Measuring error rates in genomic perturbation screens: gold standards for human functional genomics

Technological advancement has opened the door to systematic genetics in mammalian cells. Genome‐scale loss‐of‐function screens can assay fitness defects induced by partial gene knockdown, using RNA interference, or complete gene knockout, using new CRISPR techniques. These screens can reveal the basic blueprint required for cellular proliferation. Moreover, comparing healthy to cancerous tissue can uncover genes that are essential only in the tumor; these genes are targets for the development of specific anticancer therapies. Unfortunately, progress in this field has been hampered by off‐target effects of perturbation reagents and poorly quantified error rates in large‐scale screens. To improve the quality of information derived from these screens, and to provide a framework for understanding the capabilities and limitations of CRISPR technology, we derive gold‐standard reference sets of essential and nonessential genes, and provide a Bayesian classifier of gene essentiality that outperforms current methods on both RNAi and CRISPR screens. Our results indicate that CRISPR technology is more sensitive than RNAi and that both techniques have nontrivial false discovery rates that can be mitigated by rigorous analytical methods.     

Ligand binding properties of horse hemoglobins containing deutero- and mesoheme.

The reactions of horse globin reconstituted with proto-, deutero-, and mesoheme have been examined by equilibrium and kinetic methods. In virtually all reactions studied, mesohemoglobin displays the more extreme functional behavior, whereas deuterohemoglobin exhibits behavior which is either very similar to native hemoglobin or intermediate between the two. Our kinetic and equilibrium results indicate that the primary effect of heme modification on the functional properties of hemoglobin is to alter the intrinsic reactivities of the deoxy and liganded conformations. Heme modification does not, however, result in substantial alterations in the conformational equilibrium between the two states. Simple inductive electronic effects of the 2- and 4-substituents of the heme moiety in deutero- and mesohemoglobin are apparently not sufficient to explain the observed equilibrium and kinetic properties completely, which indicates that steric effects of these substituents may also play a role in determining the functional behavior of the hemoglobin molecule.     

Escherichia coli release factor 3: resolving the paradox of a typical G protein structure and atypical function with guanine nucleotides.

Escherichia coli release factor 3 (RF3) is a G protein involved in the termination of protein synthesis that stimulates the activity of the stop signal decoding release factors RF1 and RF2. Paradoxically for a G protein, both GDP and GTP have been reported to modulate negatively the activity of nucleotide-free RF3 in vitro. Using a direct ribosome binding assay, we found that RF3xGDPCP, a GTP analogue form of RF3, has a 10-fold higher affinity for ribosomes than the GDP form of the protein, and that RF3xGDPCP binds to the ribosome efficiently in the absence of the decoding release factors. These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest that the paradoxical inhibitory effect of GTP on RF3 activity in vitro is most likely due to untimely and unproductive ribosome-mediated GTP hydrolysis. Nucleotide-free RF3 has an intermediate activity and its binding to the ribosome exhibits positive cooperativity with RF2. This cooperativity is absent, however, in the presence of GDPCP. The observed activities of nucleotide-free RF3 suggest that it mimics a transition state of RF3 in which the protein interacts with the decoding release factor while it enhances the efficiency of the termination reaction.     

HIV-1 Subtype C-Infected Individuals Maintaining High Viral Load as Potential Targets for the “Test-and-Treat” Approach to Reduce HIV Transmission

The first aim of the study is to assess the distribution of HIV-1 RNA levels in subtype C infection. Among 4,348 drug-naïve HIV-positive individuals participating in clinical studies in Botswana, the median baseline plasma HIV-1 RNA levels differed between the general population cohorts (4.1–4.2 log₁₀) and cART-initiating cohorts (5.1–5.3 log₁₀) by about one log₁₀. The proportion of individuals with high (≥50,000 (4.7 log₁₀) copies/ml) HIV-1 RNA levels ranged from 24%–28% in the general HIV-positive population cohorts to 65%–83% in cART-initiating cohorts. The second aim is to estimate the proportion of individuals who maintain high HIV-1 RNA levels for an extended time and the duration of this period. For this analysis, we estimate the proportion of individuals who could be identified by repeated 6- vs. 12-month-interval HIV testing, as well as the potential reduction of HIV transmission time that can be achieved by testing and ARV treating. Longitudinal analysis of 42 seroconverters revealed that 33% (95% CI: 20%–50%) of individuals maintain high HIV-1 RNA levels for at least 180 days post seroconversion (p/s) and the median duration of high viral load period was 350 (269; 428) days p/s. We found that it would be possible to identify all HIV-infected individuals with viral load ≥50,000 (4.7 log₁₀) copies/ml using repeated six-month-interval HIV testing. Assuming individuals with high viral load initiate cART after being identified, the period of high transmissibility due to high viral load can potentially be reduced by 77% (95% CI: 71%–82%). Therefore, if HIV-infected individuals maintaining high levels of plasma HIV-1 RNA for extended period of time contribute disproportionally to HIV transmission, a modified “test-and-treat” strategy targeting such individuals by repeated HIV testing (followed by initiation of cART) might be a useful public health strategy for mitigating the HIV epidemic in some communities.     

CHOK1SV GS-KO SSI expression system: A combination of the Fer1L4 locus and glutamine synthetase selection

There are an ever-increasing number of biopharmaceutical candidates in clinical trials fueling an urgent need to streamline the cell line development process. A critical part of the process is the methodology used to generate and screen candidate cell lines compatible with GMP manufacturing processes. The relatively large amount of clone phenotypic variation observed from conventional “random integration” (RI)-based cell line construction is thought to be the result of a combination of the position variegation effect, genome plasticity and clonal variation. Site-specific integration (SSI) has been used by several groups to temper the influence of the position variegation effect and thus reduce variability in expression of biopharmaceutical candidates. Following on from our previous reports on the application of the Fer1L4 locus for SSI in CHOK1SV (10E9), we have combined this locus and a CHOK1SV glutamine synthetase knockout (GS-KO) host to create an improved expression system. The host, CHOK1SV GS-KO SSI (HD7876), was created by homology directed integration of a targetable landing pad flanked with incompatible Frt sequences in the Fer1L4 gene. The targeting vector contains a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line. SSI clones expressing four antibody candidates, selected in a streamlined cell line development process, have mAb titers which rival RI (1.0–4.5 g/L) and robust expression stability (100% of clones stable through the 50 generation “manufacturing window” which supports commercial manufacturing at 12,000 L bioreactor scale).     

arouser reveals a role for synapse number in the regulation of ethanol sensitivity

A reduced sensitivity to the sedating effects of alcohol is a characteristic associated with alcohol use disorders (AUDs). A genetic screen for ethanol sedation mutants in Drosophila identified arouser (aru), which functions in developing neurons to reduce ethanol sensitivity. Genetic evidence suggests that aru regulates ethanol sensitivity through its activation by Egfr/Erk signaling and its inhibition by PI3K/Akt signaling. The aru mutant also has an increased number of synaptic terminals in the larva and adult fly. Both the increased ethanol sensitivity and synapse number of the aru mutant are restored upon adult social isolation, suggesting a causal relationship between synapse number and ethanol sensitivity. We thus show that a developmental abnormality affecting synapse number and ethanol sensitivity is not permanent and can be reversed by manipulating the environment of the adult fly.     

A biocultural investigation of the weanling's dilemma in Kathmandu, Nepal: do universal recommendations for weaning practices make sense?

The primary objective of this report is to use data from a study of infant growth and weaning practices in Kathmandu, Nepal, to investigate universal recommendations about exclusive breast-feeding up to 6 months postpartum. A secondary objective is to demonstrate the complexity of the biocultural nature of infant feeding practices. A sample of 283 children under 5 years of age and their 228 mothers living in a peri-urban district of Kathmandu participated in this study. The children's height/length and weight were measured three times over 9 months. At each session, a demographic, child health and infant feeding survey was administered; between sessions, in-depth interviews were conducted with mothers regarding infant feeding practices. While a few of the infants under 2 months were receiving non-breast milk foods, at 3 months of age half of the sample had been introduced to non-breast milk foods and by 7 months all infants were eating non-breast milk foods. A comparison of growth indices and velocities between exclusively and partially breast-fed infants from birth to 7 months of age shows no evidence for a difference in nutritional status between the two groups. Although there are cultural rules about breast-feeding that vary by ethnic group, all mothers followed a feeding method that depended on their assessment of whether the child was getting enough breast milk. The conclusion is that exclusive breast-feeding up to 6 months may not be appropriate for all infants. In this sample, breast-feeding duration is not shortened by the early introduction of non-breast milk foods, as the median age of breast-feeding cessation is 36 months. One of the main reasons for severance was the onset of another pregnancy. Investigation of infant feeding practices must be contextualized in the local ecology of the population. While cultural beliefs about breast-feeding are relevant, mothers' individual assessments of their children's nutritional needs and demographic events in parents' lives must also be considered.     

4-Aryl-5-cyano-2-aminopyrimidines as VEGF-R2 inhibitors: synthesis and biological evaluation

A novel series of 4-aryl-5-cyano-2-aminopyrimidines were synthesized and found to have potent VEGF-R2 kinase inhibitory activity. Structure-activity relationships were investigated and compound 14a was shown to be efficacious in a mouse model of corneal neovascularization.