MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids ¹. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes ². Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 10⁸ TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line ^{3, 4}. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.     

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

Eating Frequency is Associated With Energy Intake but Not Obesity in Midlife Women

Midlife women tend to gain weight with age, thus increasing risk of chronic disease. The purpose of this study was to examine associations between overweight/obesity and behavioral factors, including eating frequency, in a cross‐sectional national sample of midlife women (n = 1,099) (mean age = 49.7 years, and BMI = 27.7 kg/m²). Eating behaviors and food and nutrient intakes were based on a mailed 1‐day food record. BMI was calculated from self‐reported height and weight, and level of physical activity was assessed by self‐reported questionnaire. After exclusion of low‐energy reporters (32% of sample), eating frequency was not associated with overweight/obesity (P > 0.05) and was not different between BMI groups (normal, 5.21 ± 1.79; overweight, 5.16 ± 1.74; obese, 5.12 ± 1.68, P = 0.769). Adjusted logistic regression showed that eating frequency, snacking frequency, breakfast consumption, eating after 10 pm and consuming meals with children or other adults were not significantly associated with overweight/obesity. Total energy intake increased as eating frequency increased in all BMI groups, however, obese women had greater energy intake compared to normal weight women who consumed the same number of meals and snacks. Intake of fruit and vegetables, whole grains, dietary fiber, dairy, and added sugars also increased as eating frequency increased. While eating frequency was not associated with overweight/obesity, it was associated with energy intake. Thus, addressing total energy intake rather than eating frequency may be more appropriate to prevent weight gain among midlife women.     

Coming of age: steroid hormones of wild immature baboons (Papio cynocephalus)

Large gaps exist in our knowledge about common patterns and variability in the endocrinology of immature nonhuman primates, and even normal hormonal profiles during that life stage are lacking for wild populations. In the present study we present steroid profiles for a wild population of baboons (Papio cynocephalus) from infancy through reproductive maturation, obtained by noninvasive fecal analyses. Fecal concentrations of glucocorticoid (fGC) and testosterone (fT) metabolites for males, and of fGC, estrogen (fE), and progestin (fP) metabolites for females were measured by radioimmunoassay (RIA). In males, infancy was characterized by high and declining levels of fGC and fT, whereas steroid concentrations were low during the juvenile years. During the months immediately prior to testicular enlargement, fT (but not fGC) concentration tended to increase. Males that matured early consistently had higher fT and fGC concentrations than those that matured late, but not significantly so at any age. Individual differences in fT concentrations were stable across ages, and average individual fT and fGC concentrations were positively correlated. For females, high and declining levels of fE characterized infancy, and values increased again after 3.5 years of age, as some females reached menarche by that age. Both fP and fGC were relatively low and constant throughout infancy and the juvenile period. During the months immediately prior to menarche, fGC concentration significantly decreased, while no changes were observed for fE levels. fP exhibited a complicated pattern of decrease that was subsequently followed by a more modest and nonsignificant increase as menarche approached. Early- (EM) and late-maturing (LM) females differed only in fP concentration; the higher fP concentrations in EM females reached significance at 4-4.5 years of age. Maternal rank at offspring conception did not predict concentrations of any hormone for either sex. Our results demonstrate the presence of individual endocrine variability, which could have important consequences for the timing of sexual maturation and subsequently for individual reproductive success. Further evaluation of the factors that affect hormone concentrations during the juvenile and adolescent periods should lead to a better understanding of mechanisms of life-history variability.     

Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte

Apoptotic-cell removal is critical for development, tissue homeostasis, and resolution of inflammation. Although many candidate systems exist, only phosphatidylserine has been identified as a general recognition ligand on apoptotic cells. We demonstrate here that calreticulin acts as a second general recognition ligand by binding and activating LDL-receptor-related protein (LRP) on the engulfing cell. Since surface calreticulin is also found on viable cells, a mechanism preventing inadvertent uptake was sought. Disruption of interactions between CD47 (integrin-associated protein) on the target cell and SIRPalpha (SHPS-1), a heavily glycosylated transmembrane protein on the engulfing cell, permitted uptake of viable cells in a calreticulin/LRP-dependent manner. On apoptotic cells, CD47 was altered and/or lost and no longer activated SIRPalpha. These changes on the apoptotic cell create an environment where "don't eat me" signals are rendered inactive and "eat me" signals, including calreticulin and phosphatidylserine, congregate together and signal for removal.     

Synthesis and cellular studies of porphyrin-cobaltacarborane conjugates

The total syntheses of five new porphyrin-cobaltacarborane conjugates (1-5) have been achieved in 88-98% yields in a single-step reaction between a nucleophilic meso-pyridyl-containing porphyrin and zwitterionic cobaltacarborane [3,3'-Co(8-C(4)H(8)O(2)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))]. These unique zwitterionic compounds have one to four cobaltabisdicarbollide anions conjugated to the porphyrin macrocycle via (CH(2)CH(2)O)(2) chains. The X-ray structure of one of these conjugates (1) is presented and discussed. The cellular uptake, cytotoxicity, and subcellular localization of cobaltacarboraneporphyrins 1-5 were investigated in human HEp2 cells. The number and distribution of cobaltacarborane residues linked to the porphyrin macrocycle has a significant effect on the cellular uptake of the conjugates.     

Regulation of trypanosome DNA glycosylation by a SWI2/SNF2-like protein

Synthesis of the modified thymine base beta-D-glucosyl-hydroxymethyluracil, or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form-specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. The mechanism of developmental and telomeric-specific regulation of J synthesis is unknown. We have previously identified a J binding protein (JBP1) involved in propagating J synthesis. We have now identified a homolog of JBP1, JBP2, containing a domain related to the SWI2/SNF2 family of chromatin remodeling proteins that is upregulated in bloodstream form cells and interacts with nuclear chromatin. We show that expression of JBP2 in procyclic form cells leads to de novo J synthesis within telomeric regions of the chromosome and that this activity is inhibited after mutagenesis of conserved residues critical for SWI2/SNF2 function. We propose a model in which chromatin remodeling by JBP2 regulates the initial sites of J synthesis within bloodstream form trypanosome DNA, with further propagation and maintenance of J by JBP1.     

U2AF binding selects for the high conservation of the C. elegans 3' splice site

Caenorhabditis elegans is unusual among animals in having a highly conserved octamer sequence at the 3' splice site: UUUU CAG/R. This sequence can bind to the essential heterodimeric splicing factor U2AF, with U2AF65 contacting the U tract and U2AF35 contacting the splice site itself (AG/R). Here we demonstrate a strong correspondence between binding to U2AF of RNA oligonucleotides with variant octamer sequences and the frequency with which such variations occur in splice sites. C. elegans U2AF has a strong preference for the octamer sequence and exerts much of the pressure for 3' splice sites to have the precise UUUUCAG/R sequence. At two positions the splice site has a very strong preference for U even though alternative bases can also bind tightly to U2AF, suggesting that evolution can select against sequences that may have a relatively modest reduction in binding. Although pyrimidines are frequently present at the first base in the exon, U2AF has a very strong bias against them, arguing there is a mechanism to compensate for weakened U2AF binding at this position. Finally, the C in the consensus sequence must remain adjacent to the AG/R rather than to the stretch of U's, suggesting this C is recognized by U2AF35.     

The Erwinia chrysanthemi type III secretion system is required for multicellular behavior

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a sigma54-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36 degrees C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.