Ensilicated tetanus antigen retains immunogenicity: in vivo study and time-resolved SAXS characterization.

Our recently developed ensilication approach can physically stabilize proteins in silica without use of a pre-formed particle matrix. Stabilisation is done by tailor fitting individual proteins with a silica coat using a modified sol-gel process. Biopharmaceuticals, e.g. liquid-formulated vaccines with adjuvants, frequently have poor thermal stability; heating and/or freezing impairs their potency. As a result, there is an increase in the prevalence of vaccine-preventable diseases in low-income countries even when there are means to combat them. One of the root causes lies in the problematic vaccine 'cold chain' distribution. We believe that ensilication can improve vaccine availability by enabling transportation without refrigeration. Here, we show that ensilication stabilizes tetanus toxin C fragment (TTCF), a component of the tetanus toxoid present in the diphtheria, tetanus and pertussis (DTP) vaccine. Experimental in vivo immunization data show that the ensilicated material can be stored, transported at ambient temperatures, and even heat-treated without compromising the immunogenic properties of TTCF. To further our understanding of the ensilication process and its protective effect on proteins, we have also studied the formation of TTCF-silica nanoparticles via time-resolved Small Angle X-ray Scattering (SAXS). Our results reveal ensilication to be a staged diffusion-limited cluster aggregation (DLCA) type reaction. An early stage (tens of seconds) in which individual proteins are coated with silica is followed by a subsequent stage (several minutes) in which the protein-containing silica nanoparticles aggregate into larger clusters. Our results suggest that we could utilize this technology for vaccines, therapeutics or other biopharmaceuticals that are not compatible with lyophilization.

Salt bridge impact on global rigidity and thermostability in thermophilic citrate synthase.

It has been suggested that structural rigidity is connected to thermostability, e.g. in enzymes from thermophilic microorganisms. We examine the importance of correctly handling salt bridges, and interactions which we term 'strong polars', when constructing the constraint network for global rigidity analysis in these systems. Through a comparison of rigidity in citrate synthases, we clarify the relationship between rigidity and thermostability. In particular, with our corrected handling of strong polar interactions, the difference in rigidity between mesophilic and thermophilic structures is detected more clearly than in previous studies. The increase in rigidity did not detract from the functional flexibility of the active site in all systems once their respective temperature range had been reached. We then examine the distribution of salt bridges in thermophiles that were previously unaccounted for in flexibility studies. We show that in hyperthermophiles these have stabilising roles in the active site; occuring in close proximity to key residues involved in catalysis and binding of the protein.

X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure-function studies of a neuronal calcium-signalling protein.

The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squid Loligo pealei showed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly, L. pealei calexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the protein has two markedly different affinities for calcium ions, the weaker of which is most likely to be associated with binding of calcium ions to the protein during neuronal excitation. In the current study, site-directed mutagenesis has been used to abolish each of the three calcium-binding sites of calexcitin, and these experiments suggest that it is the single calcium-binding site in the C-terminal domain of the protein which is likely to have a sensory role in the neuron.

Inhibition of HIV-1 protease: the rigidity perspective.

MOTIVATION: HIV-1 protease is a key drug target due to its role in the life cycle of the HIV-1 virus. Rigidity analysis using the software First is a computationally inexpensive method for inferring functional information from protein crystal structures. We evaluate the rigidity of 206 high-resolution (2 Å or better) X-ray crystal structures of HIV-1 protease and compare the effects of different inhibitors binding to the enzyme. RESULTS: Inhibitor binding has little effect on the overall rigidity of the protein homodimer, including the rigidity of the active site. The principal effect of inhibitor binding on rigidity is to constrain the flexibility of the ß-hairpin flaps, which move to allow access to the active site of the enzyme. We show that commercially available antiviral drugs which target HIV-1 protease can be divided into two classes, those which significantly affect flap rigidity and those which do not. The non-peptidic inhibitor tipranavir is distinctive in its consistently strong effect on flap rigidity. CONTACT: jack.heal@warwick.ac.uk; r.roemer@warwick.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Rapid simulation of protein motion: merging flexibility, rigidity and normal mode analyses.

Protein function frequently involves conformational changes with large amplitude on timescales which are difficult and computationally expensive to access using molecular dynamics. In this paper, we report on the combination of three computationally inexpensive simulation methods--normal mode analysis using the elastic network model, rigidity analysis using the pebble game algorithm, and geometric simulation of protein motion--to explore conformational change along normal mode eigenvectors. Using a combination of ElNemo and First/Froda software, large-amplitude motions in proteins with hundreds or thousands of residues can be rapidly explored within minutes using desktop computing resources. We apply the method to a representative set of six proteins covering a range of sizes and structural characteristics and show that the method identifies specific types of motion in each case and determines their amplitude limits.

Comparative analysis of rigidity across protein families.

We present a comparative study in which 'pebble game' rigidity analysis is applied to multiple protein crystal structures, for each of six different protein families. We find that the main-chain rigidity of a protein structure at a given hydrogen bond energy cutoff is quite sensitive to small structural variations, and conclude that the hydrogen bond constraints in rigidity analysis should be chosen so as to form and test specific hypotheses about the rigidity of a particular protein. Our comparative approach highlights two different characteristic patterns ('sudden' or 'gradual') for protein rigidity loss as constraints are removed, in line with recent results on the rigidity transitions of glassy networks.

Quadrupolar ordering in LaMnO3 revealed from scattering data and geometric modeling.

Many strongly correlated materials display quadrupolar (Jahn-Teller) distortion of the local octahedral structural units. It is common for these distortions to be observed by probes of local structure but absent in the crystallographic average structure. The ordering of these quadrupoles is important in determining the properties of manganites and cuprates, and the nature of the disorder in these structures has been an unsolved problem. We combine high resolution scattering data and novel geometrical modeling techniques to obtain a detailed picture of the local atomic structure, and also to extract the quadrupolar order parameter associated with the distorted octahedra. We show that in LaMnO3, quadrupoles undergo a strong first-order phase transition at 730 K, but with nonzero order parameter remaining in the high-temperature phase.

Geometric simulation of perovskite frameworks with Jahn-Teller distortions: applications to the cubic manganites.

A new approach is presented for modeling perovskite frameworks with disordered Jahn-Teller (JT) distortions and has been applied to study the elastic response of the LaMnO3 structure to defects in the JT ordering. Surprisingly, antiphase domain boundary defects in the pattern of ordered JT octahedra, along the [110] and [110] bonding directions, are found to produce 1D stripe patterns rotated 45 degrees along a* directions, similar to stripe structures observed in these systems. Geometric simulation is shown to be an efficient and powerful approach for finding relaxed atomic structures in the presence of disorder in networks of corner-shared JT-distorted octahedra such as the perovskites. Geometric modeling rapidly relaxes large supercells (thousands of octahedra) while preserving the local coordination chemistry, and shows great promise for studying these complex systems.

The future of surgical research: the role of the American College of Surgeons Oncology Group.

The American College of Surgeons Oncology Group (ACOSOG) was established in 1997; it is funded by the National Cancer Institute (NCI) and American College of Surgeons (ACS) for the purpose of conducting multicenter phases II and III clinical trials in the field of surgical oncology. After eight years, ACOSOG has successfully completed seven trials and has five studies currently open to accrual for patients with brain, breast, gastrointestinal, head and neck, and lung cancers. The history of randomized controlled trials in surgery and the structure and function of ACOSOG are discussed. ACOSOG is establishing an extensive specimen bank for current and future correlative science studies, providing unique educational opportunities for surgeons in clinical research, and pursuing collaborative relationships in order to conduct trials with private industries. Also, ACOSOG has expanded its membership to include international sites, which contribute to the success of ACOSOG studies and enhance the portfolio of future protocols. The participation of general surgeons and surgical oncologists in clinical trials is essential to the improvement of treatment options for cancer patients.

Guidelines for diagnosis and therapy of MEN type 1 and type 2.

This is a consensus statement from an international group, mostly of clinical endocrinologists. MEN1 and MEN2 are hereditary cancer syndromes. The commonest tumors secrete PTH or gastrin in MEN1, and calcitonin or catecholamines in MEN2. Management strategies improved after the discoveries of their genes. MEN1 has no clear syndromic variants. Tumor monitoring in MEN1 carriers includes biochemical tests yearly and imaging tests less often. Neck surgery includes subtotal or total parathyroidectomy, parathyroid cryopreservation, and thymectomy. Proton pump inhibitors or somatostatin analogs are the main management for oversecretion of entero-pancreatic hormones, except insulin. The roles for surgery of most entero-pancreatic tumors present several controversies: exclusion of most operations on gastrinomas and indications for surgery on other tumors. Each MEN1 family probably has an inactivating MEN1 germline mutation. Testing for a germline MEN1 mutation gives useful information, but rarely mandates an intervention. The most distinctive MEN2 variants are MEN2A, MEN2B, and familial medullary thyroid cancer (MTC). They vary in aggressiveness of MTC and spectrum of disturbed organs. Mortality in MEN2 is greater from MTC than from pheochromocytoma. Thyroidectomy, during childhood if possible, is the goal in all MEN2 carriers to prevent or cure MTC. Each MEN2 index case probably has an activating germline RET mutation. RET testing has replaced calcitonin testing to diagnose the MEN2 carrier state. The specific RET codon mutation correlates with the MEN2 syndromic variant, the age of onset of MTC, and the aggressiveness of MTC; consequently, that mutation should guide major management decisions, such as whether and when to perform thyroidectomy.

Medullary carcinoma of the thyroid gland.

Medullary thyroid carcinoma (MTC) is an uncommon thyroid tumor that has attracted a great deal of interest because of its frequent presentation as a familial tumor and its primary involvement in the type II multiple endocrine neoplasia (MEN) syndromes MEN-IIA and MEN-IIB and familial medullary thyroid carcinoma (FMTC). The MTC tumor cells secrete the polypeptide hormone calcitonin, which serves as an excellent tumor marker, useful for defining the presence of disease, preoperatively or following thyroidectomy. The discovery that mutations in the RET proto-oncogene are associated with MEN-II syndromes was highly significant in that it demonstrated a clear correlation between genotype and phenotype; and most importantly it provided a mechanism whereby family members at risk could be identified by direct DNA analysis. Virtually all patients with MEN-IIA, MEN-IIB, and FMTC develop MTC; therefore there is a clear rationale for performing thyroidectomy as soon as a RET mutation has been identified. Because MTC appears to be much more aggressive in patients with MEN-IIB, thyroidectomy is performed during the first year of life in this setting, whereas in patients with MEN-IIA, where the tumor appears to be more indolent, the procedure can be safety delayed until age 5 years. Reoperative neck exploration in patients with evidence of persistent or recurrent MTC has been effective in a significant number of patients, although the success of the operation requires careful patient selection and preoperative assessment. MTC, as expressed in the MEN-II syndromes, is an excellent model to evaluate the usefulness of interventional therapy in patients demonstrated to have a genetic predisposition for cancer.

The importance of molecular biology to cancer surgery.

Over the last two decades there have been striking advances in molecular biology, which now have, and will continue to have, great impact on the diagnosis and treatment of patients with either benign or malignant diseases. The translation of these new findings from the laboratory to the clinic has just begun and promises to revolutionize the care of patients with certain neoplastic diseases. The most significant advances relate to molecular genetics, particularly the identification of inherited germline mutations that can be identified in kindred members by direct DNA testing. The surgical oncologist is in a pivotal position to define prophylactic interventional strategies for patients who are destined to develop certain solid tumor malignancies. The most immediate opportunities will be in the patients with hereditary disorders, such as breast cancer, large bowel cancer, and the endocrine neoplasia syndromes.

The health beliefs, values, and practices of gay adolescents.

Barriers to optimal health in the gay adolescent population include a lack of recognition or acceptance by healthcare providers, homophobic attitudes, and an absence of awareness regarding the healthcare needs of this vulnerable population. The literature suggests that gay youths experience such problems as lack of self-esteem, school truancy and dropout, runaway behavior and subsequent homelessness, drug and alcohol abuse, prostitution and sexually transmitted diseases, depression, and suicide. Advanced practice nurses have the opportunity to improve the health of gay youths through recognition, education, outreach, and advocacy.

Compartment-mediated dissection for papillary thyroid cancer.

There is considerable controversy surrounding the appropriate treatment of papillary thyroid carcinoma (PTC), most of which centers around the extent of thyroidectomy. Despite the advocation of less than total thyroidectomy by many surgeons, there is a renewed interest by others, mainly in Europe and Japan, in the performance of routine total thyroidectomy and extensive lymph-node dissection for PTC. This has been shown to be an effective strategy for medullary thyroid carcinoma, which is not responsive to thyroid suppression or radioactive iodine treatment. PTC, however, is well treated by these adjuvant modalities and, in general, has an excellent prognosis. The benefit of extensive operations for routine cases of PTC has not been proven, and this practice is not employed by most surgeons in the United States. Node dissection is reserved for those patients with palpable adenopathy.

Germline mutations in the multiple endocrine neoplasia type 1 gene: evidence for frequent splicing defects.

Multiple endocrine neoplasia type 1 (MEN 1) is a familial cancer syndrome characterized by parathyroid hyperplasia, pituitary adenomas, and neuroendocrine tumors of the pancreas and duodenum. In 1997, the MEN1 tumor suppressor gene was identified, and numerous germline mutations have been reported to be distributed throughout the gene. We used single strand conformational variant (SSCV) analysis to search for germline mutations in the members of 33 kindreds with a confirmed diagnosis of MEN 1. SSCV analysis revealed 25 conformational variants representing germline mutations that are predicted to result in loss of normal menin function. Twenty different disease-associated mutations were identified: five resulting in potential abnormal RNA splicing, two missense mutations, seven nonsense mutations, and six frameshift mutations. The aberrant splice products were identified and confirmed by RT-PCR and direct sequence analysis for two of the five splice mutations. Sixteen of the 20 (80%) mutations identified have not been previously reported. Mutations were not identified in eight kindreds with signs and symptoms consistent with MEN 1. The SSCV analysis revealed mutations in 76% (25 of 33) of the kindreds investigated, thus showing SSCV analysis to be a reliable mutation detection strategy. One-fifth of the mutations identified in this study involve intron sequences, therefore, highlighting the importance of including intron sequences in the search for germline mutations in the MEN1 gene. The need to investigate the entire gene when characterizing new MEN 1 families presents challenges in the translation of genetic studies to efficient clinical diagnostic tests.

A specific CpG methylation pattern of the MGMT promoter region associated with reduced MGMT expression in primary colorectal cancers.

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) protects cells from the cytotoxic and mutagenic effects of alkylating agents. Approximately 20% of tumor cell lines lack MGMT activity and are highly sensitive to alkylating agents. In established cancer cell lines, MGMT expression appears to be correlated with methylation of residues in both the promoter and the body of the gene. The effect of methylation of the MGMT promoter on gene expression and carcinogenesis in primary tumors is unknown. We investigated methylation of the MGMT promoter region in primary colorectal cancers and normal colonic mucosa. We used five methylation-sensitive restriction enzymes (BssHII, SacII, Eagl, Nael, and Smal) and Southern blot analysis to assess methylation in 46 cancers and 22 controls. Methylation of Eagl and Nael sites was seen in 12 tumors but in none of the 22 normal colorectal mucosa specimens. This difference was statistically significant (P<0.01). Methylation-sensitive single-nucleotide primer extension analysis of four additional cytosine residues confirmed methylation of the promoter region in the tumors identified by Eagl and Nael digestions and served to further quantitate the extent of methylation. Western blot analysis of 21 tumors revealed statistically significant lower MGMT expression in the eight tumors with methylation of the Eagl and Nael sites and nt -128 than in the 13 tumors lacking the methylation pattern (P<0.05). MGMT activity was lower in tumors with methylation than in tumors that were not methylated. The difference was not, however, statistically significant. We conclude that a subset of colorectal tumors is characterized by a specific methylation pattern in the MGMT promoter associated with reduced MGMT expression.