Single Subclass Research Articles

Noncanonical HPV carcinogenesis drives radiosensitization of head and neck tumors

We analyzed transcriptional data from 104 HPV+ (Human papillomavirus) HNSCC (head and neck squamous cell carcinoma) tumors together with two publicly available sources to identify highly robust transcriptional programs (modules) which could be detected consistently despite heterogeneous sequencing and quantification methodologies. Among 22 modules identified, we found a single module that naturally subclassifies HPV+ HNSCC tumors based on a bimodal pattern of gene expression, clusters all atypical features of HPV+ HNSCC biology into a single subclass, and predicts patient outcome in four independent cohorts. The subclass-defining gene set was strongly correlated with Nuclear factor kappa B (NF-κB) target expression. Tumors with high expression of this NF-κB module were rarely associated with activating PIK3CA alterations or viral integration, and also expressed higher levels of HPHPV E2 and had decreased APOBEC mutagenesis. Alternatively, they harbored inactivating alterations of key regulators of NF-κB, TNF receptor associated factor 3 (TRAF3), and cylindromatosis (CYLD), as well as retinoblastoma protein (RB1). HPV+ HNSCC cells in culture with experimental depletion of TRAF3 or CYLD displayed increased expression of the subclass-defining genes, as well as robust radio-sensitization, thus recapitulating both the tumor transcriptional state and improved treatment response observed in patient data. Across all gene sets investigated, methylation to expression correlations were the strongest for the subclass-defining, NF-κB-related genes. Increased tumor-infiltrating CD4+ T cells and increased Estrogen receptors alpha (ERα) expression were identified in NF-κB active tumors. Based on the relatively high rates of cure in HPV+ HNSCC, deintensification of therapy to reduce treatment-related morbidity is being studied at many institutions. Tumor subclassification based on oncogenic subtypes may help guide the selection of therapeutic intensity or modality for patients with HPV+ HNSCC.

Development and validation of a rapid LC-MS/MS method for the detection of 182 novel psychoactive substances in whole blood.

IntroductionThe analysis of novel psychoactive substances (NPS) represents a challenge in forensic toxicology, due to the high number of compounds characterized by different structures and physicochemical properties both among different subclasses and within a single subclass of NPS. The aim of the present work is the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC‐MS/MS) method for the detection of NPS in whole blood.Materials and methodsA protein‐precipitation based LC‐MS/MS method for the detection of more than 180 NPS was developed and validated by assessing the following parameters: selectivity, linearity, accuracy, precision, limit of detection (LOD) and of quantification (LOQ) recovery, and matrix effect. Then, the method was applied to real forensic samples.ResultsThe method allowed the identification of 132 synthetic cannabinoids, 22 synthetic opioids, and 28 substances among synthetic cathinones, stimulants, and other drugs. Validation was successfully achieved for most of the compounds. Linearity was in the range of 0.25–10 ng/ml for synthetic cannabinoids and 0.25–25 ng/ml for other drugs. Accuracy and precision were acceptable according to international guidelines. Three cases tested positive for fentanyl and ketamine, in the setting of emergency room administration.ConclusionsThe present methodology represents a fast, not expensive, wide‐panel method for the analysis of more than 180 NPS by LC‐MS/MS, which can be profitably applied both in a clinical context and in postmortem toxicology.

Single-cell co-expression analysis reveals that transcriptional modules are shared across cell types in the brain.

Gene-gene relationships are commonly measured via the co-variation of gene expression across samples, also known as gene co-expression. Because shared expression patterns are thought to reflect shared function, co-expression networks describe functional relationships between genes, including co-regulation. However, the heterogeneity of cell types in bulk RNA-seq samples creates connections in co-expression networks that potentially obscure co-regulatory modules. The brain initiative cell census network (BICCN) single-cell RNA sequencing (scRNA-seq) datasets provide an unparalleled opportunity to understand how gene-gene relationships shape cell identity. Comparison of the BICCN data (500,000 cells/nuclei across 7 BICCN datasets) with that of bulk RNA-seq networks (2,000 mouse brain samples across 52 studies) reveals a consistent topology reflecting a shared co-regulatory signal. Differential signals between broad cell classes persist in driving variation at finer levels, indicating that convergent regulatory processes affect cell phenotype at multiple scales.

Neurological disorders-associated anti-glycosphingolipid IgG-antibodies display differentially restricted IgG subclass distribution

Antibodies against several self-glycans on glycosphingolipids are frequently detected in different neurological disorders. Their pathogenic role is profusely documented, but the keys for their origin remain elusive. Additionally, antibodies recognizing non-self glycans appear in normal human serum during immune response to bacteria. Using HPTLC-immunostaining we aimed to characterize IgM and IgG subclass antibody responses against glycosphingolipids carrying self glycans (GM1/GM2/GM3/GD1a/GD1b/GD3/GT1b/GQ1b) and non-self glycans (Forssman/GA1/“A” blood group/Nt7) in sera from 27 randomly selected neurological disorder patients presenting IgG reactivity towards any of these antigens. Presence of IgG2 (p = 0.0001) and IgG1 (p = 0.0078) was more frequent for IgG antibodies against non-self glycans, along with less restricted antibody response (two or more simultaneous IgG subclasses). Contrariwise, IgG subclass distribution against self glycans showed clear dominance for IgG3 presence (p = 0.0017) and more restricted IgG-subclass distributions (i.e. a single IgG subclass, p = 0.0133). Interestingly, anti-self glycan IgG antibodies with simultaneous IgM presence had higher proportion of IgG2 (p = 0.0295). IgG subclass frequencies were skewed towards IgG1 (p = 0.0266) for “anti-self glycan A” subgroup (GM2/GM1/GD1b) and to IgG3 (p = 0.0007) for “anti-self glycan B” subgroup (GM3/GD1a/GD3/GT1b/GQ1b). Variations in players and/or antigenic presentation pathways supporting isotype (M-G) and IgG-subclass pattern differences in the humoral immune response against glycosphingolipids carrying non-self versus self-glycans are discussed.

Association of host immunity with Helicobacter pylori infection in recurrent gastric cancer

BackgroundHelicobacter pylori infection is associated with the incidence of gastric cancer. Endoscopic resection has been developed as a proper technique to treat early stage of gastric cancer. However, some patients develop recurrent gastric cancer within 5 years after endoscopic treatment. The aim of the present study is to explore a biomarker for detecting people who has high risk of gastric cancer recurrence.MethodsWe analyzed the Interleukin-10 (IL-10) single nucleotide polymorphism (SNP) and IgG subclass responses to the bacteria in patients with early gastric cancer and recurrent gastric cancer.ResultsPatients with hetero-type in the 1082 SNP and CC genotype in the 592 SNP were at high risk of recurrence of gastric cancer. In patients with genotype carrying high risk of recurrence, IgG1 level tended to be higher than that in patients with other genotypes.ConclusionsDominance of T helper 2 (Th2) immunity controlled by IL-10 cytokine may be associated with H. pylori-associated gastric cancer recurrence.

Absolute Quantitation of Glycoforms of Two Human IgG Subclasses Using Synthetic Fc Peptides and Glycopeptides.

Immunoglobulins, such as immunoglobulin G (IgG), are of prime importance in the immune system. Polyclonal human IgG comprises four subclasses, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitative method for these subclasses and their Fc N-glycoforms, (glyco)peptides were synthesized using a solid-phase approach and used as internal standards. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR standards, respectively. For IgG1, a similar glycopeptide where tyrosine (Y) was isotopically labelled was used to quantify monoclonal IgG1 that had been treated with the enzyme Endo-F2, i.e., yielding tryptic glycopeptide EEQYN(GlcNAc)STYR. The next step was to quantify single subclasses within polyclonal human IgG samples. Although ion abundances in the MALDI spectra often showed higher signals for IgG2 than IgG1, depending on the spotting solvent used, determination of amounts using the newly developed quantitative method allowed to obtain accurate concentrations where IgG1 species were predominant. It was observed that simultaneous analysis of IgG1 and IgG2 yielded non-quantitative results and that more success was obtained when subclasses were quantified one by one. More experiments served to assess the respective extraction and ionization efficiencies of EEQYNSTYR/EEQFNSTFR and EEQYN(GlcNAc)STYR/EEQFN(GlcNAc)STFR mixtures under different solvent and concentration conditions. Graphical Abstract ᅟ.

Membranous Glomerulopathy With Light Chain–Restricted Deposits: A Clinicopathological Analysis of 28 Cases

IntroductionMembranous glomerulopathy (MG) is a common cause of nephrotic syndrome that results from the formation of immune complexes along the subepithelial aspect of the glomerular basement membranes. Although it is most frequently caused by polytypic deposits, cases with light chain isotype−restricted deposits are rarely seen.MethodsWe conducted a retrospective analysis of 28 cases of MG that showed light chain isotype restriction.ResultsThe mean age at diagnosis was 62.2 years and the male-to-female ratio was 1. All patients presented with proteinuria (73.1% nephrotic range), and the mean serum creatinine was 1.5 mg/dl. Six patients had an underlying lymphoproliferative disorder (LPD), 2 had autoimmune disease, and 1 patient was positive for both hepatitis B and syphilis. Only 1 of the patients with an LPD had a detectable monoclonal Ig. Four patients (14.3%) showed focal proliferation or crescents, 3 of whom had an underlying LPD. Kappa (κ) restriction was seen in 26 of 28 patients (85.7%). Staining for IgG subclasses was performed in 19 cases, 14 of which showed positive staining for a single subclass. PLA2R was positive in 7 of 27 cases. 30% of PLA2R-negative patients and 28.6% of those with positive staining for a single IgG subclass had an associated LPD.DiscussionThe majority of MG cases with light chain isotype−restricted deposits lack a recognizable secondary etiology. However, the absence of PLA2R positivity, positive staining for a single IgG subclass, and presence of focal proliferation are worrisome histopathologic features that should prompt a thorough clinical workup to exclude the presence of an underlying LPD.

Quantitation of Site-Specific Glycosylation in Manufactured Recombinant Monoclonal Antibody Drugs.

During the development of recombinant monoclonal antibody (rMAb) drugs, glycosylation receives particular focus because changes in the attached glycans can have a significant impact on the antibody effector functions. The vast heterogeneity of structures that exist across glycosylation sites hinders the in-depth analysis of glycan changes specific to an individual protein within a complex mixture. In this study, we established a sensitive and specific method for monitoring site-specific glycosylation in rMAbs using multiple reaction monitoring (MRM) on an ultrahigh-performance liquid chromatography-triple quadrupole MS (UHPLC-QqQ-MS). Our results showed that irrespective of the IgG subclass expressed in the drugs, the N-glycopeptide profiles are nearly the same but differ in abundances. In all rMAb drugs, a single subclass of IgG comprised over 97% of the total IgG content and showed over 97% N-glycan site occupancy. This study demonstrates the utility of an MRM-based method to rapidly characterize over 130 distinct glycopeptides and determine the extent of site occupancy within minutes. Such multilevel structural characterization is important for the successful development of therapeutic antibodies.

The year 2010 classification of the agglutinated foraminifera

A reclassification of the agglutinated foraminifera (within the Class Foraminiferea) is presented, consisting of three subclasses, seven orders, 18 suborders, 32 superfamilies, 122 families, 148 subfamilies, and containing a total of 832 valid genera is presented. The classification is modified from the suprageneric scheme used by Kaminski (2004a), and incorporates all the new agglutinated genera described up to and including the year 2010. The major differences from the Kaminski (2004a) classification are (1) the subdivision of the Foraminifera into three supraordinal groups according to the molecular studies of Pawlowski and co-workers (2) the inclusion of the allogromiids in the classification alongside the astrorhizids (3) the inclusion of the xenophyophorids within the Astrorhizida at superfamily rank, (4) the merging of all pseudo-bilocular and pseudo-multichambered forms within a single subclass and (5) the revision of the schlumberigerinids to encompass all miliolidswith an agglutinated wall. Numerous minor corrections and updates have been made to the generic classification based on the recent literature.

Complex behaviour in a dengue model with a seasonally varying vector population

In recent decades, dengue fever and dengue haemorrhagic fever have become a substantial public health concern in many subtropical and tropical countries throughout the world. Many of these regions have strong seasonal patterns in rainfall and temperature which are directly linked to the transmission of dengue through the mosquito vector population. Our study focuses on the development and analysis of a strongly seasonally forced, multi-subclass dengue model. This model is a compartment-based system of first-order ordinary differential equations with seasonal forcing in the vector population and also includes host population demographics. Our analysis of this model focuses particularly on the existence of deterministic chaos in regions of the parameter space which potentially hinders application of the model to predict and understand future outbreaks. The numerically efficient 0–1 test for deterministic chaos suggested by Gottwald and Melbourne (2004) [18] is used to analyze the long-term behaviour of the model as an alternative to Lyapunov exponents. Various solutions types were found to exist within the studied parameter range. Most notable are the existence of isola n-cycle solutions before the onset of deterministic chaos. Analysis of the seasonal model with the 0–1 test revealed the existence of three disconnected regions in parameter space where deterministic chaos exists in the single subclass model. Knowledge of these regions and how they relate to the parameters of the model gives greater confidence in the predictive power of the seasonal model.

Influence of illumination on the characterization of banana ripening

For its commercialization, both in internal and external markets, fruits are classified according to the degree of ripening, mainly on the basis of peel color. For a correct classification, using artificial lighting, it is necessary it provides a perception of color that is as similar as possible to natural. This paper compares the use of nine different commercial lamps (incandescent, fluorescent e LED) in the classification of banana ripening (subclasses C1 to C7 of Von Loesecke). During the analysis, it was observed that the coordinate of chromaticity a* of the CIELAB system was highly influenced by the use of different sources in the illumination system. The differences in color were visually perceived when a single subclass of ripening was observed under different lamps, mistakenly influencing the grading during packing, thus making it necessary to standardize the illumination used in the systems of visual inspection for the commercialization of fruits.

Contribution of Distinct Homeodomain DNA Binding Specificities to Drosophila Embryonic Mesodermal Cell-Specific Gene Expression Programs

Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I–HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks directing mesoderm development.

The disproportionate economic burden associated with severe and complicated obesity: a systematic review

Burden of disease studies typically classify individuals with a body mass index (BMI) ≥ 30 kg m(-2) as a single group ('obese') and make comparisons to those with lower BMIs. Here, we review the literature on the additional economic burden associated with severe obesity or classes 3 and 4 obesity (BMI ≥ 40 kg m(-2) ), the fastest growing category of obesity, with the aim of exploring and disaggregating differences in resource use as BMI increases beyond 40 kg m(-2) . We recognize the importance of comparing classes 3 and 4 obesity to less severe obesity (classes 1 and 2) as well as quantifying the single sub-class impacts (classes 3 and 4). Although the latter analysis is the aim of this review, we include results, where found in the literature, for movement between the recognized subclasses and within classes 3 and 4 obesity. Articles presenting data on the economic burden associated with severe obesity were identified from a search of Ovid MEDLINE, EMBASE, EBSCO CINAHL and Cochrane Library databases. Data were extracted on the direct costs, productivity costs and resource use associated with severe obesity along with estimates of the multiplier effects associated with increasing BMI. Fifteen studies were identified, of which four disaggregated resource use for BMI ≥ 40 kg m(-2) . The multiplier effects derived for a variety of different types of costs incurred by the severely obese compared with those of normal weight (18.5 kg m(-2) < BMI < 25 kg m(-2) ) ranged from 1.5 to 3.9 for direct costs, and from 1.7 to 8.0 for productivity costs. There are few published data on the economic burden of obesity disaggregated by BMI ≥ 40 kg m(-2) . By grouping people homogenously above a threshold of BMI 40 kg m(-2) , the multiplier effects for those at the highest end of the spectrum are likely to be underestimated. This will, in turn, impact on the estimates of cost-effectiveness for interventions and policies aimed at the severely obese.

Stem cell‐derived motoneurons: Tools for studying motoneuron disease and motoneuron development

Ten years ago, Tom Jessell and colleagues demonstrated for the first time that mouse embryonic stem (ES) cells can be directed to differentiate into motoneurons by simply treating them in culture with two mitogens, retinoic acid and a sonic hedgehog (Shh) agonist. Since this seminal discovery there has been an explosion of research using motoneurons derived from human and mouse ES cells, as well as induced pluripotent stem (iPS) cells, to study motoneuron disease and development. My presentation will first focus on developmental studies where we exploited the fact that mouse ES cell‐derived motoneurons differentiate into a single subclass of motoneurons that selectively innervate postural muscles lining the vertebral column. We show that ES cell‐derived motoneurons are an ideal model system to examine intracellular signaling cascades regulating motor axon guidance during embryogenesis. Second, I will describe a novel in vitro model system to study synapse formation at the neuromuscular junction using ES cell‐derived motoneurons and chick myotube co‐cultures. Finally, I will explore the use of motoneurons directly derived from fibroblasts as a tool to study motoneuron diseases.

Imaging of opioid receptors in the central nervous system

In vivo functional imaging by means of positron emission tomography (PET) is the sole method for providing a quantitative measurement of μ-, κ and δ-opioid receptor-mediated signalling in the central nervous system. During the last two decades, measurements of changes to the regional brain opioidergic neuronal activation—mediated by endogenously produced opioid peptides, or exogenously administered opioid drugs—have been conducted in numerous chronic pain conditions, in epilepsy, as well as by stimulant- and opioidergic drugs. Although several PET-tracers have been used clinically for depiction and quantification of the opioid receptors changes, the underlying mechanisms for regulation of changes to the availability of opioid receptors are still unclear. After a presentation of the general signalling mechanisms of the opioid receptor system relevant for PET, a critical survey of the pharmacological properties of some currently available PET-tracers is presented. Clinical studies performed with different PET ligands are also reviewed and the compound-dependent findings are summarized. An outlook is given concluding with the tailoring of tracer properties, in order to facilitate for a selective addressment of dynamic changes to the availability of a single subclass, in combination with an optimization of the quantification framework are essentials for further progress in the field of in vivo opioid receptor imaging.

The ancient subclasses of Arabidopsis ACTIN DEPOLYMERIZING FACTOR genes exhibit novel and differential expression

The Actin Depolymerizing Factor (ADF) gene family of Arabidopsis thaliana encodes 11 functional protein isovariants in four ancient subclasses. We report the characterization of the tissue-specific and developmental expression of all Arabidopsis ADF genes and the subcellular localization of several protein isovariants. The four subclasses exhibited distinct expression patterns as examined by qRT-PCR and histochemical assays of a GUS reporter gene under the control of individual ADF regulatory sequences. Subclass I ADFs were expressed strongly and constitutively in all vegetative and reproductive tissues except pollen. Subclass II ADFs were expressed specifically in mature pollen and pollen tubes or root epidermal trichoblast cells and root hairs, and these patterns evolved from an ancient dual expression pattern comprised of both polar tip growth cell types, still observed in the monocot Oryza sativa. Subclass III ADFs were expressed weakly in vegetative tissues, but were strongest in fast growing and/or differentiating cells including callus, emerging leaves, and meristem regions. The single subclass IV ADF was constitutively expressed at moderate levels in all tissues, including pollen. Immunocytochemical analysis with subclass-specific monoclonal antibodies demonstrated that subclass I isovariants localize to both the cytoplasm and the nucleus of leaf cells, while subclass II isovariants predominantly localize to the cytoplasm at the tip region of elongating root hairs and pollen tubes. The distinct expression patterns of the ADF subclasses support a model of ADF s co-evolving with the ancient and divergent actin isovariants.

Dose-effect study on the antioxidant properties of leaves and outer bracts of extracts obtained from Violetto di Toscana artichoke

Artichoke ( Cynara scolymus L.) is an edible vegetable largely used in the Mediterranean diet and in folk medicine. The present paper discusses the analysis of the polyphenol content of leaves and outer bracts of Violetto di Toscana artichoke using different extraction procedures with the aim of establishing a correlation between polyphenol subclasses and antioxidant activity measured on human LDL oxidized by copper ions. HPLC/DAD and HPLC/MS analyses revealed that both the matrixes contain identical polyphenol subclasses, with mainly quantitative differences. The antioxidant effect of four artichoke extracts decreases in the following order when the sum of total phenolic compounds was considered: ethanolic extract from leaves (IC 50 = 2.92 ± 0.46 μM); ethanolic extract from outer bracts (IC 50 = 4.04 ± 0.21 μM); ethyl acetate extract from leaves (IC 50 = 4.91 ± 0.11 μM); ethyl acetate extract from outer bracts (IC 50 = 10.18 ± 1.6 μM). IC 50 were also calculated considering the concentrations of single polyphenol subclasses. In both cases, the potency of antioxidant properties was not related to the amount of total polyphenols or the single subclasses.

Étude de la fonctionnalité des sous classes d'IgG

Children who have recurrent infections require laboratory investigation for eventual immunodeficiency. We measured specific IgG antibodies against tetanus toxoid, diphteria toxoid, pneumococcal capsular polysaccharide and Haemophilus influenzae b in serum of 70 children without pulmonary symptoms. We compared to the study of humoral immune function in 128 children with obstructive bronchopneumopathies.We observed that functional antibody deficiency may be present despite normal IgG subclass levels and conversely deficiency levels of a single subclass of IgG may be found in individuals who have effective specific antibody production and are clinically normal. Measurement of specific antibody response to bacterial antigens and the identification of children with selective antibody deficiency are important for diagnostic and therapeutic management.

A New No-Lyse, No-Wash Flow-Cytometric Method for the Determination of CD4 T Cells in Blood Samples

Background: Commonly used flow-cytometric methods for immunophenotyping are based on erythrocyte lysing reagents. It is known from the literature that these reagents result in a significant loss of leucocytes caused by membrane destruction. Although the dual-platform method should compensate this phenomenon, the subset-specific individual differences in sensitivity to lysing reagents lead to incorrect values. In order to overcome this problem we introduce a no-lyse, no-wash procedure in combination with absolute true volumetric counting (TVC) for the enumeration of CD4 T cells. Material and Methods: Whole-blood samples of 50 blood donors and 20 samples of patients with acquired immunodeficiency syndrome (AIDS) were treated with both a lyse, no-wash and a nolyse, no-wash procedure. Then, CD4 T cells were counted with a TVC flow cytometer (CyFlow Counter). 30 blood samples of blood donors were treated with a lyse and wash protocol and measured by the CyFlow Counter and FACS-Calibur system (reference method). A new gating strategy was used for the data analysis for both, the lyse and no-lyse method and compared to the traditional gating procedure. Results: The TVC method showed a good reproducibility for both, the lyse (CV 1.9%) and the no-lyse procedure (CV 1.5%). CD4 counts measured by the no-lyse procedure are on average 10% higher than by using the lysing protocol. The comparison of the CyFlow Counter and FACS-Calibur results showed a correlation of r = 0.961. The simplified gating strategy shows a good correlation to the traditional gating procedure (r = 0.998). Conclusion: Erythrocyte lysing procedures cause substantial cell loss with individual values for every single subclass and patient. Therefore, the use of a no-lyse procedure is recommended. The new gating strategy is fully comparable to the traditional one and simplifies enumeration of CD4 T cells. Using the no-lyse procedure in combination with the new gating strategy, it is possible to reduce the costs per sample from EUR 30.– to below EUR 2.–.

Constrained modeling of spin-labeled major coat protein mutants from M13 bacteriophage in a phospholipid bilayer.

The family of three-dimensional molecular structures of the major coat protein from the M13 bacteriophage, which was determined in detergent micelles by NMR methods, has been analyzed by constrained geometry optimization in a phospholipid environment. A single-layer solvation shell of dioleoyl phosphatidylcholine lipids was built around the protein, after replacing single residues by cysteines with a covalently attached maleimide spin label. Both the residues substituted and the phospholipid were chosen for comparison with site-directed spin labeling EPR measurements of distance and local mobility made previously on membranous assemblies of the M13 coat protein purified from viable mutants. The main criteria for identifying promising candidate structures, out of the 300 single-residue mutant models generated for the membranous state, were 1) lack of steric conflicts with the phospholipid bilayer, 2) good match of the positions of spin-labeled residues along the membrane normal with EPR measurements, and 3) a good match between the sequence profiles of local rotational freedom and a structural restriction parameter for the spin-labeled residues obtained from the model. A single subclass of structure has been identified that best satisfies these criteria simultaneously. The model presented here is useful for the interpretation of future experimental data on membranous M13 coat protein systems. It is also a good starting point for full-scale molecular dynamics simulations and for the design of further site-specific spectroscopic experiments.