The rockefeller university

Proteasome-mediated degradation of intracellular proteins is essential for cell function and survival. The proteasome-binding protein PI31 (Proteasomal Inhibitor of 31kD) promotes 265 assembly and functions as an adapter for proteasome transport in axons. As localized protein synthesis and degradation is especially critical in neurons, we generated a conditional loss of PI31 in spinal motor neurons (MNs) and cerebellar Purkinje cells (PCs). A cKO of PI31 in these neurons caused axon degeneration, neuronal loss, and progressive spinal and cerebellar neurological dysfunction. For both MNs and PCs, markers of proteotoxic stress preceded axonal degeneration and motor dysfunction, indicating a critical role for PI31 in neuronal homeostasis. The time course of the loss of MN and PC function in developing mouse central nervous system suggests a key role for PI31 in human neurodegenerative diseases.

Background Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. Results We report mass spectrometry-based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. Conclusions Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth.

The zebra finch has been used as a valuable vocal learning animal model for human spoken language. It is representative of vocal learning songbirds specifically, which comprise half of all bird species, and of Neoaves broadly, which comprise 95% of all bird species. Although transgenesis in the zebra finch has been accomplished, it is with a very low efficiency of germ-line transmission and far from the efficiency with a more genetically tractable but vocal nonlearning species, the chicken (a Galloanseriformes). To improve germ-line transmission in the zebra finch, we identified and characterized its primordial germ cells (PGCs) and compared them with chicken. We found striking differences between the 2 species, including that zebra finch PGCs were more numerous, more widely distributed in early embryos before colonization into the gonads, had slower timing of colonization, and had a different developmental gene-expression program. We improved conditions for isolating and culturing zebra finch PGCs in vitro and were able to transfect them with gene-expression vectors and incorporate them into the gonads of host embryos. Our findings demonstrate important differences in the PGCs of the zebra finch and advance the first stage of creating PGC-mediated germ-line transgenics of a vocal learning species.

Correlation between nuclear and cell size, the nucleocytoplasmic ratio, is a cellular phenomenon that has been reported throughout eukaryotes for more than a century but the mechanisms that achieve it are not well understood. Here, we review work that has shed light on the cellular processes involved in nuclear size control. These studies have implicated nucleocytoplasmic transport, LINC complexes, RNA processing, regulation of nuclear envelope expansion and partitioning of importin a in nuclear size control, moving us closer to a mechanistic understanding of this phenomenon.

Sex and ovarian function contribute to hypertension susceptibility, however, the mechanisms are not well understood. Prior studies show that estrogens and neurogenic factors, including hypothalamic glutamatergic NMDA receptor plasticity, play significant roles in rodent hypertension. Here, we investigated the role of sex and ovarian failure on AMPA receptor plasticity in estrogen-sensitive paraventricular nucleus (PVN) neurons in naive and angiotensin II (AngII) infused male and female mice and female mice at early and late stages of accelerated ovarian failure (AOF). High-resolution electron microscopy was used to assess the subcellular distribution of AMPA GluA1 in age-matched male and female estrogen receptor beta (ER beta) enhanced green fluorescent protein (EGFP) reporter mice as well as female ER beta-EGFP mice treated with 4-vinylcyclohexene diepoxide. In the absence of AngII, female mice at a late stage of AOF displayed higher levels of GluA1 on the plasma membrane, indicative of functional protein, in ER beta-expressing PVN dendrites when compared to male, naive female and early stage AOF mice. Following slow-pressor AngII infusion, males, as well as early and late stage AOF females had elevated blood pressure. Significantly, only late stage-AOF female mice infused with AngII had an increase in GluA1 near the plasma membrane in dendrites of ER beta-expressing PVN neurons. In contrast, prior studies reported that plasmalemmal NMDA GluN1 increased in ER beta-expressing PVN dendrites in males and early, but not late stage AOF females. Together, these findings reveal that early and late stage AOF female mice display unique molecular signatures of long-lasting synaptic strength prior to, and following hypertension. (C) 2019 IBRO. Published by Elsevier Ltd. All rights reserved.

The B cell receptors (BCRs) for antigen express variable (V) regions that are enormously diverse, thus serving as markers on individual B cells. V region-derived idiotypic (Id) peptides can be displayed as pId:MHCII complexes on B cells for recognition by CD4(+) T cells. It is not known if naive B cells spontaneously display pId:MHCII in vivo or if BCR ligation is required for expression, thereby enabling collaboration between Id(+) B cells and Id-specific T cells. Here, using a mouse model, we show that naive B cells do not express readily detectable levels of pId:MHCII. However, BCR ligation by Ag dramatically increases physical display of pId:MHCII, leading to activation of Id-specific CD4(+) T cells, extrafollicular T-B cell collaboration and some germinal center formation, and production of Id(+) IgG. Besides having implications for immune regulation, the results may explain how persistent activation of self-reactive B cells induces the development of autoimmune diseases and B cell lymphomas.

Alcohol relapse is a treatment goal for alcohol dependence and the target for medications' development. Clinically utilized nalfurafine (NFF) is a potent and selective kappa- opioid receptor (KOP-r) agonist, with fewer side effects (e.g., sedation or anhedonia) than classic KOP-r full agonists. We have recently found that NFF reduces excessive alcohol drinking in mice via a KOP-r-mediated mechanism. Here, we further investigated whether NFF alone (1-10 mu g/kg) or in combination with naltrexone (NTX, mu-opioid receptor [MOP-r] antagonist) altered alcohol relapse-like drinking using a mouse alcohol deprivation effect (ADE) paradigm to mimic the relapse episodes in human alcoholics. Nalmefene (NMF, clinically utilized KOP-r partial agonist with MOP-r antagonism) was used as a reference compound for the effects on mouse ADE of new NFF + NTX combination. After exposed to 3-week intermittent- access alcohol drinking (two-bottle choice, 24-h access every other day), both male and female mice displayed excessive alcohol intake and then pronounced ADE after 1-week abstinence. NFF prevented the ADE in a dose-dependent manner in both male and female mice. A combination of NFF with NTX reduced the ADE without sex differences at doses lower than those individual effective ones, suggesting synergistic effects between the two compounds. NMF prevented the ADE in both sexes, while selective KOP-r antagonist nor-BNI had no effect. Our new study suggests that a combination of clinically-utilized, potent KOP-r agonist NFF with low-dose NTX has therapeutic potential in alcohol "relapse" treatment.

To evaluate the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae fecal carriers at admission in a Portuguese hospital and to determine the epidemiology and antimicrobial resistance patterns of ESBL-producing isolates. During a 2-month period, rectal swabs were collected at hospital admission from 151 at-risk patients. In addition, 48 rectal swabs were obtained from weekly screenings of 37 patients hospitalized for > 48 h. All ESBL/carbapenemase-producing isolates were tested for antimicrobial susceptibility and characterized by PFGE and MLST. The prevalence of ESBL producers at hospital admission was 17% and 24% among at-risk patients hospitalized for > 48 h, while the prevalence of carbapenemase producers was 3% in both cases. Most of the isolates were Escherichia coli (54%) and Klebsiella pneumoniae (41%). The most common ESBL identified was CTX-M-15 (n = 17/34; 50%), followed by CTX-M-27 (n = 10; 29%), CTX-M-33 (n = 4; 12%), SHV-12 (n = 2), and CTX-M-55 (n = 1). The 20 E. coli isolates were distributed into 16 PFGE types and nine sequence types (ST), with 60% of the isolates belonging to ST131. The 15 K. pneumoniae were grouped into 12 PFGE types and nine STs, with three STs (ST17, ST449, ST147) corresponding to 60% of the isolates. A high proportion of isolates showed resistance to ciprofloxacin (86%), trimethoprim-sulfamethoxazole (68%), tobramycin (57%), and gentamicin (43%). All isolates remained susceptible to fosfomycin. A high prevalence of ESBL-producing Enterobacteriaceae was found at hospital admission among at-risk patients and > 50% of the isolates showed resistance to first-line antibiotics for the treatment of lower urinary tract infections, leaving fosfomycin as an alternative.

The two sides of the nervous system coordinate and integrate information via commissural neurons, which project axons across the midline. Commissural neurons in the spinal cord are a highly heterogeneous population of cells with respect to their birthplace, final cell body position, axonal trajectory, and neurotransmitter phenotype. Although commissural axon guidance during development has been studied in great detail, neither the developmental origins nor the mature phenotypes of commissural neurons have been characterized comprehensively, largely due to lack of selective genetic access to these neurons. Here, we generated mice expressing Cre recombinase from the Robo3 locus specifically in commissural neurons. We used Robo3(Cre) mice to characterize the transcriptome and various origins of developing commissural neurons, revealing new details about their extensive heterogeneity in molecular makeup and developmental lineage. Further, we followed the fate of commissural neurons into adulthood, thereby elucidating their settling positions and molecular diversity and providing evidence for possible functions in various spinal cord circuits. Our studies establish an important genetic entry point for further analyses of commissural neuron development, connectivity, and function.