Codon optimality modulates protein output by tuning translation initiation.

The impact of synonymous codon choice on protein output has important implications for understanding endogenous gene expression and design of synthetic mRNAs. Previously, we used a neural network model to design a series of synonymous fluorescent reporters whose protein output in yeast spanned a seven-fold range corresponding to their predicted translation speed. Here, we show that this effect is not due primarily to the established impact of slow elongation on mRNA stability, but rather, that an active mechanism further decreases the number of proteins made per mRNA. We combine simulations and careful experiments on fluorescent reporters to argue that translation initiation is limited on non-optimally encoded transcripts. Using a genome-wide CRISPRi screen to discover factors modulating the output from non-optimal transcripts, we identify a set of translation initiation factors including multiple subunits of eIF3 whose depletion restored protein output of a non-optimal reporter. Our results show that codon usage can directly limit protein production, across the full range of endogenous variability in codon usage, by limiting translation initiation.

Regulatory Programs of B-cell Activation and Germinal Center Reaction Allow B-ALL Escape from CD19 CAR T-cell Therapy.

Chimeric antigen receptor (CAR) T-cell therapy has led to tremendous successes in the treatment of B-cell malignancies. However, a large fraction of treated patients relapse, often with disease expressing reduced levels of the target antigen. Here, we report that exposing CD19+ B-cell acute lymphoblastic leukemia (B-ALL) cells to CD19 CAR T cells reduced CD19 expression within hours. Initially, CD19 CAR T cells caused clustering of CD19 at the T cell-leukemia cell interface followed by CD19 internalization and decreased CD19 surface expression on the B-ALL cells. CD19 expression was then repressed by transcriptional rewiring. Using single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing, we demonstrated that a subset of refractory CD19low cells sustained decreased CD19 expression through transcriptional programs of physiologic B-cell activation and germinal center reaction. Inhibiting B-cell activation programs with the Bruton's tyrosine kinase inhibitor ibrutinib increased the cytotoxicity of CD19 CAR T cells without affecting CAR T-cell viability. These results demonstrate transcriptional plasticity as an underlying mechanism of escape from CAR T cells and highlight the importance of combining CAR T-cell therapy with targeted therapies that aim to overcome this plasticity. See related Spotlight by Zhao and Melenhorst, p. 1040.

Insights into transgenerational epigenetics from studies of ciliates.

Epigenetics, a term with many meanings, can be broadly defined as the study of dynamic states of the genome. Ciliates, a clade of unicellular eukaryotes, can teach us about the intersection of epigenetics and evolution due to the advantages of working with cultivable ciliate lineages, plus their tendency to express extreme phenotypes such as heritable doublet morphology. Moreover, ciliates provide a powerful model for studying epigenetics given the presence of dimorphic nuclei - a somatic macronucleus and germline micronucleus - within each cell. Here, we exemplify the power of studying ciliates to learn about epigenetic phenomena. We highlight "classical" examples from morphology and physiology including cortical inheritance, mating type determination, and serotype expression. In addition, we detail molecular studies of epigenetic phenomena, including: DNA elimination; alternative processing and unscrambling; and copy number determination. Based on the implications of these studies, we discuss epigenetics as a possible functional mechanism for rapid speciation in ciliates.

Unusual features of non-dividing somatic macronuclei in the ciliate class Karyorelictea.

Genome structure and nuclear organization have been intensely studied in model ciliates such as Tetrahymena and Paramecium, yet few studies have focused on nuclear features of other ciliate clades including the class Karyorelictea. In most ciliates, both the somatic macronuclei and germline micronuclei divide during cell division and macronuclear development only occurs after conjugation. However, the macronuclei of Karyorelictea are non-dividing (i.e. division minus (Div-)) and develop anew from micronuclei during each asexual division. As macronuclei age within Karyorelictea, they undergo changes in morphology and DNA content until they are eventually degraded and replaced by newly developed macronuclei. No less than two macronuclei and one micronucleus are present in karyorelictid species, which suggests that a mature macronucleus 1) might be needed to sustain the cell while a new macronucleus is developing and 2) likely plays a role in guiding the development of the new macronucleus. Here we use a phylogenetic framework to compile information on the morphology and development of nuclei in Karyorelictea, largely relying on the work of Dr. Igor Raikov (1932-1998). We synthesize data to speculate on the functional implications of key features of Karyorelictea including the presence of at least two macronuclei in each cell and the inability for macronuclei to divide.

Implementation of repeat HIV testing during pregnancy in southwestern Kenya: progress and missed opportunities.

INTRODUCTION: Repeat HIV testing during the late antenatal period is crucial to identify and initiate treatment for pregnant women with incident HIV infection to prevent perinatal HIV transmission and keep mothers alive. In 2012, the Kenya Ministry of Health adopted international guidelines suggesting that pregnant women be offered retesting three months after an initial negative HIV test. Our objectives were to determine the current rate of antenatal repeat HIV testing; identify successes, missed opportunities and factors associated with retesting; and estimate the incidence of HIV during pregnancy. METHODS: Retrospective analysis of longitudinal data was conducted for a cohort of 2145 women attending antenatal care clinic at a large district hospital in southwestern Kenya. Data were abstracted from registers for all women who attended the clinic from the years 2011 to 2014. RESULTS: Although 90.2% of women first came to clinic prior to their third trimester and 27.5% had at least four clinic visits, 58.0% of all women went to delivery without a retest. Missed opportunities for retesting included not returning to clinic at all, not returning when eligible, or late gestational age (>28 weeks) at first clinic visit making them ineligible for retesting (accounting for 14.2%, 26.8% and 9.6% of all clinic attendees respectively); and failure to be retested even when eligible at one or more visits (accounting for 73.2% of eligible returnees). Being unmarried and aged 20 or younger was associated with an increase in mean gestational age of first visit by 2.52 weeks (95% CI: 1.56, 3.48) and a 2.59 increased odds (95% CI: 1.90, 3.54) of failing to return to clinic, compared to those who were married and over 20 years of age. On retest, two women tested HIV positive, suggesting an incidence rate of 4.4 per 100 person-years. After adjusting for potential confounders, only later year of last menstrual period (2013 vs. 2012 and 2011) was associated with retesting. CONCLUSIONS: Adoption of retesting guidelines in 2012 appears to have successfully increased retesting rates, but missed opportunities to identify incident HIV infection during pregnancy may contribute to continuing high rates of perinatal HIV transmission in southwestern Kenya.