Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.

Pro-angiogenic cellular and genomic expression patterns within glioblastoma influences dynamic susceptibility weighted perfusion MRI.

AIM: To investigate whether quantitative dynamic susceptibility-weighted contrast-enhanced (DSC) perfusion magnetic resonance imaging (MRI) metrics are influenced by cellular and genomic expression patterns of glioblastoma angiogenesis. MATERIALS AND METHODS: Twenty-five stereotactic neurosurgical tissue samples were prospectively obtained from enhancing and non-enhancing tumour regions from 10 patients with treatment-naïve glioblastoma. Using monoclonal antibodies, histopathological features of angiogenesis were examined: total microvascular density, vascular morphology, and hypoxia. Angiogenic expression patterns of tissue samples were investigated using RNA microarrays. DSC perfusion MRI metrics were measured from the tissue sampling sites. MRI and histopathological variables were compared using Pearson's correlations. Microarray analysis was performed using false discovery rate (FDR) statistics. RESULTS: Thirteen enhancing and 12 non-enhancing MR image-guided tissue specimens were prospectively obtained. Enhancing tumour regions demonstrated a significant difference in DSC perfusion and histopathological metrics of angiogenesis when compared to non-enhancing regions. Four angiogenic pathways (vascular endothelial growth factor [VEGF], hypoxia inducible factor [HIF], platelet-derived growth factor [PDGF], fibroblast growth factor [FGF]; 25 individual genes) were significantly up-regulated within enhancing regions when compared to non-enhancing regions (adjusted p<0.05, FDR <0.05). A statistically significant correlation was observed between VEGF-A expression, microvascular density, microvascular morphology, and DSC perfusion MRI metrics (p<0.05). CONCLUSION: Pro-angiogenic genomic and cellular expression patterns of treatment-naïve primary glioblastoma significantly influences morphological and physiological DSC perfusion metrics suggesting that expression levels of therapeutically relevant genetic signatures can be quantified using MRI.

Ising Model Reprogramming of a Repeat Protein's Equilibrium Unfolding Pathway.

Repeat proteins are formed from units of 20-40 aa that stack together into quasi one-dimensional non-globular structures. This modular repetitive construction means that, unlike globular proteins, a repeat protein's equilibrium folding and thus thermodynamic stability can be analysed using linear Ising models. Typically, homozipper Ising models have been used. These treat the repeat protein as a series of identical interacting subunits (the repeated motifs) that couple together to form the folded protein. However, they cannot describe subunits of differing stabilities. Here we show that a more sophisticated heteropolymer Ising model can be constructed and fitted to two new helix deletion series of consensus tetratricopeptide repeat proteins (CTPRs). This analysis, showing an asymmetric spread of stability between helices within CTPR ensembles, coupled with the Ising model's predictive qualities was then used to guide reprogramming of the unfolding pathway of a variant CTPR protein. The designed behaviour was engineered by introducing destabilising mutations that increased the thermodynamic asymmetry within a CTPR ensemble. The asymmetry caused the terminal α-helix to thermodynamically uncouple from the rest of the protein and preferentially unfold. This produced a specific, highly populated stable intermediate with a putative dimerisation interface. As such it is the first step in designing repeat proteins with function regulated by a conformational switch.

Adrenal gland volume in major depression. Increase during the depressive episode and decrease with successful treatment.

BACKGROUND: Hyperactivity of the pituitary-adrenocortical axis is the most prominent neuroendocrine abnormality in major depression. It is state-related, returning to normal with resolution of the depressive episode. Adrenal gland enlargement also has been reported in patients with major depression and has been hypothesized as an index of cumulative lifetime depression. However, whether or not adrenal enlargement decreases with successful treatment of depression has not yet been studied, to our knowledge. We, therefore, determined adrenal gland volume in patients with major depression before and after treatment and in matched normal controls, and compared adrenal size with functional indexes of pituitary-adrenocortical activity. METHODS: Adrenal volumes were measured by magnetic resonance imaging in nine adult and two adolescent patients with major depression during their illness and during full remission when medication had been stopped for at least 1 month, and in nine adult and two adolescent normal control subjects individually matched to the patients. Basal, 4 to 7 PM plasma corticotropin 1-39 and cortisol levels, and corticotropin 1-39 and cortisol responses to administration of ovine corticorelin and lowdose cosyntropin also were measured. RESULTS: Mean adrenal gland volume was significantly larger, by about 70% in the patients while depressed than after successful treatment, and it also was significantly larger, again by about 70%, than the mean adrenal gland volume of their matched controls. After treatment, the mean adrenal volume of the patients decreased and was no longer significantly different from that of their controls at baseline. The magnitude of the decrease was significantly positively correlated with the duration of the depressive episode. Basal, late-afternoon plasma corticotropin 1-39 levels were significantly lower in the patients while depressed than in their matched controls, but basal plasma cortisol levels did not differ significantly among the three groups, nor did the corticotropin 1-39 and cortisol responses to corticorelin or the cortisol response to cosyntropin. Correlations between adrenal gland volume and basal corticotropin and cortisol levels, and the corticotropin and cortisol responses to hormone challenge, were not consistently in the expected direction in any of the three groups of subjects. CONCLUSIONS: Adrenal gland enlargement occurring during an episode of major depression appears to be state-dependent, in that it reverts to the normal size range during remission after treatment. It thus does not appear to be an index of cumulative lifetime depression. The lack of a discernible relationship between adrenal volume and pituitary-adrenocortical activity remains to be explained and might be related to noncorticotropin influences on the adrenal gland, including other tropic hormones and/or neural mechanisms.

Planar and SPECT ventilation/perfusion imaging and computed tomography for the diagnosis of pulmonary embolism: A systematic review and meta-analysis of the literature, and cost and dose comparison.

Diagnosing acute pulmonary embolism (PE) is an indication for scintillation V/Q imaging (planar and SPECT) and/or CTPA. This study reviews, compares and aggregates the published diagnostic performance of each modality and assesses the short-term consequences in terms of diagnostic outcomes, monetary cost, and radiation burden. We performed a formal literature review of available data and aggregated the finding using a summary receiver operating characteristic. A decision tree approach was used to estimate cost and dose per correct diagnosis. The review found 19 studies, which comprised 27 data sets (6393 examinations, from 5923 patients). The results showed that planar V/Q was significantly inferior to both V/Q SPECT and CTPA with no difference between the latter two. CTPA represents best value; £129 per correct diagnosis compared to £243 (SPECT) and £226 (planar). In terms of radiation burden V/Q SPECT was the most effective with a dose of 2.12 mSv per correct diagnosis compared with 3.46 mSv (planar) and 4.96 (CTPA) mSv. These findings show no performance difference between V/Q SPECT and CTPA; planar V/Q is inferior. CTPA is clearly the most cost effective technique. V/Q SPECT should be considered in situations where radiation dose is of concern or CTPA is inappropriate.

Adrenal gland volume in major depression: relationship to basal and stimulated pituitary-adrenal cortical axis function.

We measured adrenal gland volume and both baseline and stimulated pituitary and adrenal cortical hormones in 35 unmedicated, major depressives and 35 individually matched normal control subjects. Mean adrenal volume in the depressives was significantly larger, by about 38%, than the adrenal volume of their matched controls. Basal plasma adrenocorticotropic hormone (ACTH)1-39 was significantly lower, and basal plasma cortisol was significantly higher, in the patients. In contrast, basal plasma ACTH determined by radioimmunoassay (RIA) was not significantly different between the two groups. The ACTH response to ovine corticotropin-releasing hormone (oCRH), whether measured specifically as ACTH1-39 or by the less-specific RIA, was highly significantly lower in the depressives than in the controls. However, neither the cortisol response to oCRH nor its response to low-dose ACTH 1-24 differed significantly between groups. In both groups of subjects, correlations between adrenal gland volume and all the hormone measures were low, and none represented more than 4% shared variance. In the patients, adrenal volume did not correlate significantly with duration of the present episode, lifetime number of episodes, melancholic subtype, Hamilton Depression Scale total score, or the Hamilton suicidality item. However, adrenal volume was significantly positively related to the somatization factor of the Hamilton scale, which was almost totally accounted for by the specific items of somatic symptoms and somatic anxiety.

TurboPrep II: an inexpensive, high-throughput plasmid template preparation protocol.

In an effort to reduce plasmid template preparation costs for large-scale genomic sequencing projects, a boiling minipreparation protocol has been developed that enables a single individual to easily prepare 768 sequence-quality templates in 8 h, without automation. The maximum throughput for one individual using one centrifuge in the manual configuration is 1920 templates in about 8 h. The most time-consuming manual steps of this method involve pipetting, which can be automated, resulting in a significant increase in throughput and about a 60% increase in yield. This method in the fully manual configuration yields sufficient double-stranded template for two sets of cycle sequencing reactions, membrane spotting for hybridization analysis and host cell transformation for the recovery of the original recombinant. The current materials cost per template using this method is less than twenty cents. The quality of the sequence generated has been evaluated by manual 35S radioactive cycle sequencing. Initial results have shown templates prepared by this method to yield greater than 300 bp of readable sequence when the radioactively labeled products were resolved on 6% modified denaturing polyacrylamide gels.

Adrenal gland volume determination by computed tomography and magnetic resonance imaging in normal subjects.

To compare the usefulness of computed tomography (CT) and magnetic resonance imaging (MRI) for the quantitative determination of adrenal gland size in human subjects, adrenal gland volumes were determined by both techniques in five female and five male, normal, nonstressed adult volunteers. Mean left and right adrenal volumes were slightly larger by CT, and the left adrenal volume was slightly larger than the right by both imaging techniques. Volume determinations by CT and MRI correlated +0.85 and +0.82 for the left and right glands, respectively. Body surface area was highly correlated, and sex moderately correlated, with left and right adrenal volumes by both techniques. Both CT and MRI somewhat overestimated the known volumes of a set of phantoms, CT by an average of 42% and MRI by an average of 20%. The results of this initial study suggest that MRI holds promise as a technique for in vivo adrenal gland measurements, and that the influence of both body surface area and sex should be considered in comparative studies of adrenal size.

MR and CT imaging of ethanol-treated liver tumors in an animal model.

To characterize the radiographic appearance of liver lesions over time following ethanol injection, seven New Zealand white rabbits underwent surgical implantation of small fragments of VX-2 carcinoma within the liver. Upon reaching 1 cm in diameter, a tumor nodule was directly injected with absolute ethanol. Another nodule in the same animal was injected with saline as a control. Imaging was performed 6-24 days after the injections by high resolution CT and MRI, and correlation obtained with the pathologic specimens. Long TR spin-echo MR sequences were found to characterize the ethanol-treated regions of liver most accurately. Liver tissue infarcted by alcohol could be differentiated from tumor and necrosis by virtue of its short T2 relaxation value. There were no distinguishing features by other imaging techniques between the ethanol-treated and control tumor nodules. Peripheral contrast enhancement was demonstrated in both, corresponding to fibrous tissue around the ethanol-injected regions, and to viable tumor in the case of controls.

Bond strength of six dentinal adhesives.

This study compared the shear bond strengths on dentine of five dentine adhesives against Scotchbond 2 as a control. Dentine specimens from 225 extracted human permanent molar teeth were used in a matched-pair design, such that 45 tooth sides were treated along with the matching control for each of the five test materials. Shear bond strength testing was conducted 24 h after the completion of each specimen. Results showed that mean force (MPa) for the materials were: XR Bond, 17.1 +/- 5.1; Scotchbond Multi-Purpose, 15.9 +/- 6.5; Syntac, 13.5 +/- 8.6; All-Bond 2, 6.2 +/- 4.1; Scotchbond 2 (control composite score), 4.9 +/- 3.0; Denthesive, 3.4 +/- 1.7. Matched pair t-test comparisons with Scotchbond 2 control were: XR Bond, P = 0.0005; Scotchbond Multi-Purpose, P = 0.0005; Syntac, P = 0.0005; All-Bond 2, P = 0.0368; Denthesive, P = 0.005. Analysis of variance determined a statistically significant difference (P = 0.05) between the means of the bond strengths, which fell into four groups: Group A, XR Bond and Scotchbond Multi-Purpose; Group B, Syntac; Group C, All-Bond 2 and Scotchbond 2; Group D, Scotchbond 2 and Denthesive. In Group C, there was no significant difference between Scotchbond 2 and All-Bond 2. In Group D there was no significant difference between Scotchbond 2 and Denthesive, however, there was significant difference between All-Bond 2 and Denthesive. It was concluded that XR Bond and Scotchbond Multi-Purpose had significantly better shear bond strength than the other dentine adhesives.

Magnetic resonance imaging of pediatric head and neck cystic hygromas.

We retrospectively reviewed the magnetic resonance imaging (MRI) findings of eight pathologically proved cystic hygromas in seven pediatric patients. All lesions showed multiple cysts, best depicted on T2-weighted images (T2WI). Six of the eight lesions had well-defined borders. Fluid-fluid levels were visualized in seven of eight lesions, with very high signal intensity of the upper fluid on T2WI. On T1-weighted images, all lesions showed a mean signal intensity greater than that of muscle (1.6 times) and cerebrospinal fluid (CSF; 4.04 times) and less than that of fat (0.5 times). On T2WI, the mean signal intensity was greater than that of CSF (1.8 times), muscle (9.45 times), and fat (2.54 times). Chemical analysis of the cystic fluid from one lesion showed high lipid content and hemorrhage, consistent with the preoperative and in vitro MRI findings on the fluid from the same patient. Lesion anatomic distribution showed frequent posterior triangle involvement; however, the epicenter of 75% of the lesions was outside the posterior triangle. Only one lesion had involvement limited to the posterior triangle. There was equal distribution of right and left sides and no midline lesion. Seven lesions displaced the sternocleidomastoid muscle laterally, whereas one displaced it posteriorly, with no evidence of muscle infiltration. Only those patients with submucosal involvement (two of seven) had respiratory symptoms. Gadolinium provided no additional information with regard to diagnosis or extent of involvement. Our experience indicates that MRI is useful in the diagnosis and treatment planning of cystic hygromas.

Cryopreservation of rooster sperm using methyl cellulose.

Experiments were designed to determine when, during the cryopreservation process, sperm lose fertilizing capacity and whether the cryoprotectant, methyl cellulose (MC), could be used in combination with glycerol to cryopreserve sperm and remain in the inseminate without reducing fertility. Semen diluted in Minnesota Avian extender (MNA) and inseminated immediately had greater fertility (75%) than semen processed for cryopreservation (12 to 60%). The largest decreases in fertility were due to addition of glycerol to sperm and to cryopreservation. In another experiment, fertility of inseminates containing 0, 1, and 2% glycerol were 82, 29, and 21%, respectively, for eggs collected 2 to 5 d after insemination. When 0.5% MC was added to the same three treatments, fertility rates were 88, 63, and 69%, respectively. Semen cryopreserved in MNA containing 9% glycerol; MC + 3% glycerol; MC + 4% glycerol; MC + 9% glycerol; or 9% glycerol with the cryoprotectant removed post-thaw by dilution and subsequent centrifugation exhibited 59, 30, 35, 60, and 69% viable cells, respectively; and 65, 38, 46, 69, and 65% motile sperm, respectively. Sperm cryopreserved with MC and either 4 or 9% glycerol exhibited similar numbers of sperm binding to chicken perivitelline layers in vitro as did fresh sperm, whereas sperm frozen with MC and 3% glycerol bound oocytes with only 31% efficiency (P < 0.05). The extent to which cryopreserved sperm penetrated the perivitelline layer in vitro was independent of glycerol concentration, but was four times more efficient than that of fresh sperm (P < 0.05). The fertility rates of fresh semen, semen frozen in 9% glycerol with the cryoprotectant removed after thawing, and semen frozen in MC with either 3 or 4% glycerol were 87.4, 27.6, 0.8, and 0.5%, respectively (P < 0.05). The MC reduces the contraceptive effects of glycerol when inseminated with fresh sperm, but does not maintain fertilizing capacity in frozen-thawed sperm when used in combination with 3 or 4% glycerol.

Influence of the mode of preparation on the long-term efficacy of homologous costal cartilage implants.

Devitalized homologous costal cartilage is widely employed as an implant in the management of the saddle nose. The tissue response induced by the implant is a combination of enveloping fibrosis and implant resorption, which will probably, ultimately, be complete. We have studied the balance between resorption and fibrosis, following different modes of cartilage preparation, in the mouse. Homologous costal cartilage was devitalized by four common methods--irradiation, formalin, glutaraldehyde and alcohol. Segments of this cartilage were inserted at separate sites in the subcutaneous plain of the tail. These implants were harvested after one year for histology. Variations in the mode of cartilage devitalization, while inducing variations in the degree of the tissue response, did not influence the balance between fibrosis and resorption. Thus the long term maintenance of tissue bulk following homologous cartilage implantation is not influenced by the mode of preparation. Evidence suggests that the ultimate cosmetic results of autologous and homologous costal cartilage implantation would be much the same, and the use of homologous cartilage must be justified on other grounds.

MRI of the upper abdomen using motion artifact suppression technique (MAST).

Magnetic resonance imaging of the upper abdomen has been hampered by motion artifacts that cause suboptimal image quality. A promising software solution to this problem, motion artifact suppression technique (MAST), is described. Using mathematically determined refocussing gradients, it has improved image clarity significantly. Unlike alternate techniques such as respiratory gating, there is no increase in scan time, and patient throughput is expedited.

Super-resolution track density imaging of glioblastoma: histopathologic correlation.

BACKGROUND AND PURPOSE: Super-resolution track density imaging generates anatomic images with submillimeter voxel resolution by using high-angular-resolution diffusion imaging and fiber-tractography. TDI within the diseased human brain has not been previously described. The purpose of this study was to correlate TDI with histopathologic features of GBM. MATERIALS AND METHODS: A total of 43 tumor specimens (24 contrast-enhancing, 12 NE, and 7 centrally necrotic regions) were collected from 18 patients with treatment-naïve GBM by use of MR imaging-guided neurosurgical techniques. Immunohistochemical stains were used to evaluate the following histopathologic features: hypoxia, architectural disruption, microvascular hyperplasia, and cellular proliferation. We reconstructed track density maps at a 0.25-mm isotropic spatial resolution by using probabilistic streamline tractography combined with constrained spheric deconvolution (model order, 8; 0.1-mm step size; 1 million seed points). Track density values were obtained from each tissue site. A P value of .05 was considered significant and was adjusted for multiple comparisons by use of the false discovery rate method. RESULTS: Track density was not significantly different between contrast-enhancing and NE regions but was more likely to be elevated within regions demonstrating aggressive histopathologic features (P < .05). Significant correlation between relative track density and hypoxia (odds ratio, 3.52; P = .01), architectural disruption (odds ratio, 3.49; P = .03), and cellular proliferation (odds ratio, 1.70; P = .05) was observed irrespective of the presence or absence of contrast enhancement. CONCLUSIONS: Numeric values of track density correlate with GBM biologic features and may be clinically useful for identification of regions of tumor infiltration within both enhancing and NE components of GBM.

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors.

Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.