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Jose MR Perez,1 Sergio G Golombek,2 Carlos Fajardo,3 Augusto Sola41Stella Maris Hospital, International Neurodevelopment Neonatal Center (CINN), Sao Paulo, Brazil; 2M Fareri Children’s Hospital, Westchester Medical Center, New York Medical College, Valhalla, NY, USA; 3University of Calgary, Calgary, Canada; 4St Jude Hospital, Fullerton, California, CA, USAIntroduction: Medical and nursing care of newborns is predicated on the delicate control and balance of several vital parameters. Closed incubators and open radiant warmers are the most widely used devices for the care of neonates in intensive care; however, several well-known limitations of these devises have not been resolved. The use of laminar flow is widely used in many fields of medicine, and may have applications in neonatal care.Objective: To describe the neonatal laminar flow unit, a new equipment we designed for care of ill newborns.Methods: The idea, design, and development of this device was completed in Sao Paulo, Brazil. The unit is an open mobile bed designed with the objective of maintaining the advantages of the incubator and radiant warmer, while overcoming some of their inherent shortcomings; these shortcomings include noise, magnetic fields and acrylic barriers in incubators, and lack of isolation and water loss through skin in radiant warmers. The unit has a pump that aspirates environmental air which is warmed by electrical resistance and decontaminated with High Efficiency Particulate Air Filter (HEPA) filters (laminar flow). The flow is directed by an air flow directioner. The unit has an embedded humidifier to increase humidity in the infant’s microenvironment and a servo control mechanism for regulation of skin temperature.Results: The laminar flow unit is open and facilitates access of care providers and family, which is not the case in incubators. It provides warming by convection at an air velocity of 0.45 m/s, much faster than an incubator (0.1 m/s). The system provides isolation 1000 class (less than 1,000 particles higher than 0.3 micron per cubic feet at all times). This is much more protection than an incubator provides and more than radiant warmers, which have no isolation whatsoever. Additionally, it provides humidification of the newborn’s microenvironment (about 60% relative humidity), which is impossible with a radiant warmer, which produces high water body loss. It has no mechanical barriers like acrylic walls, its magnetic field is lower than an incubator (0.25 µt versus 1.2 µt), and the noise is minimal compared to incubators. The unit is also able to provide controlled total body hypothermia, which is not possible with either of the other two units.Conclusion: The laminar flow unit for neonatal care is a novel device which we recently developed. The introduction of laminar flow technology represents a real innovation in the neonatal field. We have described the various components of the unit and the potential advantages for management of ill neonates. This will hopefully lead to improved clinical outcomes and more effective neonatal management and safety.Keywords: laminar flow, newborn intensive care, incubator, radiant warmer

Aneurysms pose life-threatening risks due to the dilatation of the arteries and carry a high risk of rupture. Despite continuous research efforts, there are still no satisfactory or clinically effective pharmaceutical treatments for this condition. Accelerated inflammatory processes during aneurysm development lead to increased levels of matrix metalloproteinases (MMPs) and destabilization of the vessel wall through the degradation of the structural components of the extracellular matrix (ECM), mainly collagen and elastin. Tissue inhibitors of metalloproteinases (TIMPs) directly regulate MMP activity and consequently inhibit ECM proteolysis. In this work, the synthesis of TIMP-1 protein was increased by the exogenous delivery of synthetic TIMP-1 encoding mRNA into aortic vessel tissue in an attempt to inhibit MMP-9. In vitro, TIMP-1 mRNA transfection resulted in significantly increased TIMP-1 protein expression in various cells. The functionality of the expressed protein was evaluated in an appropriate ex vivo aortic vessel model. Decreased MMP-9 activity was detected using in situ zymography 24 h and 48 h post microinjection of 5 µg TIMP-1 mRNA into the aortic vessel wall. These results suggest that TIMP-1 mRNA administration is a promising approach for the treatment of aneurysms.

Maturational changes in the gut start in utero and rapidly progress after birth, with some functions becoming fully developed several months or years post birth including the acquisition of a full gut microbiome, which is made up of trillions of bacteria of thousands of species. Many factors influence the normal development of the neonatal and infantile microbiome, resulting in dysbiosis, which is associated with various interventions used for neonatal morbidities and survival. Extremely low gestational age neonates (<28 weeks' gestation) frequently experience recurring arterial oxygen desaturations, or apneas, during the first few weeks of life. Apnea, or the cessation of breathing lasting 15-20 s or more, occurs due to immature respiratory control and is commonly associated with intermittent hypoxia (IH). Chronic IH induces oxygen radical diseases of the neonate, including necrotizing enterocolitis (NEC), the most common and devastating gastrointestinal disease in preterm infants. NEC is associated with an immature intestinal structure and function and involves dysbiosis of the gut microbiome, inflammation, and necrosis of the intestinal mucosal layer. This review describes the factors that influence the neonatal gut microbiome and dysbiosis, which predispose preterm infants to NEC. Current and future management and therapies, including the avoidance of dysbiosis, the use of a human milk diet, probiotics, prebiotics, synbiotics, restricted antibiotics, and fecal transplantation, for the prevention of NEC and the promotion of a healthy gut microbiome are also reviewed. Interventions directed at boosting endogenous and/or exogenous antioxidant supplementation may not only help with prevention, but may also lessen the severity or shorten the course of the disease.

Loss of elastin due to aging, disease, or injury can lead to impaired tissue function. In this study, de novo tropoelastin (TE) synthesis is investigated in vitro and in vivo using different TE-encoding synthetic mRNA variants after codon optimization and nucleotide modification. Codon optimization shows a strong effect on protein synthesis without affecting cell viability in vitro , whereas nucleotide modifications strongly modulate translation and reduce cell toxicity. Selected TE mRNA variants (3, 10, and 30 μg) are then analyzed in vivo in porcine skin after intradermal application. Administration of 30 μg of native TE mRNA with a me 1 Ψ modification or 10 and 30 μg of unmodified codon-optimized TE mRNA is required to increase TE protein expression in vivo . In contrast, just 3 μg of a codon-optimized TE mRNA variant with the me 1 Ψ modification is able to increase protein expression. Furthermore, skin toxicity is investigated in vitro by injecting 30 μg of mRNA of selected TE mRNA variants into a human full-thickness skin model, and no toxic effects are observed. Thereby, for the first time, an increased dermal TE synthesis by exogenous administration of synthetic mRNA is demonstrated in vivo . Codon optimization of a synthetic mRNA can significantly increase protein expression and therapeutic outcome., Competing Interests: The authors have no competing interests to declare., (© 2023 The Authors.)

Background: The molecular signature response classifier (MSRC) predicts tumor necrosis factor-ɑ inhibitor (TNFi) non-response in rheumatoid arthritis. This study evaluates decision-making, validity, and utility of MSRC testing., Methods: This comparative cohort study compared an MSRC-tested arm (N = 627) from the Study to Accelerate Information of Molecular Signatures (AIMS) with an external control arm (N = 2721) from US electronic health records. Propensity score matching was applied to balance baseline characteristics. Patients initiated a biologic/targeted synthetic disease-modifying antirheumatic drug, or continued TNFi therapy. Odds ratios (ORs) for six-month response were calculated based on clinical disease activity index (CDAI) scores for low disease activity/remission (CDAI-LDA/REM), remission (CDAI-REM), and minimally important differences (CDAI-MID) ., Results: In MSRC-tested patients, 59% had a non-response signature and 70% received MSRC-aligned therapy . In TNFi-treated patients, the MSRC had an 88% PPV and 54% sensitivity. MSRC-guided patients were significantly (p < 0.0001) more likely to respond to b/tsDMARDs than those treated according to standard care (CDAI-LDA/REM: 36.0% vs 21.9%, OR 2.01[1.55-2.60]; CDAI-REM: 10.4% vs 3.6%, OR 3.14 [1.94-5.08]; CDAI-MID: 49.5% vs 32.8%, OR 2.01[1.58-2.55])., Conclusion: MSRC clinical validity supports high clinical utility: guided treatment selection resulted in significantly superior outcomes relative to standard care; nearly three times more patients reached CDAI remission.

Background: Excessive weight loss enhances the incidence of neonatal hypernatremic dehydration (NHD). We compared the effect of a new breastfeeding policy against an old breastfeeding policy on neonatal weight change and the incidence of NHD., Methods: This was a QA project between two sets of breastfeeding (BF) protocols for exclusively BF newborns. Under our old BF policy, a number of neonates had a significant loss of weight after birth and were admitted to the NICU due to NHD. We implemented a new BF policy that was used when a newborn loses > 5% of previously recorded weight within a 24-h interval. Two groups were compared: the preintervention group (old BF policy) and postintervention group (new BF policy). Additionally, characteristics of newborns admitted to NICU were separately compared with the subgroup of pre- and post intervention dehydration groups., Results: Preintervention = 1320 and postintervention = 1450. Neonates with weight loss of ≥ 5% within the first 24-h time interval were higher in the postintervention group (19.7%) as compared to the preintervention group (10.2%) ( P  < .05). However, the number of infants diagnosed to have NHD was lower in the postintervention group (0.68%) than in the preintervention group (1.66%), ( P  < .03). Neonatal characteristics were comparable between subgroups of dehydration., Conclusion: An intervention at ≥ 5% neonatal weight loss markedly reduces the incidence of NHD-associated NICU admissions., Competing Interests: We declare that none of the authors have any conflict of interest or have received support for any of the products described., (© 2021 Publishing services provided by Elsevier B.V. on behalf of King Faisal Specialist Hospital & Research Centre (General Organization), Saudi Arabia.)

Congenital heart disease (CHD) is among the four most common causes of infant mortality in Latin America. Pulse oximetry screening (POS) is useful for early diagnosis and improved outcomes of critical CHD. Here, we describe POS implementation efforts in Latin American countries guided and/or coordinated by the Ibero American Society of Neonatology (SIBEN), as well as the unique challenges that are faced for universal implementation. SIBEN collaborates to improve the neonatal quality of care and outcomes. A few years ago, a Clinical Consensus on POS was finalized. Since then, we have participated in 12 Latin American countries to educate neonatal nurses and neonatologists on POS and to help with its implementation. The findings reveal that despite wide disparities in care that exist between and within countries, and the difficulties and challenges in implementing POS, significant progress has been made. We conclude that universal POS is not easy to implement in Latin America but, when executed, has not only been of significant value for babies with CHD, but also for many with other hypoxemic conditions. The successful and universal implementation of POS in the future is essential for reducing the mortality associated with CHD and other hypoxemic conditions and will ultimately lead to the survival of many more Latin American babies. POS saves newborns' lives in Latin America., Competing Interests: Conflicts of InterestA.S. works part time, as VP for Medical Affairs, Education and Research in Neonatology, with an annual salary at Masimo Corporation (Irvine, CA, USA). None of the other co-authors have a conflict of interest., (© 2020 by the authors.)

The application of synthetic modified messenger RNA (mRNA) is a promising approach for the treatment of a variety of diseases and vaccination. In the past few years, different modifications of synthetic mRNA were applied to render the mRNA more stable and less immunogenic. However, the repeated application of synthetic mRNA still requires the suppression of immune activation to avoid cell death and to allow a sufficient production of exogenous proteins. Thus, the addition of type I interferon (IFN) inhibiting recombinant protein B18R is often required to avoid IFN response. In this study, the ability of B18R encoding mRNA to prevent the immune response of cells to the delivered synthetic mRNA was analyzed. The co-transfection of enhanced green fluorescent protein (eGFP) mRNA transfected fibroblasts with B18R encoding mRNA over 7-days resulted in comparable cell viability and eGFP protein expression as in the cells transfected with eGFP mRNA and incubated with B18R protein. Using qRT-PCR, significantly reduced expression of interferon-stimulated gene Mx1 was detected in the cells transfected with B18R mRNA and stimulated with IFNβ compared to the cells without B18R mRNA transfection. Thereby, it was demonstrated that the co-transfection of synthetic mRNA transfected cells with B18R encoding mRNA can reduce the IFN response-related cell death and thus, improve the protein expression., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Musculoskeletal disorders, such as osteoarthritis and intervertebral disc degeneration are causes of morbidity, which concomitantly burdens the health and social care systems worldwide, with massive costs. Link N peptide has recently been described as a novel anabolic stimulator for intervertebral disc repair. In this study, we analyzed the influence on anabolic response, by delivering synthetic Link N encoding mRNA into primary human chondrocytes and mesenchymal stromal cells (SCP1 cells), Furthermore, both cell types were seeded on knitted titanium scaffolds, and the influence of Link N peptide mRNA for possible tissue engineering applications was investigated. Synthetic modified Link N mRNA was efficiently delivered into both cell types and cell transfection resulted in an enhanced expression of aggrecan , Sox 9 , and type II collagen with a decreased expression of type X collagen . Interestingly, despite increased expression of BMP2 and BMP7 , BMP signaling was repressed and TGFβ signaling was boosted by Link N transfection in mesenchymal stromal cells, suggesting possible regulatory mechanisms. Thus, the exogenous delivery of Link N peptide mRNA into cells augmented an anabolic response and thereby increased extracellular matrix synthesis. Considering these findings, we suppose that the cultivation of cells on knitted titanium scaffolds and the exogenous delivery of Link N peptide mRNA into cells could mechanically support the stability of tissue-engineered constructs and improve the synthesis of extracellular matrix by seeded cells. This method can provide a potent strategy for articular cartilage and intervertebral disc regeneration.

The application of endothelial progenitor cells (EPCs) for the revascularization of ischemic tissues, such as after myocardial infarction, stroke, and acute limb ischemia, has a huge clinical potential. However, the low retention and engraftment of EPCs as well as the poor survival of migrated stem cells in ischemic tissues still hamper the successful clinical application. Thus, in this study, we engineered, for the first time, murine EPCs with synthetic mRNAs to transiently produce proangiogenic factors vascular endothelial growth factor-A (VEGF-A), stromal cell-derived factor-1α (SDF-1α), and angiopoietin-1 (ANG-1). After the transfection of cells with synthetic mRNAs, significantly increased VEGF-A, SDF-1α, and ANG-1 protein levels were detected compared to untreated EPCs. Thereby, mRNA-engineered EPCs showed significantly increased chemotactic activity versus untreated EPCs and resulted in significantly improved attraction of EPCs. Furthermore, ANG-1 mRNA-transfected EPCs displayed a strong wound-healing capacity. Already after 12 hr, 94% of the created wound area in the scratch assay was closed compared to approximately 45% by untreated EPCs. Moreover, the transfection of EPCs with ANG-1 or SDF-1α mRNA also significantly improved the in vitro tube formation capacity; however, the strongest effect could be detected with EPCs simultaneously transfected with VEGF-A, SDF-1α, and ANG-1 mRNA. In the in vivo chicken chorioallantoic membrane (CAM) assay, EPCs transfected with ANG-1 mRNA revealed the strongest angiogenetic potential with significantly elevated vessel density and total vessel network length. In conclusion, this study demonstrated that EPCs can be successfully engineered with synthetic mRNAs encoding proangiogenic factors to improve their therapeutic angiogenetic potential in patients experiencing chronic or acute ischemic disease., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Hemocompatibility of blood-contacting biomaterials is one of the most important criteria for their successful in vivo applicability. Thus, extensive in vitro analyses according to ISO 10993-4 are required prior to clinical applications. In this review, we summarize essential aspects regarding the evaluation of the hemocompatibility of biomaterials and the required in vitro analyses for determining the blood compatibility. Static, agitated, or shear flow models are used to perform hemocompatibility studies. Before and after the incubation of the test material with fresh human blood, hemolysis, cell counts, and the activation of platelets, leukocytes, coagulation and complement system are analyzed. Furthermore, the surface of biomaterials are evaluated concerning attachment of blood cells, adsorption of proteins, and generation of thrombus and fibrin networks.

Elastin is one of the most important and abundant extracellular matrix (ECM) proteins that provide elasticity and resilience to tissues and organs, including vascular walls, ligaments, skin, and lung. Besides hereditary diseases, such as Williams-Beuren syndrome (WBS), which results in reduced elastin synthesis, injuries, aging, or acquired diseases can lead to the degradation of existing elastin fibers. Thus, the de novo synthesis of elastin is required in several medical conditions to restore the elasticity of affected tissues. Here, we applied synthetic modified mRNA encoding tropoelastin (TE) for the de novo synthesis of elastin and determined the mRNA-mediated elastin synthesis in cells, as well as ex vivo in porcine skin. EA.hy926 cells, human fibroblasts, and mesenchymal stem cells (MSCs) isolated from a patient with WBS were transfected with 2.5 μg TE mRNA. After 24 hr, the production of elastin was analyzed by Fastin assay and dot blot analyses. Compared with untreated cells, significantly enhanced elastin amounts were detected in TE mRNA transfected cells. The delivered synthetic TE mRNA was even able to significantly increase the elastin production in elastin-deficient MSCs. In porcine skin, approximately 20% higher elastin amount was detected after the intradermal delivery of synthetic mRNA by microinjection. In this study, we demonstrated the successful applicability of synthetic TE encoding mRNA to produce elastin in elastin-deficient cells as well as in skin. Thus, this auspicious mRNA-based integration-free method has a huge potential in the field of regenerative medicine to induce de novo elastin synthesis, e.g., in skin, blood vessels, or alveoli., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

In recent years, synthetic mRNA-based applications to produce desired exogenous proteins in cells have been gaining importance. However, systemic delivery of synthetic mRNA can result in unspecific uptake into undesired cells or organs and, thereby, fail to target desired cells. Thus, local and targeted delivery of synthetic mRNA becomes increasingly important to reach the desired cell types and tissues. In this study, intradermal delivery of synthetic mRNA using a hollow microneedle injection-based method was evaluated. Furthermore, an ex vivo porcine skin model was established to analyze synthetic mRNA-mediated protein expression in the skin following intradermal delivery. Using this model, highly efficient delivery of synthetic mRNA was demonstrated, which resulted in detection of high levels of secretable humanized Gaussia luciferase (hGLuc) protein encoded by the microinjected synthetic mRNA. Interestingly, synthetic mRNA injected without transfection reagent was also able to enter the cells and resulted in protein expression. The established ex vivo porcine skin model can be used to evaluate the successful production of desired proteins after intradermal delivery of synthetic mRNAs before starting with in vivo experiments. Furthermore, the use of microneedles enables patient-friendly, painless, and efficient delivery of synthetic mRNAs into the dermis; thus, this method could be applied for local treatment of different skin diseases as well as for vaccination and immunotherapy., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The application of synthetic messenger RNA (mRNA) exhibits various advantages, such as expression of desired proteins in cells without genomic integration. In the field of tissue engineering, synthetic mRNAs could be also used to modulate the protein expression in implanted cells. Therefore, in this study, we incorporated synthetic humanized Gaussia luciferase (hGLuc) mRNA into alginate, chitosan, or chitosan-alginate hybrid hydrogels and analyzed the release of hGLuc mRNA from these hydrogels. After 3 weeks, 79% of the incorporated mRNA was released from alginate hydrogels, approximately 42% was released from chitosan hydrogels, and about 70% was released from chitosan-alginate hydrogels. Due to the injectability, chitosan-alginate hybrid hydrogels were selected for further investigation of the bioactivity of embedded hGLuc mRNA and the stability of these hydrogels was examined after the incorporation of synthetic mRNA by rheometric analysis. Therefore, HEK293 cells were incorporated into chitosan-alginate hydrogels containing mRNA transfection complexes and the luciferase activity in the supernatants was detected for up to 3 weeks. These results showed that the biodegradable chitosan-alginate hybrid hydrogels are promising delivery systems for sustained delivery of synthetic mRNAs into cells. Since chitosan-alginate hybrid hydrogels are injectable, the hydrogels can be simultaneously loaded with cells and the desired synthetic mRNA for exogenous protein synthesis and can be administered by minimally invasive local injection for tissue engineering applications., Competing Interests: The authors declare no conflict of interest.

Depolarized mitochondria are degraded by mitophagy in a process that depends on the Parkinson's disease gene products PINK1 and Parkin. This is accompanied by ubiquitylation of several mitochondrial substrates. The roles of E2 ubiquitin-conjugating enzymes (UBE2) in mitophagy are poorly understood. Here, we investigate a set of UBE2 enzymes that might regulate Parkin-mediated mitophagy. Knockdown of the E2 enzymes UBE2N, UBE2L3 or UBE2D2 and UBE2D3 (UBE2D2/3) significantly reduced autophagic clearance of depolarized mitochondria. However, this did not interfere with mitochondrial PINK1 stabilization and Parkin translocation. UBE2N knockdown prevented specifically K63-linked ubiquitylation at mitochondrial sites. Nevertheless, polyubiquitin and p62 (officially known as SQSTM1) were still found on mitochondria after individual UBE2 knockdown. Knockdown of all of these UBE2s together significantly reduced mitochondrial polyubiquitylation and p62 recruitment. Moreover, reduced ubiquitylation of mitofusins, the mitochondrial import receptor subunits TOM20 and TOM70, the voltage-dependent anion channel protein 1 and Parkin was observed in cells silenced for all of these UBE2s. A version of Parkin with a mutation in the active site (C431S) failed to ubiquitylate these mitochondrial substrates even in the presence of UBE2s. We conclude that UBE2N, UBE2L3 and UBE2D2/3 synergistically contribute to Parkin-mediated mitophagy., (© 2014. Published by The Company of Biologists Ltd.)

Non-invasive magnetic resonance imaging (MRI) is gaining significant attention in the field of tissue engineering, since it can provide valuable information on in vitro production parameters and in vivo performance. It can e.g. be used to monitor the morphology, location and function of the regenerated tissue, the integrity, remodeling and resorption of the scaffold, and the fate of the implanted cells. Since cells are not visible using conventional MR techniques, ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are routinely employed to label and monitor the cells embedded in tissue-engineered implants. We here set out to optimize cell labeling procedures with regard to labeling efficiency, biocompatibility and in vitro validation during bioreactor cultivation, using flavin mononucleotide (FMN)-coated fluorescent USPIO (FLUSPIO). Efficient FLUSPIO uptake is demonstrated in three different cell lines, applying relatively short incubation times and low labeling concentrations. FLUSPIO-labeled cells were successfully employed to visualize collagen scaffolds and tissue-engineered vascular grafts. Besides promoting safe and efficient cell uptake, an exquisite property of the non-polymeric FMN-coating is that it renders the USPIO fluorescent, providing a means for in vitro, in vivo and ex vivo validation via fluorescence microscopy and fluorescence reflectance imaging (FRI). FLUSPIO cell labeling is consequently considered to be a suitable tool for theranostic tissue engineering purposes.

The small subunit of a Vicia faba ADP-glucose pyrophosphorylase (AGP) cDNA was expressed in antisense orientation in Vicia narbonensis under the control of the seed-specific legumin B4 promoter. From several independent transgenic lines both ADP-glucose pyrophosphorylase AGP-mRNA and AGP enzyme activity were reduced by up to 95% in the cotyledons during the mid- to late-maturation phase. Starch was moderately decreased and sucrose was increased. In two of three lines, transcripts encoding the large subunit of AGP and the storage protein vicilin were increased, whereas legumin B-mRNA was decreased. Transcripts of other storage-associated genes were not altered. The cotyledons contained more protein and total nitrogen. Despite the reduction in starch, total carbon was not decreased and dry weight was unchanged. Compared to the wild type, transgenic seeds contained more water and accumulated dry weight during a longer period, and therefore had a prolonged seed-filling period. Transgenic cotyledon cells of comparable age to the wild type were more highly vacuolated and contained smaller starch grains, indicating a delay in cellular differentiation. We conclude that a specific alteration in carbon metabolism can have pleiotropic effects on water and nitrogen content and induces temporal changes in seed development.

To analyze the role of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) during seed development, two cDNA clones encoding two isoforms of PEPCase were isolated from a seed-specific library of Vicia faba. The two sequences (VfPEPCase1 and VfPEPCase2) have a sequence identity of 82 and 89% on the nucleotide and amino acid levels. The VfPEPCase1 mRNA was found to be predominantly expressed in roots and developing cotyledons whereas the VfPEPCase2 mRNA was more abundant in green and maternal tissues. In the cotyledons, PEPCase mRNAs accumulated from early to mid cotyledon stage and decreased thereafter. The PEPCase activity increased continuously during cotyledon development. The enzyme was strongly activated by glucose-6-phosphate, but not by glucose, fructose or sucrose. Asparagine was weakly activating whereas malate, aspartate and glutamate were inhibitory. The inhibitors became less effective with increasing pH. Aspartate was a much stronger inhibitor of cotyledonary PEPCase than glutamate at both pH 7.0 and 7.5. The sensitivity of PEPCase to malate inhibition decreased from early to mid cotyledon stage at a time when storage proteins are synthesized. This indicates activation on the protein level, possibly by protein phosphorylation. Nitrogen starvation in the presence of hexoses but not sucrose decreased mRNA levels of VfPEPCase1 and enzyme activity, indicating control on the mRNA level by both carbon and nitrogen. It is concluded that in developing cotyledons PEPCase is probably important for the synthesis of organic acids to provide carbon skeletons for amino acid synthesis.

In plants the carbohydrate state provides signals to adjust metabolism to specific physiological conditions. Storage-active sink organs like seeds often contain high levels of sucrose. In order to change the sugar status during seed development a yeast-derived invertase gene was expressed in Vicia narbonensis under control of the LeguminB4 promoter. A signal sequence targeted the invertase to the apoplast in maturing embryos. In the cotyledons, sucrose was decreased whereas hexoses strongly accumulated. There was a major reduction of starch whereas proteins were less affected. Vacuoles of cotyledon cells were enlarged and dry seeds wrinkled. Transcripts and enzyme activity of sucrose synthase, the small and large subunit of ADP-glucose pyrophosphorylase as well as vicilin were downregulated. Sucrose phosphate synthase and legumin-mRNAs were not affected. Analysing single seeds with different sucrose levels revealed a positive correlation of sucrose concentration to mRNA levels of sucrose synthase and most pronounced to ADP-glucose pyrophosphorylase-mRNA levels as well as to starch content. Glucose on the other hand did not show any correlation. After feeding 14C-sucrose in vitro, the invertase-expressing cotyledons partitioned less carbon into starch compared to the wild-type. In the transgenic cotyledons, a relatively higher amount was directed into proteins compared to starch. We conclude that starch accumulation in developing cotyledons could be a function of sucrose concentration. Our results are consistent with a possible sucrose-mediated induction of storage-associated differentiation indicated by upregulation of specific genes of the starch synthesis pathway.

Autoantibodies may play an important role in the pathogenesis of central nervous system (CNS) disease in systemic lupus erythematosus (SLE). We obtained cerebrospinal fluid (CSF) and, in some cases, sera from 19 SLE patients with CNS lupus and from 12 SLE patients without CNS lupus. Autoantibodies to saline soluble cellular antigens were detected in the CSF of lupus patients and reflected those present in the serum. These antibodies were distinct from the previously described antineuronal antibodies. Analysis of the fine specificities of the anti-saline soluble cellular antigen antibodies revealed that the antiribosomal P protein antibody was present in 4 of 4 patients with lupus psychosis and was enriched in the CSF of 1 patient. Sera containing antiribosomal P protein showed prominent cytoplasmic staining of human cortical neurons, as well as an epithelial cell substrate. These observations, together with the increase in intrathecal IgG synthesis detected in 71% of patients tested, suggest that several populations of antibodies may contribute to the enhanced immunologic activity in the CSF of CNS lupus patients.