Safety and efficacy of the tumor-selective adenovirus enadenotucirev, in combination with nivolumab, in patients with advanced/metastatic epithelial cancer: a phase I clinical trial (SPICE).

BACKGROUND: Novel combination therapies to overcome anti-PD-1 resistance are required. Enadenotucirev, a tumor-selective blood stable adenoviral vector, has demonstrated a manageable safety profile and ability to increase tumor immune-cell infiltration in phase I studies in solid tumors. METHODS: We conducted a phase I multicenter study of intravenous enadenotucirev plus nivolumab in patients with advanced/metastatic epithelial cancer not responding to standard therapy. Co-primary objectives were safety/tolerability and maximum tolerated dose and/or maximum feasible dose (MTD/MFD) of enadenotucirev plus nivolumab. Additional endpoints included response rate, cytokine responses, and anti-tumor immune responses. RESULTS: Overall, 51 heavily pre-treated patients were treated, 45/51 (88%) of whom had colorectal cancer (35/35 patients with information available were microsatellite instability-low/microsatellite stable) and 6/51 (12%) had squamous cell carcinoma of the head and neck. The MTD/MFD of enadenotucirev plus nivolumab was not reached, with the highest dose level tested (1×1012 vp day 1; 6×1012 vp days 3 and 5) shown to be tolerable. Overall, 31/51 (61%) patients experienced a grade 3-4 treatment-emergent adverse event (TEAE), most frequently anemia (12%), infusion-related reaction (8%), hyponatremia (6%), and large intestinal obstruction (6%). Seven (14%) patients experienced serious TEAEs related to enadenotucirev; the only serious TEAE related to enadenotucirev occurring in >1 patient was infusion-related reaction (n=2). Among the 47 patients included in efficacy analyses, median progression-free survival was 1.6 months, objective response rate was 2% (one partial response for 10 months), and 45% of patients achieved stable disease. Median overall survival was 16.0 months; 69% of patients were alive at 12 months. Persistent increases in Th1 and related cytokines (IFNγ, IL-12p70, IL-17A) were seen from ~day 15 in two patients, one of whom had a partial response. Among the 14 patients with matching pre-tumor and post-tumor biopsies, 12 had an increase in intra-tumoral CD8+ T-cell infiltration and 7 had increased markers of CD8 T-cell cytolytic activity. CONCLUSIONS: Intravenously dosed enadenotucirev plus nivolumab demonstrated manageable tolerability, an encouraging overall survival and induced immune cell infiltration and activation in patients with advanced/metastatic epithelial cancer. Studies of next-generation variants of enadenotucirev (T-SIGn vectors) designed to further re-program the tumor microenvironment by expressing immune-enhancer transgenes are ongoing. TRIAL REGISTRATION NUMBER: NCT02636036.

Target engagement and cellular fate of otelixizumab: a repeat dose escalation study of an anti-CD3ε mAb in new-onset type 1 diabetes mellitus patients.

AIMS: This paper describes the pharmacological findings from a study where otelixizumab, an anti-CD3ɛ mAb, was dosed in new onset Type 1 diabetes mellitus (NOT1DM) patients. This is the first time that the full dose-response of an anti-CD3ɛ mAb has been investigated in the clinic. The data have been validated using a previously developed pharmacokinetic/pharmacodynamic (PK/PD) model of otelixizumab to simulate the interplay between drug administration, CD3ɛ target engagement and downmodulation. METHODS: Patients were randomized to control or active treatment with otelixizumab (1:4), administered via infusion over 6 days, in a dose-ascending study consisted of three cohorts (n = 10 per cohort) at doses of 9, 18 or 27 mg respectively. The study allowed quantification of otelixizumab PK, CD3ɛ target engagement and its pharmacodynamic effect (CD3ε/TCR modulation on circulating T lymphocytes). RESULTS: Otelixizumab concentrations increased and averaged to 364.09 (54.3), 1625.55 (72.5) and 2781.35 (28.0) ng ml-1 (Geom.mean, %CV) at the 9, 18 and 27 mg dose respectively. CD3ɛ target engagement was found to be rapid (within the first 30 min), leading to a receptor occupancy of ~60% within 6 h of dosing in all three doses. A dose-response relationship was observed with the two highest doses achieving a ~90% target engagement and consequential CD3ɛ/TCR downmodulation by Day 6. CONCLUSIONS: Data from this study revealed maximum target engagement and CD3ɛ/TCR modulation is achieved at doses of 18, 27 mg of otelixizumab. These findings can be useful in guiding dose selection in clinical trials with anti-CD3ɛ mAbs.

Acetosyringone treatment duration affects large T-DNA molecule transfer to rice callus.

BACKGROUND: Large T-DNA fragment transfer has long been a problem for Agrobacterium-mediated transformation. Although vector systems, such as the BIBAC series, were successfully developed for the purpose, low transformation efficiencies were consistently observed. RESULTS: To gain insights of this problem in monocot transformation, we investigated the T-strand accumulation of various size of T-DNA in two kinds of binary vectors (one copy vs. multi-copy) upon acetosyringone (AS) induction and explored ways to improve the efficiency of the large T-DNA fragment transfer in Agrobacterium-mediated rice transformation. By performing immuno-precipitation of VirD2-T-strands and quantitative real-time PCR assays, we monitored the accumulation of the T-strands in Agrobacterium tumeficiens after AS induction. We further demonstrated that extension of AS induction time highly significantly improved large-size T-DNA transfer to rice cells. CONCLUSIONS: Our data provide valuable information of the T-strand dynamics and its impact on large T-DNA transfer in monocots, and likely dicots as well.

Best practices in performing flow cytometry in a regulated environment: feedback from experience within the European Bioanalysis Forum.

Flow cytometry is a powerful tool that can be used for the support of (pre)clinical studies. Although various white papers are available that describe the set-up and validation of the instrumentation (the flow cytometer) and validation of flow cytometry methods, to date no guidelines exist that address the requirements for performing flow cytometry in a regulated environment. In this manuscript, the European Bioanalysis Forum presents additional practice guidance on the use of flow cytometry in the support of drug development programs and addresses areas that are not covered in the previous publications. The concepts presented here are based on the consensus of discussions in the European Bioanalysis Forum Topic Team 32, in meetings in Barcelona, Limelette and multiple telephone conferences.

Association Between Midlife Cardiorespiratory Fitness and Risk of Stroke: The Cooper Center Longitudinal Study.

BACKGROUND AND PURPOSE: Low cardiorespiratory fitness (CRF) is associated with an increased risk of stroke. However, the extent to which this association is explained by the development of stroke risk factors such as diabetes mellitus, hypertension, and atrial fibrillation is unknown. We evaluated the relationship between midlife CRF and risk of stroke after the age of 65 years, independent of the antecedent risk factor burden. METHODS: Linking participant data from the Cooper Center Longitudinal Study with Medicare claims files, we studied 19 815 individuals who survived to receive Medicare coverage from 1999 to 2009. CRF estimated at baseline by Balke treadmill time was analyzed as a continuous variable (in metabolic equivalents) and according to age- and sex-specific quintiles (Q1=low CRF). Associations between midlife CRF and stroke hospitalization after the age of 65 years were assessed by applying a proportional hazards recurrent events model to the failure time data with hypertension, diabetes mellitus, and atrial fibrillation as time-dependent covariates. RESULTS: After 129 436 person-years of Medicare follow-up, we observed 808 stroke hospitalizations. After adjustment for baseline risk factors, higher midlife CRF was associated with a lower risk of stroke hospitalization (hazard ratio [HR], 0.61; 95% confidence interval [CI], 0.49-0.76; quintiles 4-5 versus 1]. This association remained unchanged after additional adjustment for burden of Medicare-identified stroke risk factors (hypertension, diabetes mellitus, and atrial fibrillation; HR, 0.63; 95% CI, 0.51-0.79; quintiles 4-5 versus 1). CONCLUSIONS: There is a strong, inverse association between midlife CRF and stroke risk in later life independent of baseline and antecedent burden of risk factors, such as hypertension, diabetes mellitus, and atrial fibrillation.

Class II Resin Composites: Restorative Options.

Tooth-coloured, resin composite restorations are amongst the most frequently prescribed forms of dental restoration to manage defects in posterior teeth. The attainment of a desirable outcome when placing posterior resin composite restorations requires the clinician to have a good understanding of the benefits (as well as the limitations) posed by this material, together with a sound knowledge of placement technique. Numerous protocols and materials have evolved to assist the dental operator with this type of demanding posterior restoration. With the use of case examples, four techniques available are reported here. CPD/Clinical Relevance: This article explores varying techniques for the restoration of Class II cavities using resin composite.

Refractory Epistaxis due to Severe Factor V Deficiency with Inhibitor.

Factor V deficiency secondary to inhibitors is extremely rare and can be caused by a wide collection of exposures such as bovine thrombin and beta lactamase antibiotics. The management of factor V deficiency with inhibitor is a condition treated based on case reports due to the rarity of this condition. We describe a complicated case of an elderly patient with severe factor V deficiency with high inhibitor titer refractory to FEIBA (anti-inhibitor coagulation complex) treated with NovoSeven concurrently with cyclosporine immunosuppression and Rituxan. Given that there are no consensus guidelines on treatment, this case offers important insight into the therapeutic approaches that can be used to treat such patients.

Atrial fibrillation ablation and left appendage closure in heart failure patients.

PURPOSE OF REVIEW: Patients with atrial fibrillation and heart failure experience an increased morbidity and mortality from the hemodynamic consequences of atrial fibrillation and an increased stroke risk. Consequently, there has been increased attention to procedural alternatives to pharmacologic rhythm control and anticoagulation for stroke prevention. This review aims to evaluate the evidence for atrial fibrillation ablation and left atrial appendage closure in heart failure patients. RECENT FINDINGS: Several randomized control trials and systematic reviews demonstrate the safety and efficacy of atrial fibrillation ablation in patients with heart failure and left ventricular systolic dysfunction. In multiple trials, these patients have shown clinical benefit from atrial fibrillation ablation including improved left ventricular systolic function, quality of life, and clinical heart failure symptoms. The evidence of clinical benefit of atrial fibrillation ablation in heart failure patients with preserved ejection fraction remains limited. Only a handful of randomized controlled trials have been performed evaluating left atrial appendage closure, and there is insufficient data regarding the safety and efficacy of these procedures in heart failure patients. SUMMARY: Atrial fibrillation ablation in heart failure patients remains well tolerated with an overall efficacy comparable to atrial fibrillation ablation in patients without heart failure. There is consistent evidence for the clinical benefit of atrial fibrillation ablation in heart failure patients with left ventricular systolic dysfunction and limited evidence for atrial fibrillation ablation in heart failure patients with preserved ejection fraction. Currently, there is insufficient data regarding the safety and efficacy of left atrial appendage closure devices in heart failure patients.

Changes in mid-life fitness predicts heart failure risk at a later age independent of interval development of cardiac and noncardiac risk factors: the Cooper Center Longitudinal Study.

AIMS: Low mid-life fitness is associated with higher risk for heart failure (HF). However, it is unclear to what extent this HF risk is modifiable and mediated by the burden of cardiac and noncardiac comorbidities. We studied the effect of cardiac and noncardiac comorbidities on the association of mid-life fitness and fitness change with HF risk. METHODS: Linking individual subject data from the Cooper Center Longitudinal Study (CCLS) with Medicare claims files, we studied 19,485 subjects (21.2% women) who survived to receive Medicare coverage from 1999 to 2009. Fitness estimated by Balke treadmill time at mean age of 49 years was analyzed as a continuous variable (in metabolic equivalents [METs]) and according to age- and sex-specific quintiles. Associations of mid-life fitness and fitness change with HF hospitalization after age of 65 years were assessed by applying a proportional hazards recurrent events model to the failure time data with each comorbidity entered as time-dependent covariates. RESULTS: After 127,110 person years of Medicare follow-up, we observed 1,038 HF hospitalizations. Higher mid-life fitness was associated with a lower risk for HF hospitalization (hazard ratio [HR] 0.82 [0.76-0.87] per MET) after adjustment for traditional risk factors. This remained unchanged after further adjustment for the burden of Medicare-identified cardiac and noncardiac comorbidities (HR 0.83 [0.78-0.89]). Each 1 MET improvement in mid-life fitness was associated with a 17% lower risk for HF hospitalization in later life (HR 0.83 [0.74-0.93] per MET). CONCLUSIONS: Mid-life fitness is an independent and modifiable risk factor for HF hospitalization at a later age.

Overexpression of ubiquitin-like LpHUB1 gene confers drought tolerance in perennial ryegrass.

HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin-like post-translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate-controlled growth chambers. Here, pasture-type and turf-type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses.

Clinical intervention for quality improvement of gastric-emptying studies.

UNLABELLED: Prompted by clinical concerns for false-negative tests, we implemented a clinical intervention consisting of a training session and an image-based verification procedure to document homogeneous radioactivity distribution in the radiolabeled meal (egg substitute per the guideline). METHODS: A technologist training session emphasized the importance of thorough mixing of (99m)Tc-sulfur colloid in the egg meal. For 6 mo after training, an image of the prepared mixed egg was acquired before patient ingestion. Consecutive gastric-emptying studies performed 6 mo before and after training were reviewed by 2 experienced physicians. RESULTS: There were 7 abnormal and 44 normal studies before and 15 abnormal and 29 normal studies after training (P < 0.05). Subjective evaluations of images for meal-mixing quality by 2 readers correlated with each other and with an objective measure of expected gastric-emptying physiology (correlation coefficients, 0.54 and 0.38, respectively). CONCLUSION: The described clinical intervention improved the accuracy of our gastric-emptying studies by decreasing false-negative studies.

A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C4 plants.

BACKGROUND: Plants that utilize the highly efficient C4 pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C4 capability and function. In mature C4 leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO2 fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C4 patterns of rbcL gene expression has been an elusive goal for many years. RESULTS: RLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C4 chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C4 plants. Comparative analysis using maize (C4) and Arabidopsis (C3) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks. CONCLUSIONS: Reductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation and binding of the RLSB binding protein to rbcL mRNA within BS chloroplasts may be one determinant leading to the characteristic cell type-specific localization of Rubisco in C4 plants. Evolutionary modification of RLSB expression, from a C3 "default" state to BS cell-specificity, could represent one mechanism by which rbcL expression has become restricted to only one cell type in C4 plants.

Hypereosinophilic syndrome with cardiac involvement in a pregnant patient with multiple sclerosis.

In hypereosinophilic syndrome, the sustained overproduction of eosinophils leads to the dysfunction of one or more organs. Symptoms vary in accordance with which organ is affected. Cardiac involvement leads to substantial morbidity and to most of the deaths that are associated with hypereosinophilic syndrome.Herein, we present the case of a 31-year-old woman, pregnant for 12 weeks and with a history of multiple sclerosis, who presented with transient vision loss and splinter hemorrhages in her fingernail beds. The diagnosis was hypereosinophilic syndrome with cardiac involvement. Echocardiography revealed 2 echodense structures: one that obliterated the left ventricular apex, and another in the basal lateral wall. The patient underwent therapy with prednisone and heparin but developed heparin-induced thrombocytopenia. This condition resolved when argatroban was substituted for heparin. Two weeks after the patient's release from the hospital, echocardiography revealed improvement in the echodense ventricular structures. The transient vision loss and the splinter hemorrhages were attributed to the hypereosinophilic syndrome.We believe that this is the 1st report of a pregnant patient with hypereosinophilic syndrome and cardiac involvement.

Development of a rapid biolistic assay to determine changes in relative levels of intracellular calcium in leaves following tetracycline uptake by pinto bean plants.

Tetracycline antibiotics, such as chlortetracycline (CTC) and tetracycline (TC), are introduced into agricultural lands through the application of manure as fertilizer. These compounds are phytotoxic to certain crop plants, including pinto beans (Phaseolus vulgaris), the species used for this investigation. While the mechanism of this toxicity is not yet understood, CTC is known to be a calcium chelator. We describe here a novel method to show that CTC is taken up by pinto bean plants and chelates calcium in leaves. Cameleon fusion proteins can provide qualitative and quantitative imaging of intracellular calcium levels, but current methodology requires stable transformation. Many plant species, including pinto beans, are not yet transformable using standard Agrobacterium-based protocols. To determine the role of calcium chelation in this plant, a rapid, biolistic method was developed to transiently express the cameleon protein. This method can easily be adapted to other plant systems. Our findings provide evidence that chelation of intracellular calcium by CTC is related to phytotoxic effects caused by this antibiotic in pinto beans. Root uptake of CTC and TC by pinto beans and their translocation to leaves were further verified by fluorescence spectroscopy and liquid chromatography/mass spectrometry, confirming results of the biolistic method that showed calcium chelation by tetracyclines in leaves.

Rubisco gene expression in C4 plants.

In leaves of most C(4) plants, ribulose 1,5 bisphosphate carboxylase (Rubisco) accumulates only in bundle sheath (bs) cells that surround the vascular centres, and not in mesophyll (mp) cells. It has been shown previously that in the C(4) dicots amaranth and Flaveria bidentis, post-transcriptional control of mRNA translation and stability mediate the C(4) expression patterns of genes encoding the large and small Rubisco subunits (chloroplast rbcL and nuclear RbcS, respectively). Translational control appears to regulate bs cell-specific Rubisco gene expression during early dicot leaf development, while control of mRNA stability appears to mediate bs-specific accumulation of RbcS and rbcL transcripts in mature leaves. Post-transcriptional control is also involved in the regulation of Rubisco gene expression by light, and in response to photosynthetic activity. Transgenic and transient expression studies in F. bidentis provide direct evidence for post-transcriptional control of bs cell-specific RbcS expression, which is mediated by the 5' and 3' untranslated regions (UTRs) of the mRNA. Comparisons of Rubisco gene expression in these dicots and in the monocot maize indicates possible commonalities in the regulation of RbcS and rbcL genes in these divergent C(4) species. Now that the role of post-transcriptional regulation in C(4) gene expression has been established, it is likely that future studies of mRNA-protein interactions will address long-standing questions about the establishment and maintenance of cell type-specificity in these plants. Some of these regulatory mechanisms may have ancestral origins in C(3) species, through modification of pre-existing factors, or by the acquisition of novel C(4) processes.

Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells.

We have previously shown that polyamine depletion decreased migration, Rac activation, and protein serine threonine phosphatase 2A activity. We have also shown that polyamine depletion increased cortical F-actin and decreased lamellipodia and stress fibers. In this study, we used staurosporine (STS), a potent, cell-permeable, and broad-spectrum serine/threonine kinase inhibitor, and studied migration. STS concentrations above 100 nM induced apoptosis. However, in polyamine-depleted cells, a lower concentration of STS (5 nM) increased attachment, spreading, Rac1 activation, and, subsequently, migration without causing apoptosis. STS-induced migration was completely prevented by a Rac1 inhibitor (NSC-23766) and dominant negative Rac1. These results imply that STS restores migration in polyamine-depleted cells through Rac1. The most important finding in this study was that polyamine depletion increased the association of phosphorylated myosin regulatory light chain (pThr(18)/Ser(19)-MRLC) at the cell periphery, which colocalized with thick cortical F-actin. Localization of pThr(18)- and pSer(19)-MRLC was found with stress fibers and nuclei, respectively. STS decreased the phosphorylation of cellular and peripheral pThr(18)-MRLC without any effect on nuclear pSer(19)-MRLC, dissolved thick cortical F-actin, and increased lamellipodia and stress fiber formation in polyamine-depleted cells. In control and polyamine-depleted cells, focal adhesion kinase (FAK) colocalized with stress fibers and the actin cortex, respectively. STS reorganized FAK, paxillin, and the cytoskeleton. These results suggest that polyamine depletion prevents the dephosphorylation of MRLC and thereby prevents the dynamic reorganization of the actin cytoskeleton and decreases lamellipodia formation resulting in the inhibition of migration.

The Lem2 gene promoter of barley directs cell- and development-specific expression of gfp in transgenic plants.

Transgenic approaches to combating fungal pathogens, such as Fusarium graminearum, require the targeting of antifungal gene expression in tissues of developing seed spikes of cereal grains, especially lemmas and epicarps. The Lem2 gene of barley encodes a lectin-like protein that is strongly up-regulated by salicylic acid and is preferentially expressed in lemmas, paleas (lemma/palea) and coleoptiles. Transient expression studies have indicated that the proximal -75/+70 region (relative to the transcription start site) determines organ specificity. In the present study, Golden Promise barley stably transformed with Morex Lem2 promoter/gfp reporter constructs displayed cell- and development-specific expression of gfp (green fluorescent protein gene). This expression corresponded to the expression seen in Northern blots of Morex organs. Under the full-length promoter, strong GFP fluorescence was observed in the lemma/palea, glumes, coleoptile, auricle and ligule. Weak GFP fluorescence was also observed in the rachis, tips of primary leaves and the leaf sheath. Unexpectedly, strong expression occurred in the epicarp, even though Lem2 is not expressed in this organ in Morex. Studies showed that the Lem2 promoter is more highly methylated in the epicarp than in the lemma of Morex. In the lemma/palea, gfp underwent a temporal shift in expression from the mesophyll to specialized epidermal cork cells. Similar to the lemma/palea, expression in the leaf sheath was localized in the cork cells. Progressive 5' deletions of the promoter to nucleotide -75 gradually reduced the level of gfp expression, but tissue- and cell-specific expression was retained.

Untranslated regions of FbRbcS1 mRNA mediate bundle sheath cell-specific gene expression in leaves of a C4 plant.

C4 photosynthesis typically requires two specialized leaf cell types, bundle sheath (bs) and mesophyll (mp), which provide the foundation for this highly efficient carbon assimilation pathway. In leaves of Flaveria bidentis, a dicotyledonous C4 plant, ribulose 1,5-bisphosphate carboxylase (rubisco) accumulates only in bs cells surrounding the vascular centers and not in mp cells. This is in contrast to the more common C3 plants, which accumulate rubisco in all photosynthetic cells. Many previous studies have focused on transcriptional control of C4 cell type-specificity; however, post-transcriptional regulation has also been implicated in the bs-specific expression of genes encoding the rubisco subunits. In this current study, a biolistic leaf transformation assay has provided direct evidence that the 5'- and 3'-untranslated regions (UTRs) of F. bidentis FbRbcS1 mRNA (from a nuclear gene encoding the rubisco small subunit), in themselves, confer strong bs cell-specific expression to gfpA reporter gene transcripts when transcribed from a constitutive CaMV promoter. In transformed leaf regions, strong bs cell-specific GFP expression was accompanied by corresponding bs cell-specific accumulation of the constitutively transcribed FbRbcS1 5'-UTR-gfpA-3'-UTR mRNAs. Control constructs lacking any RbcS mRNA sequences were expressed in all leaf cell types. These findings demonstrate that characteristic cell type-specific FbRbcS1 expression patterns in C4 leaves can be established entirely by sequences contained within the transcribed UTRs of FbRbcS1 mRNAs. We conclude that selective transcript stabilization (in bs cells) or degradation (in mp cells) plays a key role in determining bs cell-specific localization of the rubisco enzyme.