Programming multicellular assembly with synthetic cell adhesion molecules.

Cell adhesion molecules are ubiquitous in multicellular organisms, specifying precise cell-cell interactions in processes as diverse as tissue development, immune cell trafficking and the wiring of the nervous system1-4. Here we show that a wide array of synthetic cell adhesion molecules can be generated by combining orthogonal extracellular interactions with intracellular domains from native adhesion molecules, such as cadherins and integrins. The resulting molecules yield customized cell-cell interactions with adhesion properties that are similar to native interactions. The identity of the intracellular domain of the synthetic cell adhesion molecules specifies interface morphology and mechanics, whereas diverse homotypic or heterotypic extracellular interaction domains independently specify the connectivity between cells. This toolkit of orthogonal adhesion molecules enables the rationally programmed assembly of multicellular architectures, as well as systematic remodelling of native tissues. The modularity of synthetic cell adhesion molecules provides fundamental insights into how distinct classes of cell-cell interfaces may have evolved. Overall, these tools offer powerful abilities for cell and tissue engineering and for systematically studying multicellular organization.

A Conserved Histidine Residue Drives Extein Dependence in an Enhanced Atypically Split Intein.

Split intein-mediated protein trans-splicing (PTS) is widely applied in chemical biology and biotechnology to carry out traceless and specific protein ligation. However, the external residues immediately flanking the intein (exteins) can reduce the splicing rate, thereby limiting certain applications of PTS. Splicing by a recently developed intein with atypical split architecture ("Cat") exhibits a stark dependence on the sequence of its N-terminal extein residues. Here, we further developed Cat using error-prone polymerase chain reaction (PCR) and a cell-based selection assay to produce Cat*, which exhibits greatly enhanced PTS activity in the presence of unfavorable N-extein residues. We then applied solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations to explore how the dynamics of a conserved B-block histidine residue (His78) contribute to this extein dependence. The enhanced extein tolerance of Cat* reported here should expand the applicability of atypically split inteins, and the mechanism highlights common principles that contribute to extein dependence.

Use of 'omics for endometrial timing: the cycle moves on.

For some years, the prospect of precise and personalized timing of the endometrial cycle for optimal embryo replacement has been held out as a potential solution to low implantation rates. It is envisaged that a receptive state can be defined and reached at a predictable time, and embryo replacement performed in synchrony. In the last century, morphological changes characteristic of the mid secretory phase were defined in precisely timed cycles in women of proven fertility, but when deviations from this standardized schedule occur, their significance for implantation has remained uncertain. 'Omics technologies have been widely advocated for staging the endometrial cycle and defining a set of biochemical requirements for implantation, but after two decades of research, improvements to pregnancy rates have not followed, and there is a striking lack of agreement regarding the molecular characterization of the receptive state. Some of the rationale underlying these problems is now emerging with the application of higher-level computational and biological methodology. Here, we consider the challenges of defining an endometrial phenotype that can support implantation and continuing pregnancy. Receptivity may be an emergent trait depending on contributions from multiple proteins that have low pathway connectivity. We recommend that authors choose language which rigorously avoids the implication that protocols for molecular staging of the mid secretory phase inherently identify a state of receptivity to the implanting blastocyst.

Trophectoderm differentiation to invasive syncytiotrophoblast is promoted by endometrial epithelial cells during human embryo implantation.

STUDY QUESTION: How does the human embryo breach the endometrial epithelium at implantation? SUMMARY ANSWER: Embryo attachment to the endometrial epithelium promotes the formation of multinuclear syncytiotrophoblast from trophectoderm, which goes on to breach the epithelial layer. WHAT IS KNOWN ALREADY: A significant proportion of natural conceptions and assisted reproduction treatments fail due to unsuccessful implantation. The trophectoderm lineage of the embryo attaches to the endometrial epithelium before breaching this barrier to implant into the endometrium. Trophectoderm-derived syncytiotrophoblast has been observed in recent in vitro cultures of peri-implantation embryos, and historical histology has shown invasive syncytiotrophoblast in embryos that have invaded beyond the epithelium, but the cell type mediating invasion of the epithelial layer at implantation is unknown. STUDY DESIGN, SIZE, DURATION: Fresh and frozen human blastocyst-stage embryos (n = 46) or human trophoblast stem cell (TSC) spheroids were co-cultured with confluent monolayers of the Ishikawa endometrial epithelial cell line to model the epithelial phase of implantation in vitro. Systems biology approaches with published transcriptomic datasets were used to model the epithelial phase of implantation in silico. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human embryos surplus to treatment requirements were consented for research. Day 6 blastocysts were co-cultured with Ishikawa cell layers until Day 8, and human TSC spheroids modelling blastocyst trophectoderm were co-cultured with Ishikawa cell layers for 48 h. Embryo and TSC morphology was assessed by immunofluorescence microscopy, and TSC differentiation by real-time quantitative PCR (RT-qPCR) and ELISA. Single-cell human blastocyst transcriptomes, and bulk transcriptomes of TSC and primary human endometrial epithelium were used to model the trophectoderm-epithelium interaction in silico. Hypernetworks, pathway analysis, random forest machine learning and RNA velocity were employed to identify gene networks associated with implantation. MAIN RESULTS AND THE ROLE OF CHANCE: The majority of embryos co-cultured with Ishikawa cell layers from Day 6 to 8 breached the epithelial layer (37/46), and syncytiotrophoblast was seen in all of these. Syncytiotrophoblast was observed at the embryo-epithelium interface before breaching, and syncytiotrophoblast mediated all pioneering breaching events observed (7/7 events). Multiple independent syncytiotrophoblast regions were seen in 26/46 embryos, suggesting derivation from different regions of trophectoderm. Human TSC spheroids co-cultured with Ishikawa layers also exhibited syncytiotrophoblast formation upon invasion into the epithelium. RT-qPCR comparison of TSC spheroids in isolated culture and co-culture demonstrated epithelium-induced upregulation of syncytiotrophoblast genes CGB (P = 0.03) and SDC1 (P = 0.008), and ELISA revealed the induction of hCGß secretion (P = 0.03). Secretory-phase primary endometrial epithelium surface transcriptomes were used to identify trophectoderm surface binding partners to model the embryo-epithelium interface. Hypernetwork analysis established a group of 25 epithelium-interacting trophectoderm genes that were highly connected to the rest of the trophectoderm transcriptome, and epithelium-coupled gene networks in cells of the polar region of the trophectoderm exhibited greater connectivity (P < 0.001) and more organized connections (P < 0.0001) than those in the mural region. Pathway analysis revealed a striking similarity with syncytiotrophoblast differentiation, as 4/6 most highly activated pathways upon TSC-syncytiotrophoblast differentiation (false discovery rate (FDR < 0.026)) were represented in the most enriched pathways of epithelium-coupled gene networks in both polar and mural trophectoderm (FDR < 0.001). Random forest machine learning also showed that 80% of the endometrial epithelium-interacting trophectoderm genes identified in the hypernetwork could be quantified as classifiers of TSC-syncytiotrophoblast differentiation. This multi-model approach suggests that invasive syncytiotrophoblast formation from both polar and mural trophectoderm is promoted by attachment to the endometrial epithelium to enable embryonic invasion. LARGE SCALE DATA: No omics datasets were generated in this study, and those used from previously published studies are cited. LIMITATIONS, REASONS FOR CAUTION: In vitro and in silico models may not recapitulate the dynamic embryo-endometrial interactions that occur in vivo. The influence of other cellular compartments in the endometrium, including decidual stromal cells and leukocytes, was not represented in these models. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the mechanism of human embryo breaching of the epithelium and the gene networks involved is crucial to improve implantation success rates after assisted reproduction. Moreover, early trophoblast lineages arising at the epithelial phase of implantation form the blueprint for the placenta and thus underpin foetal growth trajectories, pregnancy health and offspring health. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by grants from Wellbeing of Women, Diabetes UK, the NIHR Local Comprehensive Research Network and Manchester Clinical Research Facility, and the Department of Health Scientist Practitioner Training Scheme. None of the authors has any conflict of interest to declare.

Gene expression signatures predict response to therapy with growth hormone.

Recombinant human growth hormone (r-hGH) is used as a therapeutic agent for disorders of growth including growth hormone deficiency (GHD) and Turner syndrome (TS). Treatment is costly and current methods to model response are inexact. GHD (n = 71) and TS patients (n = 43) were recruited to study response to r-hGH over 5 years. Analysis was performed using 1219 genetic markers and baseline (pre-treatment) blood transcriptome. Random forest was used to determine predictive value of transcriptomic data associated with growth response. No genetic marker passed the stringency criteria for prediction. However, we identified an identical set of genes in both GHD and TS whose expression could be used to classify therapeutic response to r-hGH with a high accuracy (AUC > 0.9). Combining transcriptomic markers with clinical phenotype was shown to significantly reduce predictive error. This work could be translated into a single genomic test linked to a prediction algorithm to improve clinical management. Trial registration numbers: NCT00256126 and NCT00699855.

The expression and activity of Toll-like receptors in the preimplantation human embryo suggest a new role for innate immunity.

STUDY QUESTION: Is the innate immunity system active in early human embryo development? SUMMARY ANSWER: The pattern recognition receptors and innate immunity Toll-like receptor (TLR) genes are widely expressed in preimplantation human embryos and the pathway appears to be active in response to TLR ligands. WHAT IS KNOWN ALREADY: Early human embryos are highly sensitive to their local environment, however relatively little is known about how embryos detect and respond to specific environmental cues. While the maternal immune response is known to be key to the establishment of pregnancy at implantation, the ability of human embryos to detect and signal the presence of pathogens is unknown. STUDY DESIGN, SIZE, DURATION: Expression of TLR family and related genes in human embryos was assessed by analysis of published transcriptome data (n = 40). Day 5 (D-5) human embryos (n = 25) were cultured in the presence of known TLR ligands and gene expression and cytokine production measured compared to controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human embryos surplus to treatment requirements were donated with informed consent from several ART centres. Embryos were cultured to Day 6 (D-6) in the presence of the TLR3 and TLR5 ligands Poly (I: C) and flagellin, with gene expression measured by quantitative PCR and cytokine release into medium measured using cytometric bead arrays. MAIN RESULTS AND THE ROLE OF CHANCE: TLR and related genes, including downstream signalling molecules, were expressed variably at all human embryo developmental stages. Results showed the strongest expression in the blastocyst for TLRs 9 and 5, and throughout development for TLRs 9, 5, 2, 6 and 7. Stimulation of Day 5 blastocysts with TLR3 and TLR5 ligands Poly (I: C) and flagellin produced changes in mRNA expression levels of TLR genes, including the hyaluronan-mediated motility receptor (HMMR), TLR5, TLR7, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and monocyte chemoattractant Protein-1 (MCP-1) (P < 0.05, P < 0.001 compared to unstimulated controls), and release into culture medium of cytokines and chemokines, notably IL8 (P = 0.00005 and 0.01277 for flagellin and Poly (I: C), respectively). LIMITATIONS, REASONS FOR CAUTION: This was a descriptive and experimental study which suggests that the TLR system is active in human embryos and capable of function, but does not confirm any particular role. Although we identified embryonic transcripts for a range of TLR genes, the expression patterns were not always consistent across published studies and expression levels of some genes were low, leaving open the possibility that these were expressed from the maternal rather than embryonic genome. WIDER IMPLICATIONS OF THE FINDINGS: This is the first report of the expression and activity of a number of components of the innate immunity TLR system in human embryos. Understanding the role of TLRs during preimplantation human development may be important to reveal immunological mechanisms and potential clinical markers of embryo quality and pregnancy initiation during natural conception and in ART. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Ministry of Higher Education, The State of Libya, the UK Medical Research Council, and the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility and the European Union's Horizon 2020 Research and Innovation Programmes under the Marie Sklodowska-Curie Grant Agreement No. 812660 (DohART-NET). In accordance with H2020 rules, no new human embryos were sacrificed for research activities performed from the EU funding, which concerned only in silico analyses of recorded time-lapse and transcriptomics datasets. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: n/a.

Pharmacogenomics applied to recombinant human growth hormone responses in children with short stature.

We present current knowledge concerning the pharmacogenomics of growth hormone therapy in children with short stature. We consider the evidence now emerging for the polygenic nature of response to recombinant human growth hormone (r-hGH). These data are related predominantly to the use of transcriptomic data for prediction. The impact of the complex interactions of developmental phenotype over childhood on response to r-hGH are discussed. Finally, the issues that need to be addressed in order to develop a clinical test are described.

Correction: A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct from Kabuki syndrome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct from Kabuki syndrome.

PURPOSE: To investigate if specific exon 38 or 39 KMT2D missense variants (MVs) cause a condition distinct from Kabuki syndrome type 1 (KS1). METHODS: Multiple individuals, with MVs in exons 38 or 39 of KMT2D that encode a highly conserved region of 54 amino acids flanked by Val3527 and Lys3583, were identified and phenotyped. Functional tests were performed to study their pathogenicity and understand the disease mechanism. RESULTS: The consistent clinical features of the affected individuals, from seven unrelated families, included choanal atresia, athelia or hypoplastic nipples, branchial sinus abnormalities, neck pits, lacrimal duct anomalies, hearing loss, external ear malformations, and thyroid abnormalities. None of the individuals had intellectual disability. The frequency of clinical features, objective software-based facial analysis metrics, and genome-wide peripheral blood DNA methylation patterns in these patients were significantly different from that of KS1. Circular dichroism spectroscopy indicated that these MVs perturb KMT2D secondary structure through an increased disordered to ɑ-helical transition. CONCLUSION: KMT2D MVs located in a specific region spanning exons 38 and 39 and affecting highly conserved residues cause a novel multiple malformations syndrome distinct from KS1. Unlike KMT2D haploinsufficiency in KS1, these MVs likely result in disease through a dominant negative mechanism.

EVI1 carboxy-terminal phosphorylation is ATM-mediated and sustains transcriptional modulation and self-renewal via enhanced CtBP1 association.

The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.

A promiscuous split intein with expanded protein engineering applications.

The protein trans-splicing (PTS) activity of naturally split inteins has found widespread use in chemical biology and biotechnology. However, currently used naturally split inteins suffer from an "extein dependence," whereby residues surrounding the splice junction strongly affect splicing efficiency, limiting the general applicability of many PTS-based methods. To address this, we describe a mechanism-guided protein engineering approach that imbues ultrafast DnaE split inteins with minimal extein dependence. The resulting "promiscuous" inteins are shown to be superior reagents for protein cyclization and protein semisynthesis, with the latter illustrated through the modification of native cellular chromatin. The promiscuous inteins reported here thus improve the applicability of existing PTS methods and should enable future efforts to engineer promiscuity into other naturally split inteins.

The in vitro functional analysis of single-nucleotide polymorphisms associated with growth hormone (GH) response in children with GH deficiency.

Response to recombinant human growth hormone (r-hGH) in the first year of therapy has been associated with single-nucleotide polymorphisms (SNPs) in children with GH deficiency (GHD). Associated SNPs were screened for regulatory function using a combination of in silico techniques. Four SNPs in regulatory sequences were selected for the analysis of in vitro transcriptional activity (TA). There was an additive effect of the alleles in the four genes associated with good growth response. For rs3110697 within IGFBP3, rs1045992 in CYP19A1 and rs2888586 in SOS1, the variant associated with better growth response showed higher TA with r-hGH treatment. For rs1024531 in GRB10, a negative regulator of IGF-I signalling and growth, the variant associated with better growth response had a significantly lower TA on r-hGH stimulation. These results indicate that specific SNP variants have effects on TA that provide a rationale for their clinical impact on growth response to r-hGH therapy.

NF-Y-dependent regulation of glutamate receptor 4 expression and cell survival in cells of the oligodendrocyte lineage.

Glutamate receptor subunit 4 (GluA4) is highly expressed by neural cells sensitive to excitotoxicity, and is the predominant subunit expressed by oligodendrocyte precursor cells (OPC) during a key period of vulnerability to hypoxic-ischemic injury. Therefore, transcriptional networks downstream of excitotoxic GluA4 activation represent a promising area for therapeutic intervention. In this work, we identify the CCAAT binding transcription factor NF-Yb as a novel transcriptional regulator of Gria4 (GluA4 gene), and a controller of excitotoxic death in the oligodendroglial lineage. We describe a novel regulatory region within Gria4 containing CCAAT sequences whose binding by NF-Yb is regulated by excitotoxicity. Excitotoxicity-induced alterations in NF-Yb binding are associated with changes in Gria4 transcription, while knockdown of NF-Yb alters the transcription of reporter constructs containing this regulatory region. Data from immortalized and primary OPC reveal that RNAi and pharmacological disruption of NF-Yb alter Gria4 transcription, with the latter inducing apoptosis and influencing a set of apoptotic genes similarly regulated during excitotoxicity. These data provide the first definition of a trans-acting mechanism regulating Gria4, and identify the NF-Y network as a potential source of pharmacological targets for promoting OPC survival.

An Atypical Mechanism of Split Intein Molecular Recognition and Folding.

Split inteins associate to trigger protein splicing in trans, a post-translational modification in which protein sequences fused to the intein pair are ligated together in a traceless manner. Recently, a family of naturally split inteins has been identified that is split at a noncanonical location in the primary sequence. These atypically split inteins show considerable promise in protein engineering applications; however, the mechanism by which they associate is unclear and must be different from that of previously characterized canonically split inteins due to unique topological restrictions. Here, we use a consensus design strategy to generate an atypical split intein pair (Cat) that has greatly improved activity and is amenable to detailed biochemical and biophysical analysis. Guided by the solution structure of Cat, we show that the association of the fragments involves a disorder-to-order structural transition driven by hydrophobic interactions. This molecular recognition mechanism satisfies the topological constraints of the intein fold and, importantly, ensures that premature chemistry does not occur prior to fragment complementation. Our data lead a common blueprint for split intein complementation in which localized structural rearrangements are used to drive folding and regulate protein-splicing activity.

Delineating differential regulatory signatures of the human transcriptome in the choriodecidua and myometrium at term labor.

Preterm deliveries remain the leading cause of neonatal morbidity and mortality. Current therapies target only myometrial contractions and are largely ineffective. As labor involves multiple coordinated events across maternal and fetal tissues, identifying fundamental regulatory pathways of normal term labor is vital to understanding successful parturition and consequently labor pathologies. We aimed to identify transcriptomic signatures of human normal term labor of two tissues: in the fetal-facing choriodecidua and the maternal myometrium. Microarray transcriptomic data from choriodecidua and myometrium following term labor were analyzed for functional hierarchical networks, using Cytoscape 2.8.3. Hierarchically high candidates were analyzed for their regulatory casual relationships using Ingenuity Pathway Analysis. Selected master regulators were then chemically inhibited and effects on downstream targets were assessed using real-time quantitative PCR (RT-qPCR). Unbiased network analysis identified upstream molecular components in choriodecidua including vimentin, TLR4, and TNFSF13B. In the myometrium, candidates included metallothionein 2 (MT2A), TLR2, and RELB. These master regulators had significant differential gene expression during labor, hierarchically high centrality in community cluster networks, interactions amongst the labor gene set, and strong causal relationships with multiple downstream effects. In vitro experiments highlighted MT2A as an effective regulator of labor-associated genes. We have identified unique potential regulators of the term labor transcriptome in uterine tissues using a robust sequence of unbiased mathematical and literature-based in silico analyses. These findings encourage further investigation into the efficacy of predicted master regulators in blocking multiple pathways of labor processes across maternal and fetal tissues, and their potential as therapeutic approaches.

Improving outcomes in adults with diabetes through an interprofessional collaborative practice program.

In 2014, the Midwest Interprofessional Practice, Education and Research Center partnered with a Federally Qualified Health Center (FQHC) to implement an interprofessional collaborative practice (IPCP) education program to improve the health of adult patients with diabetes and to improve practice efficiency. This partnership included integrating an interprofessional team of students with the practice team. Twenty-five students and 20 staff engaged in the IPCP program, which included completion of educational modules on IPCP and implementation of daily huddles, focus patient visits, phone calls, team-based case presentations, medication reconciliation, and student-led group diabetes education classes. This study used a sequential mixed methods design. Tools used for collecting data from staff and students included demographic forms, the Interdisciplinary Education Perception Scale (IEPS), the Entry-level Interprofessional Questionnaire, the Collaborative Practice Assessment Tool, and pre/post module knowledge tests completed at baseline and at one-year post implementation. Patient clinical indicators included HgbA1c, glucose, lipid panel laboratory assessments, body mass index, blood pressure, and documentation of annual dental, foot, and eye examinations. Practice efficiency was measured by the average number of patients seen per provider per hour. Both students and staff showed significant knowledge gains in IPCP on Team Dynamics and Tips for Behavioural Changes knowledge tests (p < .05). Patients who had an HgbA1c of ≥ 7% significantly decreased their HgbA1c (p < .05) and glucose (p < .01). However, BMI and annual dental and eye examinations did not improve. Providers demonstrated an increase in the number of patients seen per hour. This IPCP intervention showed improvement in practice efficiencies and select patient outcomes in a family practice clinic.

Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis.

Naturally split inteins have found widespread use in chemical biology due to their ability to drive the ligation of separately expressed polypeptides through a process termed protein trans-splicing (PTS). In this study, we harness PTS by rendering association of split intein fragments conditional upon the presence of a user-defined protease. We show that these intein "zymogens" can be used to create protein sensors and actuators that respond to the presence of various stimuli, including bacterial pathogens, viral infections, and light. We also show that this design strategy is compatible with several orthogonal split intein pairs, thereby opening the way to the creation of multiplexed sensor systems.

NOMAD spectrometer on the ExoMars trace gas orbiter mission: part 2-design, manufacturing, and testing of the ultraviolet and visible channel.

NOMAD is a spectrometer suite on board the ESA/Roscosmos ExoMars Trace Gas Orbiter, which launched in March 2016. NOMAD consists of two infrared channels and one ultraviolet and visible channel, allowing the instrument to perform observations quasi-constantly, by taking nadir measurements at the day- and night-side, and during solar occultations. Here, in part 2 of a linked study, we describe the design, manufacturing, and testing of the ultraviolet and visible spectrometer channel called UVIS. We focus upon the optical design and working principle where two telescopes are coupled to a single grating spectrometer using a selector mechanism.

Design of a Split Intein with Exceptional Protein Splicing Activity.

Protein trans-splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The durability and efficiency of Cfa should improve current intein based technologies and may provide a platform for the development of new protein chemistry techniques.

Metabolites involved in glycolysis and amino acid metabolism are altered in short children born small for gestational age.

BACKGROUND: Later life metabolic dysfunction is a well-recognized consequence of being born small for gestational age (SGA). This study has applied metabolomics to identify whether there are changes in these pathways in prepubertal short SGA children and aimed to compare the intracellular and extracellular metabolome in fibroblasts derived from healthy children and SGA children with postnatal growth impairment. METHODS: Skin fibroblast cell lines were established from eight SGA children (age 1.8-10.3 y) with failure of catch-up growth and from three healthy control children. Confluent cells were incubated in serum-free media and the spent growth medium (metabolic footprint), and intracellular metabolome (metabolic fingerprint) were analyzed by gas-chromatography mass spectrometry. RESULTS: Nineteen metabolites were significantly altered between SGA and control cell lines. The greatest fold difference (FD) was seen for alanine (fingerprint FD, SGA: control 0.3, P = 0.01 and footprint FD = 0.19, P = 0.01), aspartic acid (fingerprint FD = 5.21, P = 0.01), and cystine (footprint FD = 1.66, P = 0.02). Network analysis of the differentially expressed metabolites predicted inhibition of insulin as well as growth (ERK) signaling in SGA cells. CONCLUSION: This study indicates that changes in cellular metabolism associated with both growth failure and insulin insensitivity are present in prepubertal short children born SGA.