Many cell-fate decisions during embryonic development are governed by a motif comprised of two transcription factors (TFs) A and B that mutually inhibit each other and may self-activate. This motif, called as a self-activating toggle switch (SATS), can typically have three stable states (phenotypes)-two corresponding to differentiated cell fates, each of which has a much higher level of one TF than the other-[Formula: see text] or [Formula: see text]-and the third state corresponding to an 'undecided' stem-like state with similar levels of both A and B-[Formula: see text]. Furthermore, two or more SATSes can be coupled together in various topologies in different contexts, thereby affecting the coordination between multiple cellular decisions. However, two questions remain largely unanswered: (a) what governs the co-existence and relative stability of these three stable states? (b) What orchestrates the decision-making of coupled SATSes? Here, we first demonstrate that the co-existence and relative stability of the three stable states in an individual SATS can be governed by the relative strength of self-activation, external signals activating and/or inhibiting A and B, and mutual degradation between A and B. Simultaneously, we investigate the effects of these factors on the decision-making of two coupled SATSes. Our results offer novel understanding into the operating principles of individual and coupled tristable self-activating toggle switches (SATSes) regulating cellular differentiation and can yield insights into synthesizing three-way genetic circuits and understanding of cellular reprogramming.

This topical report covers the year 2 of the subject 3-year grant, evaluating the relationship between fracture-related dolomite and dolomite constrained by primary rock fabric in the 3 most prolific reservoir intervals in the Michigan Basin (Ordovician Trenton-Black River Formations; Silurian Niagara Group; and the Devonian Dundee Formation). The characterization of select dolomite reservoirs has been the major focus of our efforts in Phase II/Year 2. Fields have been prioritized based upon the availability of rock data for interpretation of depositional environments, fracture density and distribution as well as thin section, geochemical, and petrophysical analyses. Structural mapping and log analysis in the Dundee (Devonian) and Trenton/Black River (Ordovician) suggest a close spatial relationship among gross dolomite distribution and regional-scale, wrench fault-related NW-SE and NE-SW structural trends. A high temperature origin for much of the dolomite in the 3 studied intervals (based upon initial fluid inclusion homogenization temperatures and stable isotopic analyses,) coupled with persistent association of this dolomite in reservoirs coincident with wrench fault-related features, is strong evidence for these reservoirs being influenced by hydrothermal dolomitization. For the Niagaran (Silurian), a comprehensive high resolution sequence stratigraphic framework has been developed for a pinnacle reef in the northern reef trend where we had 100% core coverage throughout the reef section. Major findings to date are that facies types, when analyzed at a detailed level, have direct links to reservoir porosity and permeability in these dolomites. This pattern is consistent with our original hypothesis of primary facies control on dolomitization and resulting reservoir quality at some level. The identification of distinct and predictable vertical stacking patterns within a hierarchical sequence and cycle framework provides a high degree of confidence at this point that results will be exportable throughout the basin. Ten petrophysically significant facies have been described in the northern reef trend, providing significantly more resolution than the standard 4-6 that are used most often in the basin (e.g. Gill, 1977). Initial petrophysical characterization (sonic velocity analysis under confining pressures) shows a clear pattern that is dependent upon facies and resulting pore architecture. Primary facies is a key factor in the ultimate diagenetic modification of the rock and the resulting pore architecture. Facies with good porosity and permeability clearly show relatively slow velocity values as would be expected, and low porosity and permeability samples exhibit fast sonic velocity values, again as expected. What is significant is that some facies that have high porosity values, either measured directly or from wireline logs, also have very fast sonic velocity values. This is due to these facies having a pore architecture characterized by more localized pores (vugs, molds or fractures) that are not in commun...

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The Circumsporozoite Protein (CSP) of Plasmodium falciparum contains an N-terminal region, a conserved Region I (RI), a junctional region, 25–42 copies of major (NPNA) and minor repeats followed by a C-terminal domain. The recently approved malaria vaccine, RTS,S/AS01 contains NPNAx19 and the C-terminal region of CSP. The efficacy of RTS,S against natural infection is low and short-lived, and mapping epitopes of inhibitory monoclonal antibodies may allow for rational improvement of CSP vaccines. Tobacco Mosaic Virus (TMV) was used here to display the junctional epitope (mAb CIS43), Region I (mAb 5D5), NPNAx5, and NPNAx20 epitope of CSP (mAbs 317 and 580). Protection studies in mice revealed that Region I did not elicit protective antibodies, and polyclonal antibodies against the junctional epitope showed equivalent protection to NPNAx5. Combining the junctional and NPNAx5 epitopes reduced immunogenicity and efficacy, and increasing the repeat valency to NPNAx20 did not improve upon NPNAx5. TMV was confirmed as a versatile vaccine platform for displaying small epitopes defined by neutralizing mAbs. We show that polyclonal antibodies against engineered VLPs can recapitulate the binding specificity of the mAbs and immune-focusing by reducing the structural complexity of an epitope may be superior to immune-broadening as a vaccine design approach. Most importantly the junctional and restricted valency NPNA epitopes can be the basis for developing highly effective second-generation malaria vaccine candidates.

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