Quantitative profiling of human translation initiation reveals regulatory elements that potently affect endogenous and therapeutically modified mRNAs.

mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5' untranslated regions and identify sequences that mediate 250-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3-6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-methylpseudouridine (m1Ψ) selectively enhances translation by specific 5' UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissect mechanisms of human translation initiation and engineer more potent therapeutic mRNAs. Highlights: Measurement of >30,000 human 5' UTRs reveals a 250-fold range of translation outputSystematic mutagenesis demonstrates the causality of short (3-6nt) regulatory elementsN1-methylpseudouridine alters translation initiation in a sequence-specific mannerOptimal modified 5' UTRs outperform those in the current class of mRNA vaccines.

Optimized protocol for quantifying 5' UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting.

Direct analysis of ribosome targeting (DART) allows investigators to measure the translation initiation potential of thousands of RNAs in parallel. Here, we describe an optimized protocol for generating active translation extract from S. cerevisiae, followed by in vitro translation, purification of ribosome-bound RNAs, and subsequent library preparation and sequencing. This protocol can be applied to a variety of cell types and will enable high-throughput interrogation of translational determinants. For complete details on the use and execution of this protocol, please refer to Niederer et al. (2022).1.

RNA modifications and structures cooperate to guide RNA-protein interactions.

An emerging body of evidence indicates that post-transcriptional gene regulation relies not only on the sequence of mRNAs but also on their folding into intricate secondary structures and on the chemical modifications of the RNA bases. These features, which are highly dynamic and interdependent, exert direct control over the transcriptome and thereby influence many aspects of cell function. Here, we consider how the coupling of RNA modifications and structures shapes RNA-protein interactions at different steps of the gene expression process.

Manipulating thyroid status alters endoplasmic reticulum calcium homeostasis in rat cerebellum.

Thyroid-related hormones regulate the efficiency and expression of sarco-endoplasmic reticulum calcium ATPases in cardiac and skeletal muscle. However, little is known about the relationship between thyroid hormones and calcium (Ca2+) homeostasis in the brain. It is hypothesized that manipulating rat thyroid hormone levels would induce significant brain Ca2+ adaptations consistent with clinical findings. Adult male Sprague-Dawley rats were assigned to one of three treatment groups for 28 days: control, hypothyroid (6-n-propyl-2-thiouracil (PTU), an inhibitor of thyroxine (T4) synthesis), and hyperthyroid (T4). Throughout, rats were given weekly behavioral tests. Ca2+ accumulation decreased in the cerebellum in both hyper- and hypothyroid animals. This was specific to different ER pools of calcium with regional heterogeneity in the response to thyroid hormone manipulation. Behavioral tasks demonstrated sensitivity to thyroid manipulation, and corresponded to alterations in calcium homeostasis. Ca2+ accumulation heterogeneity in chronic hyper- and hypothyroid animals potentially explains clinical manifestations of altered thyroid status.

Physiological responses in swine treated with water containing sodium bicarbonate as a prophylactic for gastric ulcers.

Maintenance of gastric pH above 4.0 aids the prevention of bile acid-mediated ulcerative damage to the pars esophageal tissue in pigs. One means of doing so is the addition of buffering compounds, such as sodium bicarbonate, to the water supply; however, any potential physiological effect of buffer consumption has yet to be determined. Experiment 1 tested the acute effects of buffer addition to the water supply on systemic acid-base and electrolyte balance in swine (BW 40.7 +/- 3.0 kg). Consumption of water calculated to a 200 mOsm solution with sodium bicarbonate for 24 h increased (P < 0.05) blood Na+, HCO3(-), and pCO2, although these effects were all within physiologically tolerable levels. Urine pH and Na+ excretion increased (P < 0.001) following the consumption of NaHCO3, with Na+ concentration almost threefold higher in treated pigs compared with controls. Experiment 2 determined the chronic systemic effects of buffer consumption by measuring blood and urine variables, with pigs consuming NaHCO3-treated water throughout. Water consumption increased (P < 0.001) during buffer consumption, although intake levels remained within normal ranges. Blood pH levels were not affected by long-term consumption of dietary buffer; however, blood HCO3(-) (P < 0.05), Na+, and pCO2 (P < 0.01) increased. Urine pH and urine Na+ concentration increased (P < 0.01) in buffer-treated compared with control animals. Results indicate that sodium bicarbonate can safely be added to the water supply for pigs, with no clinically relevant alterations in acid-base balance because the animals readily compensate for buffer intake.

A phase III study of radiation therapy plus carmustine with or without recombinant interferon-alpha in the treatment of patients with newly diagnosed high-grade glioma.

BACKGROUND: The current study was conducted to determine whether the addition of interferon-alpha (IFN-alpha) to treatment with radiation therapy and carmustine (BCNU) improves time to disease progression or overall survival in patients with high-grade glioma. METHODS: Patients with anaplastic astrocytoma, anaplastic oligoastrocytoma, glioblastoma multiforme, or gliosarcoma received radiation therapy plus BCNU as initial therapy. Subsequently, patients without tumor progression at the completion of radiation therapy were stratified by age, extent of surgery, tumor grade and histology, Eastern Cooperative Oncology Group performance status, and treating institution, and then were randomly assigned to receive either BCNU alone (200 mg/m(2) on Day 1) or BCNU (150 mg/m(2) on Day 3) plus IFN--alpha (12 million U/m(2) on Days 1-3, Weeks 1, 3, and 5) every 7 weeks for a maximum of 6 cycles. RESULTS: Of the 383 patients enrolled in the study, 275 eligible patients were randomized. There was no significant difference with regard to time to disease progression or overall survival between the two groups. Patients receiving IFN-alpha experienced more fever, chills, myalgias, and neurocortical symptoms including somnolence, confusion, and exacerbation of neurologic deficits. Cox multivariate regression models confirmed known favorable prognostic variables including younger age, Grade 3 tumor (according to World Health Organization criteria), and greater extent of surgery. Cox and classification and regression tree analysis models also demonstrated that a normal baseline Folstein mini-mental status examination (MMSE) score was associated with better prognosis. CONCLUSIONS: IFN-alpha does not appear to improve time to disease progression or overall survival in patients with high-grade glioma and appears to add significantly to toxicity. The baseline MMSE score may serve as an independent prognostic factor and warrants further investigation.

Can breast cancer be prevented?

Breast cancer is the most common invasive cancer in American women and is second only to carcinoma of the lung in cancer deaths. The results of the National Surgical Adjuvant Breast and Bowel Project Breast Cancer Prevention Trial (BCPT) were released in April 1998. In the BCPT, 13,388 women at increased risk for the development of breast cancer were randomized to receive tamoxifen or placebo for 5 years, resulting in a 49% reduction in invasive breast cancer and a 50% reduction in noninvasive breast cancer. In May 1998, the preliminary results from the Multiple Outcomes of Raloxifene Evaluation (MORE) trial were reported to the American Society of Clinical Oncology. The MORE trial was evaluating the drug raloxifene for the prevention and treatment of osteoporosis; however, a secondary outcome was a reduction in breast cancer risk in raloxifene-treated women. Based upon the results of the BCPT and MORE trials, a second-generation breast cancer prevention trial has been initiated. The Study of Tamoxifen and Raloxifene (STAR) was initiated in June 1999. Ochsner Clinic and Alton Ochsner Medical Foundation, active participants in the BCPT, were named a clinical center for the STAR trial.

Recent advances in the systemic therapy of breast cancer.

A number of advances have been made over the last decade in the systemic therapy of breast cancer, including 1) the introduction of the taxanes, paclitaxel and docetaxel, into the treatment regimes for both early and advanced breast cancer; 2) a greater understanding of the use of high-dose chemotherapy, supplemented with stem cell rescue; 3) the development of newer parenteral and oral chemotherapeutic drugs; 4) the first biologic therapy for breast cancer; 5) the first demonstration of effective drug therapy to prevent the development of breast cancer; and 6) effective agents to palliate and/or prevent complications from bone metastasis. These and other developments are allowing both the extension of life and a better quality of life for those stricken with the breast cancer.

Soybean hulls as a dietary fiber source for dogs.

In Exp. 1, soybean hull samples were obtained from nine sources across the United States and analyzed for nutrient content to determine their suitability for inclusion in dog diets. Compositional data revealed variation in both the amount of total dietary fiber (TDF; 63.8 to 81.2%) in the soybean hulls and the ratio of insoluble:soluble fiber (5.0:1 to 15.4:1). Crude protein content varied widely among sources, ranging from 9.2 to 18.7%. An in vivo trial (Exp. 2) was conducted using a premium dog diet containing 3.0, 4.5, 6.0, 7.5, or 9.0% soybean hulls (DM basis). There was a negative linear effect (P < .05) of soybean hull inclusion in the diet on DM, OM, TDF, and GE total-tract digestibilities, as well as on calculated ME. Crude protein and fat digestibilities were unaffected by treatment. Based on these results, ileally cannulated dogs were fed diets containing 6.0, 7.5, or 9.0% soybean hulls (DM basis) in addition to diets containing either 0% supplemental fiber or 7.5% beet pulp (Exp. 3). Nutrient digestion at the ileum was unaffected by inclusion of supplemental fiber. Total tract digestion of DM, OM, and GE was lower ( P < .05) for diets containing supplemental fiber when compared with the diet containing 0% fiber. Crude protein and fat digestibilities were unaffected by treatment. There was no difference in nutrient digestibility between those diets containing soybean hulls and a diet containing beet pulp. Soybean hull inclusion in the diet resulted in a negative linear effect (P < .05) on calculated ME, in addition to lowering ME (P < .05) when compared with the 0% fiber control diet. Calculated ME for dogs fed a 7.5% beet pulp-containing diet was lower (P < .05) than that for dogs fed the soybean hull-containing diets. Results indicate that soybean hulls can be an effective dietary fiber source in dog diets.

Sepsis due to coryneform group A-4 in an immunocompromised host.

Corynebacteria are more commonly being recognized as significant human pathogens. We describe a case of Coryneform group A-4 sepsis secondary to infection of a Hickman catheter in an immunocompromised man; the organism was identified by biochemical analysis conducted at the Louisiana State Reference Laboratory.

Postsplenectomy infections.

Serious postsplenectomy infection is a significant threat to patients. Although the incidence of such infection is low, the resultant mortality is extremely high. This susceptibility to infection and ensuing mortality results from multiple immunologic changes that occur after splenectomy. These changes include alteration in immunoglobulin levels, loss of serum opsonizing proteins, and alterations in antigen clearance and cellular immunity.

Acute myelomonocytic leukemia presenting as a benign-appearing cutaneous eruption.

Aleukemic leukemia cutis is a rare condition in which patients have skin lesions containing leukemic cells before evidence of leukemia can be detected in the peripheral blood. There are only 23 cases of this phenomenon documented in the English literature. We describe a 62-year-old woman who developed a diffuse, clinically benign-appearing cutaneous eruption, which histologically showed an atypical infiltrate of cells, 4 months before leukemic cells were found in her peripheral blood and the diagnosis of acute myelomonocytic leukemia was made by bone marrow aspiration. This case illustrates the difficulty in diagnosing leukemia cutis from examination of routine histologic sections and the importance of specialized marker studies in determining the cause of an atypical cellular infiltrate of the skin. It also illustrates how leukemia cutis can masquerade as a clinically benign-appearing cutaneous eruption in a seemingly healthy patient with normal blood parameters.

The effect in vitro of 2'-deoxycytidine on the metabolism and cytotoxicity of 2',3'-dideoxycytidine.

We examined in vitro the effect of high, but clinically achievable and non-toxic, concentrations of 2'-deoxycytidine (dCyd) (greater than or equal to 100 mumols/l on the metabolism and cytotoxicity of 2',3'-dideoxycytidine (DDC) in normal human bone marrow mononuclear cells (BMMCs) and a cultured T-lymphocyte (HUT-102) cell line. Colony formation in semi-solid medium by bone marrow progenitor cells (CFU-GM and CFU-GEMM) was significantly protected by dCyd against the cytotoxic effects of high doses of DDC. In contrast, in HIV-infected HUT-102 cells, anti-HIV effect of DDC (10 mumols/l) was preserved in the presence of 100 mumols/l dCyd but partially reversed by higher levels of dCyd. dCyd reduced the generation of DDC triphosphate (DDC-TP) relative to dCyd triphosphate (dCTP) pools to a significantly greater extent in BMMCs versus HUT-102 cells. This might explain dCyd-mediated abrogation of DDC cytotoxicity against marrow progenitor cells with relative preservation of its anti-HIV-1 activity in HUT-102 cells.

Amniotic fluid levels of alpha-fetoprotein and other proteins in a case of Turner's syndrome of the fetus.

A 34-year-old primigravida underwent genetic amniocentesis at 20 weeks gestation and the fetus was diagnosed as having Turner's syndrome. The concentration of alpha-fetoprotein in amniotic fluid was greatly elevated. Normal concentrations of amniotic fluid total protein, albumin and immunoglobulin indicated that the elevated level of alpha-fetoprotein was not the result of leakage through a hygroma, which has been previously suggested without supporting data in cases of Turner's syndrome of the fetus.