Increasing emphasis on the use of real-world evidence (RWE) to support clinical policy and regulatory decision-making has led to a proliferation of guidance, advice, and frameworks from regulatory agencies, academia, professional societies, and industry. A broad spectrum of studies use real-world data (RWD) to produce RWE, ranging from randomized trials with outcomes assessed using RWD to fully observational studies. Yet, many proposals for generating RWE lack sufficient detail, and many analyses of RWD suffer from implausible assumptions, other methodological flaws, or inappropriate interpretations. The Causal Roadmap is an explicit, itemized, iterative process that guides investigators to prespecify study design and analysis plans; it addresses a wide range of guidance within a single framework. By supporting the transparent evaluation of causal assumptions and facilitating objective comparisons of design and analysis choices based on prespecified criteria, the Roadmap can help investigators to evaluate the quality of evidence that a given study is likely to produce, specify a study to generate high-quality RWE, and communicate effectively with regulatory agencies and other stakeholders. This paper aims to disseminate and extend the Causal Roadmap framework for use by clinical and translational researchers; three companion papers demonstrate applications of the Causal Roadmap for specific use cases.

BACKGROUND: IGTS is a rare phenomenon of paradoxical germ cell tumor (GCT) growth during or following treatment despite normalization of tumor markers. We sought to evaluate the frequency, clinical characteristics and outcome of IGTS in patients in 21 North-American and Australian institutions. METHODS: Patients with IGTS diagnosed from 2000-2017 were retrospectively evaluated. RESULTS: Out of 739 GCT diagnoses, IGTS was identified in 33 patients (4.5%). IGTS occurred in 9/191 (4.7%) mixed-malignant GCTs, 4/22 (18.2%) immature teratomas (ITs), 3/472 (0.6%) germinomas/germinomas with mature teratoma, and in 17 secreting non-biopsied tumours. Median age at GCT diagnosis was 10.9 years (range 1.8-19.4). Male gender (84%) and pineal location (88%) predominated. Of 27 patients with elevated markers, median serum AFP and Beta-HCG were 70 ng/mL (range 9.2-932) and 44 IU/L (range 4.2-493), respectively. IGTS occurred at a median time of 2 months (range 0.5-32) from diagnosis, during chemotherapy in 85%, radiation in 3%, and after treatment completion in 12%. Surgical resection was attempted in all, leading to gross total resection in 76%. Most patients (79%) resumed GCT chemotherapy/radiation after surgery. At a median follow-up of 5.3 years (range 0.3-12), all but 2 patients are alive (1 succumbed to progressive disease, 1 to malignant transformation of GCT). CONCLUSION: IGTS occurred in less than 5% of patients with GCT and most commonly after initiation of chemotherapy. IGTS was more common in patients with IT-only on biopsy than with mixed-malignant GCT. Surgical resection is a principal treatment modality. Survival outcomes for patients who developed IGTS are favourable.

Inheritance of resistance to diclofop was studied in three wild oat biotypes (designated B, C., and H) from the Willamette Valley of Oregon. Cultivated oat (cultivar ‘Monida’) was crossed, including reciprocals, to three wild oat biotypes. Leaves of each F1 plant were spotted with diclofop as a nondestructive test for resistance or susceptibility. All F1 hybrids were resistant, indicating that resistance is dominant and is under nuclear control. The F2 plants where Monida was the maternal parent were screened with diclofop, and F2 plants of the Monida/C cross were screened with fenoxaprop because the parent C biotype was resistant to fenoxaprop. At lower doses, a 3:1 (R:S) segregation ratio in F2 was observed and at higher doses a 1:3 (R:S) segregation ratio was often observed. The F2:3 families segregated in a 1:2:1 (all resistant : segregating resistant and susceptible all susceptible) ratio when treated with a 1.1-kg ae ha−1 dose of diclofop. This confirms that resistance to diclofop in the B, C, and H biotypes is primarily under monogenic control, with resistance being dominant to susceptibility at lower herbicide doses. At increased doses, susceptibility becomes dominant. Knowledge of the inheritance of resistance may help in the development of containment measures to prevent the spread of herbicide-resistance genes.

The DNA sequence of a 196 base pair (bp) region of the acetolactate synthase (ALS) genes of three weed species, kochia, prickly lettuce, and Russian thistle, was determined. This region encompasses the coding sequence for Domain A, a region of the amino acid sequence previously demonstrated to play a pivotal role in conferring resistance to herbicides that inhibit ALS. The Domain A DNA sequence from a chlorsulfuron-resistant (R) prickly lettuce biotype from Idaho differed from that of a chlorsulfuron-susceptible (S) biotype by a single point mutation, which substituted a histidine for a proline. The Domain A DNA sequence from an R kochia biotype from Kansas also differed from that of an S biotype by a single point mutation in the same proline codon. This point mutation, however, conferred substitution of threonine for proline. Two different ALS-homologous sequences were isolated from an R biotype of Russian thistle. Neither sequence encoded amino acid substitutions in Domain A that differed from the consensus S sequence. The DNA sequence variation among the R and S kochia biotypes was used to characterize six Ada County, Idaho, kochia collections for correlation between phenotypic chlorsulfuron susceptibility and restriction digest patterns (RFLPs) of polymerase chain reaction amplification products. Most collections showed excellent correspondence between the RFLP patterns and the phenotypic response to chlorsulfuron application. However, one entirely R collection had the RFLP pattern of the S biotype, suggesting that resistance was not due to mutation in the proline codon.

A combination of microscopical methods (TEM, SEM, freeze fracture, confocal, immunoEM) are utilized to demonstrate functional interactions between somatic cells and the germline in the model organism, C. elegans.A variety of signaling mechanisms are required to coordinate activity of maturing oocytes and the somatic sheath of the gonad. Communication via gap junctions allows direct coordination of sheath cell motility and oocyte development. Gap junctions link adjoining sheath cells, and link the sheath to the most mature oocyte. Yolk protein produced in the intestine passes from the pseudocoelom through the basal lamina of the proximal gonad and then through fenestrations (sheath pores) in the gonad sheath before being endocytosed into the most mature oocytes. Myoepithelial specializations anchored by hemi-adherens junctions in the proximal sheath act to squeeze the primary oocyte out of the gonad to the spermatheca for fertilization.