciparum erythrocyte membrane protein 1 variants and identifies targets for interventions to treat or prevent cerebral malaria.

Anesthesia in infancy impairs performance in recognition memory tasks in mammalian animals, but it is unknown if this occurs in humans. Successful recognition can be based on stimulus familiarity or recollection of event details. Several brain structures involved in recollection are affected by anesthesia-induced neurodegeneration in animals. Therefore, we hypothesized that anesthesia in infancy impairs recollection later in life in humans and rats. Twenty eight children ages 6-11 who had undergone a procedure requiring general anesthesia before age 1 were compared with 28 age- and gender-matched children who had not undergone anesthesia. Recollection and familiarity were assessed in an object recognition memory test using receiver operator characteristic analysis. In addition, IQ and Child Behavior Checklist scores were assessed. In parallel, thirty three 7-day-old rats were randomized to receive anesthesia or sham anesthesia. Over 10 months, recollection and familiarity were assessed using an odor recognition test. We found that anesthetized children had significantly lower recollection scores and were impaired at recollecting associative information compared with controls. Familiarity, IQ, and Child Behavior Checklist scores were not different between groups. In rats, anesthetized subjects had significantly lower recollection scores than controls while familiarity was unaffected. Rats that had undergone tissue injury during anesthesia had similar recollection indices as rats that had been anesthetized without tissue injury. These findings suggest that general anesthesia in infancy impairs recollection later in life in humans and rats. In rats, this effect is independent of underlying disease or tissue injury.

During apoptosis mediated by the intrinsic pathway, BAX/BAK triggers mitochondrial permeabilization and the release of cytochrome- c , followed by a dramatic remodelling of the mitochondrial network that results in mitochondrial herniation and the subsequent release of pro-inflammatory mitochondrial components. Here, we show that mitochondrial herniation and subsequent exposure of the inner mitochondrial membrane (IMM) to the cytoplasm, initiates a unique form of mitophagy to deliver these damaged organelles to lysosomes. IMM-induced mitophagy occurs independently of canonical PINK1/Parkin signalling and is driven by ubiquitination of the IMM. Our data suggest IMM-induced mitophagy is an additional safety mechanism that cells can deploy to contain damaged mitochondria. It may have particular relevance in situations where caspase activation is incomplete or inhibited, and in contexts where PINK1/Parkin-mitophagy is impaired or overwhelmed.

Background Home-delivered meal recipients often present with complex nutritional and functional needs that place them at elevated risk for health decline and potential institutionalization. To address these complex needs, clinical services—namely registered dietitian and occupational therapy services—may be warranted to reduce the risk of health decline and maximize outcomes of this vulnerable older adult population. Accordingly, this study will explore the feasibility of testing four different clinical service models with home-delivered meal recipients. In particular, this study will determine the feasibility of recruiting and retaining participants from one home-delivered meal provider, determine challenges and opportunities to improve data collection procedures, assess resources needed to conduct study activities, and identify if service models can be implemented as intended. Methods This is a feasibility RCT with 1:1:1:1 allocation to four service arms: (a) meals only, (b) meals + registered dietitian services, (c) meals + occupational therapy services, or (d) meals + registered dietitian services + occupational therapy services. Study activities will be conducted in collaboration with one, large community-based agency in the Midwest United States. We will recruit 60 participants who meet the following inclusion criteria: is eligible to receive home-delivered meals funded through Title 3 of the Older Americans Act, has a self-reported diagnosis of diabetes and/or heart disease, is at risk for falling, and can store and reheat up to 14 frozen meals per week. Data collection will occur at baseline and at 3 months after informed consent to assess malnutrition risk, self-management of health conditions, and fall risk. Discussion While tailored dietitian and occupational therapy services may be warranted to address the nutritional and functional needs of home-delivered meal recipients, the effect of these services on recipient outcomes has yet to be rigorously examined. This feasibility study will identify the degree to which our service models can be tested with one community-based agency and identify opportunities to improve study procedures prior to conducting a definitive, stage III randomized controlled trial. Trial registration NCT06059404.

The 1994 Back to Sleep public education campaign resulted in dramatic reductions in sleep-related infant deaths, but comparable progress in recent years has been elusive. We conducted qualitative analyses of recent safe sleep campaigns from 13 U.S. cities. Goals were to (a) determine whether the campaigns reflect the full range of American Academy of Pediatrics (AAP) 2011 safe sleep recommendations, (b) describe tone and framing of the messages (e.g., use of fear appeals), (c) describe targeting/tailoring of messages to priority populations, and (d) ascertain whether the campaigns have been evaluated for reach and/or effectiveness. Methods included computer-assisted analyses of campaign materials and key informant interviews. All campaigns included “ABC” (Alone, Back, Crib) messaging; many ignored other AAP recommendations such as breastfeeding, room-sharing, immunizations, and avoiding smoke exposure. Campaigns frequently targeted priority populations such as African Americans. Fear appeals were used in three quarters of the campaigns, and 60% of the fear-based campaigns used guilt/blame messaging. We did not find published evaluation data for any of the campaigns. More attention is needed in public education campaigns to the full range of AAP recommendations, and evaluations are needed to determine the impact of these interventions on knowledge, behavior, and health outcomes.