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Neural crest (NC) is a unique vertebrate cell type arising from the border of the neural plate and epidermis that gives rise to diverse tissues along the entire body axis. Roberto Mayor and colleagues have made major contributions to our understanding of NC induction, delamination, and migration. We report that a truncating mutation of the classical tumor suppressor Adenomatous Polyposis Coli (apc) disrupts craniofacial development in zebrafish larvae, with a marked reduction in the cranial neural crest (CNC) cells that contribute to mandibular and hyoid pharyngeal arches. While the mechanism is not yet clear, the altered expression of signaling molecules that guide CNC migration could underlie this phenotype. For example, apcmcr/mcr larvae express substantially higher levels of complement c3, which Mayor and colleagues showed impairs CNC cell migration when overexpressed. However, we also observe reduction in stroma-derived factor 1 (sdf1/cxcl12), which is required for CNC migration into the head. Consistent with our previous work showing that APC directly enhances the activity of glycogen synthase kinase 3 (GSK-3) and, independently, that GSK-3 phosphorylates multiple core mRNA splicing factors, we identify 340 mRNA splicing variations in apc mutant zebrafish, including a splice variant that deletes a conserved domain in semaphorin 3f (sema3f), an axonal guidance molecule and a known regulator of CNC migration. Here, we discuss potential roles for apc in CNC development in the context of some of the seminal findings of Mayor and colleagues.

A sports medicine physician manages musculoskeletal (MSK) injuries and sport-related medical and MSK conditions of patients of all ages and abilities. Physical medicine and rehabilitation physicians (physiatrists) must be adequately trained to provide this care for all patients including, but not limited to, athletes participating in organized sports, the weekend warrior as well as athletes with disabilities. Accreditation Council of Graduate Medical Education core requirements and basic guidelines help physiatry residency training programs develop and implement residency curriculums. The goal of this article is to provide suggested curricular guidelines to optimize physiatrist training in MSK and sports medicine.

In this issue of Cell Chemical Biology, Cheng et al. (2021) identify a class of drugs that activate a mitotic stress-dependent signaling cascade, which culminates in p53 activation and Wnt pathway inhibition (PAWI). PAWI compounds may therefore be effective in cancers associated with loss of p53 and activation of Wnt signaling.

Musculoskeletal (MSK) and sports-related conditions are relatively common in the pediatric population. Pediatric residencies should provide residents with the knowledge and skills to assess and manage both acute and chronic MSK and sports injuries and complaints. Residents should develop the competencies and attitudes to safeguard and promote a healthy and active lifestyle for youth. Programs can use a variety of educational tools, both in the clinic and on the field, to provide a well-rounded MSK curriculum throughout the residency years. This article provides a review of general pediatric sports medicine curriculum guidelines and suggested implementation strategies.

Musculoskeletal and sports medicine conditions are common in the emergency department (ED). Emergency physicians may not be receiving adequate education to achieve clinical competency in musculoskeletal medicine during residency training. This article aims to provide a standardized musculoskeletal and sports medicine curriculum for emergency medicine training. Broad curriculum goals include proficiency in evaluating and managing patients presenting to the ED with acute and chronic musculoskeletal complaints and other medical conditions related to or affected by physical exertion, sports participation, or environmental exposure. Specific objectives focus on knowledge of these disorders, physical examination skills, procedural skills including musculoskeletal ultrasound, appropriate consultation and referral, and patient education for these conditions. Educational methods will consist of didactics; online self-directed learning modules; simulation; and supervised clinical experiences in the ED, primary care sports medicine clinics, and orthopedic clinics if available. Curriculum implementation is expected to vary across programs due to differences in residency program structure and resources.

Significance COVID-19 is taking a major toll on personal health, healthcare systems, and the global economy. With three betacoronavirus epidemics in less than 20 y, there is an urgent need for therapies to combat new and existing coronavirus outbreaks. Our analysis of clinical data from over 300,000 patients in three major health systems demonstrates a 50% reduced risk of COVID-19 in patients taking lithium, a direct inhibitor of glycogen synthase kinase-3 (GSK-3). We further show that GSK-3 is essential for phosphorylation of the SARS-CoV-2 nucleocapsid protein and that GSK-3 inhibition blocks SARS-CoV-2 infection in human lung epithelial cells. These findings suggest an antiviral strategy for COVID-19 and new coronaviruses that may arise in the future., The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35–0.74], P = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type–dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.

Patients present to primary care physicians with musculoskeletal complaints more often than they do for upper respiratory infections, hypertension, or diabetes. Despite this, instruction in musculoskeletal medicine for internal medicine residents represents less than 1% of their total didactic and clinical education time. We recognize the immense breadth of knowledge and skill required to train residents in the practice of internal medicine. This curriculum guideline defines a recommended training strategy, and supplies relevant resources, to improve musculoskeletal education among internal medicine residents to optimize patient care. This curriculum guideline was created by internists who are sports medicine specialists. Sports medicine physicians promote overall health and well-being while providing expertise in acute and chronic musculoskeletal conditions, as well as how disease affects exercise and using exercise as medicine for people with chronic disease.

Arterial stiffening is the progressive loss of elasticity of the large vessels resulting from age. Increased arterial stiffening has been correlated with hypertension and other cardiovascular co-morbidities. KDM6A is a histone 3 lysine demethylase whose activity increases expression of target genes via removal of silencing methylation marks on lysine 27 of histone 3 (H3K27). The precise role of H3K27me3, or its absence in aortic vascular smooth muscle cells (VSMCs), remains to be investigated. The objectives of this study were to determine the effects of loss of KDM6A on arterial stiffness, blood pressure, and vascular cell function. We generated VSMC-specific Kdm6a knockout mice of C57BL/6 background and confirmed knockout by western blot. Knockout of Kdm6a resulted in increased resting systolic blood pressure and pulse wave velocity in male mice. Pulse wave velocity (PWV) is the gold-standard method for determination of arterial stiffness. PWVs of the knockout mice were significantly increased compared to controls four weeks after Kdm6a knockout (Controls 2.62 + 0.69 mm/ms; KO 3.13 + 0.30 mm/ms; P=0.0228). Systolic blood pressure was directly obtained for each mouse via carotid cannulation. The systolic blood pressures of knockout mice were significantly increased. Mesenteric arteries were isolated from three control and three Kdm6a knockout mice and mounted onto a wire myograph. Vessels were pre-constricted with norepinephrine and vasodilation was measured by treatment with increasing doses of acetylcholine and sodium nitroprusside to assess endothelial and VSMC function. Interestingly, despite Kdm6a knockout in VSMCs, vasodilation of VSMCs was not different between control and knockout arteries, but relaxation in response to acetylcholine, which stimulates the release of nitric oxide from endothelial cells, was decreased. This suggests endothelial dysfunction. These results indicate that loss of KDM6A in VSMCs leads to elevated systolic blood pressure and arterial stiffness.

The American Medical Society for Sports Medicine (AMSSM) convened a group of experts to develop a position statement addressing the problem of sexual violence in sport. The AMSSM Sexual Violence in Sport Task Force held a series of meetings over 7 months, beginning in July 2019. Following a literature review, the task force used an iterative process and expert consensus to finalize the position statement. The objective of this position statement is to raise awareness of this critical issue among sports medicine physicians and to declare a commitment to engage in collaborative, multidisciplinary solutions to reduce sexual violence in sport.

Acute myeloid leukemia (AML) is a hematologic malignancy that affects predominantly older patients, with a median age of diagnosis around 67. Overall prognosis is poor; however, novel targeted therapies that can potentially improve outcomes in these patients have emerged in recent years. Mutations in isocitrate dehydrogenase (IDH) occur in 20% of AML diagnoses. IDH2 performs a crucial role in cellular metabolism, and when this enzyme is inhibited, the cell cannot rid itself of endogenous products and is thus marked for apoptosis. The US Food and Drug Administration (FDA) approved the first mutant IDH2 inhibitor, enasidenib, for patients with relapsed or refractory IDH2-mutated AML detected by an FDA-approved test.

Objective To identify factors associated with entry into primary care sports medicine (PCSM) fellowship programs. Design Primary care sports medicine fellowship directors (FDs) and fellowship faculty were surveyed regarding preferences for accepting applicants into their programs. Setting Survey study. Participants Primary care sports medicine FDs and fellowship faculty. Assessment of risk factors Questions were designed to delineate factors [clinical experience, letters of recommendation (LOR), scholarship, service commitment, interview performance, etc] perceived to be associated with entry into PCSM fellowship (1-10 scale; 10 = highest value). Weighted mean ± SD were calculated for each question. Main outcome measures Determination of most valued factors for entry into PCSM fellowship. Results Responses were provided by 242/2332 (10.4%) of the American Medical Society for Sports Medicine members, including 77 of 175 (44%) FDs. The top 3 factors for entry into PCSM fellowships for all respondents were as follows: interview performance (9.17 ± 1.13), LOR from SM fellowship faculty (8.20 ± 1.67), and high school game/event coverage (7.83 ± 1.70). Musculoskeletal ultrasound experience (4.50 ± 2.23) and residency training in pediatrics (4.58 ± 2.54), internal medicine (4.48 ± 2.44), emergency medicine (4.44 ± 2.59), and physical medicine and rehabilitation (4.40 ± 2.83) received the lowest scores. Conclusions Applicants seeking entry into SM fellowships should prioritize performance during interviews, LOR from SM fellowship faculty, and team game/event coverage experiences.

Acute myeloid leukemia (AML) is a hematologic malignancy that affects predominantly older patients, with a median age of diagnosis around 67. Overall prognosis is poor; however, novel targeted therapies that can potentially improve outcomes in these patients have emerged in recent years. Mutations in isocitrate dehydrogenase (IDH) occur in 20% of AML diagnoses. IDH2 performs a crucial role in cellular metabolism, and when this enzyme is inhibited, the cell cannot rid itself of endogenous products and is thus marked for apoptosis. The US Food and Drug Administration (FDA) approved the first mutant IDH2 inhibitor, enasidenib, for patients with relapsed or refractory IDH2-mutated AML detected by an FDA-approved test.

Organic synthesis is changing; in a world where budgets are constrained and the environmental impacts of practice are scrutinized, it is increasingly recognized that the efficient use of human resource is just as important as material use. New technologies and machines have found use as methods for transforming the way we work, addressing these issues encountered in research laboratories by enabling chemists to adopt a more holistic systems approach in their work. Modern developments in this area promote a multi-disciplinary approach and work is more efficient as a result. This Review focuses on the concepts, procedures and methods that have far-reaching implications in the chemistry world. Technologies have been grouped as topics of opportunity and their recent applications in innovative research laboratories are described.

Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3–dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3–dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3–dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3β phosphorylated these proteins in vitro. RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3–dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing.

The thapsigargins are a family of complex guaianolides with potent and selective Ca 2+ -modulating properties. This article documents the evolution of a synthetic route through several iterations to a final practical and scaleable synthetic route capable of generating both unnatural and natural products based around the guaianolide skeleton.

Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement1, increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively2, 3. Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement4. Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP2, 3. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.

Summary Centrosomes associate with spindle poles; thus, the presence of two centrosomes promotes bipolar spindle assembly in normal cells. Cancer cells often contain supernumerary centrosomes, and to avoid multipolar mitosis and cell death, these are clustered into two poles by the microtubule motor protein HSET. We report the discovery of an allosteric inhibitor of HSET, CW069, which we designed using a methodology on an interface of chemistry and biology. Using this approach, we explored millions of compounds in silico and utilized convergent syntheses. Only compound CW069 showed marked activity against HSET in vitro. The inhibitor induced multipolar mitoses only in cells containing supernumerary centrosomes. CW069 therefore constitutes a valuable tool for probing HSET function and, by reducing the growth of cells containing supernumerary centrosomes, paves the way for new cancer therapeutics., Highlights • Hybrid approach to compound discovery using chemogenomics, chemistry, and biology • Design of an allosteric HSET inhibitor, CW069, a synthetic compound • CW069 selectively inhibits HSET activity both in vitro and in cells • CW069 induces multipolar anaphases by blocking centrosome clustering, Inhibition of HSET provides a unique opportunity to selectively target malignant cells with supernumerary centrosomes. Watts et al. develop an allosteric HSET inhibitor that does not disrupt bipolar division in normal cells but leads to multipolar mitoses in cancer cells with extra centrosomes, causing apoptosis.

The antiproliferative activity on ovarian cancer (SK-OV-3) cells of a series of triazole-bridged combretastatin analogues ( 37 , 38 , 40–43 ) containing dihalogenation of the A-ring is reported, and compared with their trimethoxy analogues ( 5 , 15 , 39 ). It was found that dihalogenation with either bromine or iodine was a tolerated modification when compared to the parent compound combretastatin (CA–4, 1 ) and had less effect than B-ring modification on potency. These compounds exhibited G 2 /M arrest, and maintained antitubulin activity. Further assays on human umbilical vein endothelial cells (HUVECs) demonstrated the potential antivascular effects of these triazoles. Of particular note was a 3,5-diiodo-4-methoxyaryl triazole ( 43 ) which had promising 7-fold selectivity for HUVECs over ovarian cancer cells.

The combretastatins have been investigated for their antimitotic and antivascular properties, and it is widely postulated that a 3,4,5-trimethoxyaryl A-ring is essential to maintain potent activity. We have synthesized new tetrazole analogues (32-34), demonstrating that 3,5-dihalogenation can consistently increase potency by up to 5-fold when compared to the equivalent trimethoxy compound on human umbilical vein endothelial cells (HUVECs) and a range of cancer cells. Moreover, this increased potency offsets that lost by installing the tetrazole bridge into combretastatin A-4 (1), giving crystalline, soluble compounds that have low nanomolar activity, arrest cells in G2/M phase, and retain microtubule inhibitory activity. Molecular modeling has shown that optimized packing within the binding site resulting in increased Coulombic interaction may be responsible for this improved activity.

The epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase (RTK) and is recognized as a key mediator of tumorigenesis in many human tumors. Currently there are five EGFR inhibitors used in oncology, two monoclonal antibodies (panitumumab, and cetuximab) and three tyrosine kinase inhibitors (erlotinib, gefitinib, and lapatinib). Both strategies of EGFR inhibition have demonstrated clinical successes, however many tumors remain non-responsive or acquire resistance during therapy. To explore potential molecular mechanisms of acquired resistance to cetuximab we previously established a series of cetuximab-resistant clones by chronically exposing the NCI-H226 NSCLC cell line to escalating doses of cetuximab. Cetuximab-resistant clones exhibited a dramatic increase in steady-state expression of EGFR, HER2, and HER3 receptors as well as increased signaling through the MAPK and AKT pathways. RNAi studies demonstrated dependence of cetuximab-resistant clones on the EGFR signaling network. These findings prompted investigation on whether or not cells with acquired resistance to cetuximab would be sensitive to the EGFR targeted TKI erlotinib. In vitro, erlotinib was able to decrease signaling through the EGFR axis, decrease cellular proliferation, and induce apoptosis. To determine if erlotinib could have therapeutic benefit in vivo, we established cetuximab-resistant NCI-H226 mouse xenografts, and subsequently treated them with erlotinib. Mice harboring cetuximab-resistant tumors treated with erlotinib exhibited either a tumor regression or growth delay as compared to vehicle controls. Analysis of the erlotinib treated tumors demonstrated a decrease in cell proliferation and increase rates of apoptosis. The work presented herein suggests that 1) cells with acquired resistance to cetuximab maintain their dependence on EGFR and 2) tumors developing resistance to cetuximab can benefit from subsequent treatment with erlotinib, providing rationale for its use in the setting of cetuximab resistance.

KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, Sprycel), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases (SFKs). We analyzed 16 CRC lines for: (1) KRAS mutation status, (2) dependence on mutant KRAS signaling and (3) expression level of epidermal growth factor receptor (EGFR) and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo and HCT116) cell lines and two KRAS wild-type cell lines (SW48 and CaCo2). In vitro, using poly-D-lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab, whereas KRAS wild-type lines showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater antiproliferative effect on KRAS mutant lines when compared with either agent alone in vitro and in vivo. To investigate potential mechanisms for this antiproliferative response in the combinatorial therapy, we performed Human Phospho-Kinase Antibody Array analysis, measuring the relative phosphorylation levels of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in the KRAS mutant lines LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the mitogen-activated protein kinase (MAPK) pathway, AKT/mammalian target of rapamycin (mTOR) pathway and the family of signal transducers and activators of transcription (STATs) when compared with the untreated control or monotherapy treatments. Next, we analyzed tumor growth with cetuximab, dasatinib or their combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an antitumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation, as measured by Ki67, and higher rates of apoptosis, as measured by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). The data presented in this study indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key signaling pathways. Furthermore, these results suggest that signaling via EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. These data strengthen the rationale for clinical trials combining cetuximab and dasatinib in the KRAS mutant CRC genetic setting.

Chiral glycolic acid equivalents are used extensively for the enantioselective synthesis of α-hydroxycarbonyl compounds. Their efficacy in enolate reactions and their utility in synthetic applications are discussed, highlighting the relevance of these methods to natural product synthesis.

Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with different regions of an FSHR/LHR chimera having only two unique LHR residues and that binds both hormones with high affinity. hCG and hFSH analogs dock with this receptor chimera in a manner similar to that in which they bind LHR and FSHR, respectively. This shows that although the FSHR does not normally bind hCG, it contains a cryptic lutropin binding site that has the potential to recognize hCG in a manner similar to the LHR. The presence of this cryptic site may explain why equine lutropins bind many mammalian FSHR and why mutations in the transmembrane domain distant from the extracellular domain enable the FSHR to bind hCG. The leucine-rich repeat domain (LRD) of the FSHR also appears to contain a cryptic FSH binding site that is obscured by other parts of the extracellular domain. This will explain why contacts seen in crystals of hFSH complexed with an LRD fragment of the human FSHR are hard to reconcile with the abilities of FSH analogs to interact with membrane G-protein coupled FSHR. We speculate that cryptic lutropin binding sites in the FSHR, which are also likely to be present in thyrotropin receptors (TSHR), permit the physiological regulation of ligand binding specificity. Cryptic FSH binding sites in the LRD may enable alternate spliced forms of the FSHR to interact with FSH.

In this Review we describe how the advent of machines is impacting on organic synthesis programs, with particular emphasis on the practical issues associated with the design of chemical reactors. In the rapidly changing, multivariant environment of the research laboratory, equipment needs to be modular to accommodate high and low temperatures and pressures, enzymes, multiphase systems, slurries, gases, and organometallic compounds. Additional technologies have been developed to facilitate more specialized reaction techniques such as electrochemical and photochemical methods. All of these areas create both opportunities and challenges during adoption as enabling technologies.

In this Review we describe how the advent of machines is impacting on organic synthesis programs, with particular emphasis on the practical issues associated with the design of chemical reactors. In the rapidly changing, multivariant environment of the research laboratory, equipment needs to be modular to accommodate high and low temperatures and pressures, enzymes, multiphase systems, slurries, gases, and organometallic compounds. Additional technologies have been developed to facilitate more specialized reaction techniques such as electrochemical and photochemical methods. All of these areas create both opportunities and challenges during adoption as enabling technologies.

Flow chemistry and continuous processing techniques are beginning to have a profound impact on the production of functional materials ranging from quantum dots, nanoparticles and metal organic frameworks to polymers and dyes. These techniques provide robust procedures which not only enable accurate control of the product material’s properties but they are also ideally suited to conducting experiments on scale. The modular nature of flow and continuous processing equipment rapidly facilitates reaction optimisation and variation in function of the products.

In the complex pathway of histidine biosynthesis, a key branch point linking amino acid and purine biosynthesis is catalyzed by the bifunctional enzyme imidazole glycerol phosphate (IGP) synthase. The first domain of IGP synthase, a triad glutamine amidotransferase, hydrolyzes glutamine to form glutamate and ammonia. Its activity is tightly regulated by the binding of the substrate PRFAR to its partner synthase domain. Recent crystal structures and molecular dynamics simulations strongly suggest that the synthase domain, a (beta/alpha)(8) barrel protein, mediates the insertion of ammonia and ring formation in IGP by channeling ammonia from one remote active site to the other. Here, we combine both mutagenesis experiments and computational investigations to gain insight into the transfer of ammonia and the mechanism of conduction. We discover an alternate route for the entrance of ammonia into the (beta/alpha)(8) barrel and argue that water acts as both agonist and antagonist to the enzymatic function. Our results indicate that the architecture of the two subdomains, most notably the strict conservation of key residues at the interface and within the (beta/alpha)(8) barrel, has been optimized to allow the efficient passage of ammonia, and not water, between the two remote active sites.

Efforts to identify the manner in which human choriogonadotropin (hCG) contacts lutropin receptors (LHR) have been stymied by the complex structure of the hormone and the likelihood that it contacts the receptor at multiple sites. During studies of hCG assembly in mammalian cells, we found that addition of a cysteine to the long disordered beta-subunit COOH terminus (betaCT) enabled it to become cross-linked by a disulfide to cysteines that are substituted for residues in loop alpha2 or in the alpha-subunit COOH terminus (alphaCT). This created a "knob" on the alpha-subunit at the location of the cysteine. Knobs of various sizes and charges were useful for probing surfaces of the alpha-subunit thought previously to contact the LHR. Attachment of the betaCT to residues in loop alpha2 facing loops beta1 and beta3 reduced hormone activity only a few fold revealing that this surface does not participate in essential high affinity receptor contacts, a finding inconsistent with our earlier view of the hCG-LHR complex. In contrast, this approach showed that the opposite surface of loop alpha2 appeared to be nearer the receptor interface. Although attachment of knobs to portions of the alphaCT reduced hormone activity substantially, this finding was difficult to interpret. As discussed, this procedure should be adapted readily to other proteins and may facilitate the introduction of fluorophores, enzymes, or other reagents at specific sites on protein surfaces. It may also permit one to cross-link proteins or to obscure specific protein surfaces during the development of "Trojan Horse" therapeutics.

All three human glycoprotein hormone heterodimers are assembled in the endoplasmic reticulum by threading the glycosylated end of α-subunit loop two (α2) beneath a disulfide “latched” strand of the β-subunit known as the “seatbelt.” This remarkable event occurs efficiently even though the seatbelt effectively blocks the reverse process, thereby stabilizing each heterodimer. Studies described here show that assembly is facilitated by the formation, disruption, and reformation of a loop within the seatbelt that is stabilized by the most easily reduced disulfide in the free β-subunit. We refer to this disulfide as the “tensor” because it shortens the seatbelt, thereby securing the heterodimer. Formation of the tensor disulfide appears to precede and facilitate seatbelt latching in most human choriogonadotropin β-subunit molecules. Subsequent disruption of the tensor disulfide elongates the seatbelt, thereby increasing the space beneath the seatbelt and the β-subunit core. This permits the formation of hydrogen bonds between backbone atoms of the β-subunit cystine knot and the tensor loop with backbone atoms in loop α2, a process that causes the glycosylated end of loop α2tobe threaded between the seatbelt and the β-subunit core. Contacts between the tensor loop and loop α2 promote reformation of the tensor disulfide, which explains why it is more stable in the heterodimer than in the uncombined β-subunit. These findings unravel the puzzling nature of how a threading mechanism can be used in the endoplasmic reticulum to assemble glycoprotein hormones that have essential roles in vertebrate reproduction and thyroid function.

The physical environment can promote the functional ability of persons with dementia. Many care facilities use environmental signage (e.g., names on doors) to facilitate adaptive behavior (e.g., room finding). However, the effects of such signage on residents’functioning are not well understood. In three experiments, we investigated if persons with moderate to severe dementia had the required skills necessary to benefit from signage. Compared to a control condition (recognition of fellow residents’ photographs), a high percentage of participants could identify written names and photographs of themselves (Experiment 1). Moreover, name and photographic labels helped participants identify belongings (Experiment 2). Training improved some participants’ recognition of their own photographs but not of their fellow residents’ photographs (Experiment 3). These findings are consistent with research on self-reference and age-related changes in face recognition and reading, and they suggest that many persons with dementia may have the requisite abilities to benefit from prosthetic signage.

Several excellent procedures for trapping tagged proteins have been devised, but many of these are expensive, cannot be used outside a limited pH range, fail to work in the presence of chaotropic agents, or are difficult to use. The chitin binding domain (CBD) of Bacillus circulans chitinase, which binds to chitin matrices prepared from inexpensive reagents isolated from crab shells, is an alternative tag that can be used under a variety of pH and denaturing conditions. Kits based on the interaction between the CBD and the chitin beads are available commercially. Here, we show that simultaneous treatment of microtiter plates with chitosan, a deacetylated form of chitin, and acetic anhydride produces a surface-bound film of chitin that also interacts tightly with the CBD. Chitin-coated microtiter well plates captured a CBD-tagged heterodimeric human glycoprotein hormone analog directly from mammalian cell culture media, even when present in trace amounts. Binding to the surface was stable in sodium dodecylsulfate and reversed only partially at low pH or in 8M urea at 37 degrees C. This technique appears well suited to surface attachment and permits biochemical or other analyses of molecules that can be tagged with a CBD.

IGP synthase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the unusual nucleotide, N(1)-[(5'-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to form 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR) and imidazole glycerol phosphate (IGP). A common feature of all glutamine amidotransferases is the upregulation of glutamine hydrolysis in the presence of an acceptor substrate. A refined assay system was developed to establish that Saccharomyces cerevisae IGP synthase shows a 4900-fold stimulation of glutaminase in the presence of the substrate acceptor PRFAR. The structure and function of IGP synthase acceptor substrate binding site were probed with competitive inhibitors that are nucleotide substrate and product analogues. In addition, these analogues were also used to establish that the normal steady-state turnover cycle involves a random sequential mechanism. Upregulation of the glutaminase active site occurs when these competitive inhibitors bind in the nucleotide site over 30 A away. One of the key structural features of IGP synthase is that the transfer of ammonia from the glutaminase site occurs through the (beta/alpha)(8) core of the protein. Upon the basis of the recent substrate-occupied structure for yeast IGP synthase (1), kinetic investigations of site-directed mutants revealed that a conserved K258 residue is key to productive binding and the overall stoichiometry of the reaction. The binding of the ribulosyl phosphate portion of the substrate PRFAR appears to be transduced through reorientation of K258 resulting in a conformational switch at the base of the (beta/alpha)(8) core that enables the passage of ammonia through the core of the protein. The overall analysis also leads to further discussion of how the residues that cover the opening of the (beta/alpha)(8) in the closed state may assist the channeling of ammonia at the interface of the two functional domains in the open state.

Derek Harold Richard Barton was born on 8 September 1918 in the Thames-side town of Gravesend in Kent, the only child of William Thomas Barton and Maude Henrietta ( née Lukes). His humble background gave no indication of the magnitude, depth and impact that his career would have on the lives of so many and the scientific community in general.

Flow chemistry and continuous processing techniques are beginning to have a profound impact on the production of functional materials ranging from quantum dots, nanoparticles and metal organic frameworks to polymers and dyes. These techniques provide robust procedures which not only enable accurate control of the product material’s properties but they are also ideally suited to conducting experiments on scale. The modular nature of flow and continuous processing equipment rapidly facilitates reaction optimisation and variation in function of the products.

Most heterodimeric proteins are stabilized by intersubunit contacts or disulfide bonds. In contrast, human chorionic gonadotropin (hCG) and other glycoprotein hormones are secured by a strand of their β-subunits that is wrapped around α-subunit loop 2 “like a seatbelt.” During studies of hCG synthesis in COS-7 cells, we found that, when the seatbelt was prevented from forming the disulfide that normally “latches” it to the β-subunit, its carboxyl-terminal end can “scan” the surface of the heterodimer and become latched by a disulfide to cysteines substituted for residues in the α-subunit. Analogs in which the seatbelt was latched to residues 35, 37, 41–43, and 56 of α-subunit loop 2 had similar lutropin activities to those of hCG; that in which it was latched to residue 92 at the carboxyl terminus had 10–20% the activity of hCG. Attachment of the seatbelt to α-subunit residues 45–51, 86, 88, 90, and 91 reduced lutropin activity substantially. These findings show that the heterodimer can form before the β-subunit has folded completely and support the notions that the carboxyl-terminal end of the seatbelt, portions of α-subunit loop 2, and the end of the α-subunit carboxyl terminus do not participate in lutropin receptor interactions. They suggest also that several different architectures could have been sampled without disrupting hormone activity as the glycoprotein hormones diverged from other cysteine knot proteins.

Background: Imidazole glycerol phosphate synthase catalyzes a two-step reaction of histidine biosynthesis at the bifurcation point with the purine de novo pathway. The enzyme is a new example of intermediate channeling by glutamine amidotransferases in which ammonia generated by hydrolysis of glutamine is channeled to a second active site where it acts as a nucleophile. In this case, ammonia reacts in a cyclase domain to produce imidazole glycerol phosphate and an intermediate of purine biosynthesis. The enzyme is also a potential target for drug and herbicide development since the histidine pathway does not occur in mammals. Results: The 2.1 A crystal structure of imidazole glycerol phosphate synthase from yeast reveals extensive interaction of the glutaminase and cyclase catalytic domains. At the domain interface, the glutaminase active site points into the bottom of the (β/α) 8 barrel of the cyclase domain. An ammonia tunnel through the (β/α) 8 barrel connects the glutaminase docking site at the bottom to the cyclase active site at the top. A conserved "gate" of four charged residues controls access to the tunnel. Conclusions: This is the first structure in which all the components of the ubiquitous (β/α) 8 barrel fold, top, bottom, and interior, take part in enzymatic function. Intimate contacts between the barrel domain and the glutaminase active site appear to be poised for crosstalk between catalytic centers in response to substrate binding at the cyclase active site. The structure provides a number of potential sites for inhibitor development in the active sites and in a conserved interdomain cavity.

The total syntheses of the bromotyrosine-derived natural products ianthelline, 5-bromoverongamine and JBIR-44 are described and their cytotoxic activity in a cervical cancer (HeLa) cell line and human umbilical vein endothelial cells (HUVECs) are reported.

Interactions of the placental glycoprotein hormone human choriogonadotropin (hCG) with lutropin receptors (LHR) are required for maintenance of early pregnancy. Knowledge of how hCG interacts with LHR is useful for understanding the mechanism of receptor function, an issue of considerable debate. A large surface of hCG remains exposed after the hormone binds the LHR and can be readily detected with monoclonal antibodies. Here we show that the surface of hCG α-subunit loop 1 furthest from the β-subunit interface can also be recognized by a monoclonal antibody when hCG is bound to the LHR. This extends the area of hCG known to be exposed in the hormone receptor complex, an observation that further restricts models of hCG–LHR interaction.

A strategy for making amides and ureas using a polymer-supported hydroxylamine resin as a traceless linker is described. The cleavage of the linker by samarium(II) iodide is reported for the first time.

A rapid and simple HPLC assay has been developed to separate and quantify N-acyl glutamates and the corresponding carboxylic acids of the acyl moiety. This method was specifically developed to assay hydrolytic activity for glutamate carboxypeptidases. Although established assays for specific substrates of such enzymes exist, they may not be amenable for examining the hydrolytic activity of new substrate probes. This assay was developed to accommodate such probes.

Minimized proteins have long been used to elicit an immune response to particular regions of a protein antigen. Most efforts to derive minimized proteins have employed synthetic peptide fragments. This approach works well for linear epitopes but poorly for conformational epitopes. Here we describe a homodimeric human chorionic gonadotropin (hCG) analog that retains the conformation of related parts of hCG and elicits high affinity specific antibodies. This novel immunogen displays the tertiary structure of selected loops of the protein but lacks structures that could elicit potentially undesirable antibodies.

Human chorionic gonadotropin (hCG) contains a beta-subunit N-terminal amino acid extension that contacts the alpha-subunit and is needed for efficient alpha and hCG beta-subunit combination. Here we report that an hCG beta-subunit analog, lacking residues 2-8, combined with the alpha-subunit more efficiently when positively charged residues between beta-subunit cysteines 10 and 11 were replaced with negatively charged residues found in the corresponding portion of follitropin. Residues 2-8 had no influence on binding of hCG to lutropin receptors. Positive charges between cysteines 10 and 11 are essential for high affinity binding of lutropins to their receptors. Therefore, the N-terminal extension found in all lutropin beta-subunits appears to have evolved to offset the inhibition of subunit combination by beta-subunit residues that are essential for lutropin activity. This beta-subunit extension is not found in follitropins or thyrotropins, hormones that have negatively charged residues between cysteines 10 and 11.