Magnetospherically driven optical and radio aurorae at the end of the stellar main sequence.

Aurorae are detected from all the magnetized planets in our Solar System, including Earth. They are powered by magnetospheric current systems that lead to the precipitation of energetic electrons into the high-latitude regions of the upper atmosphere. In the case of the gas-giant planets, these aurorae include highly polarized radio emission at kilohertz and megahertz frequencies produced by the precipitating electrons, as well as continuum and line emission in the infrared, optical, ultraviolet and X-ray parts of the spectrum, associated with the collisional excitation and heating of the hydrogen-dominated atmosphere. Here we report simultaneous radio and optical spectroscopic observations of an object at the end of the stellar main sequence, located right at the boundary between stars and brown dwarfs, from which we have detected radio and optical auroral emissions both powered by magnetospheric currents. Whereas the magnetic activity of stars like our Sun is powered by processes that occur in their lower atmospheres, these aurorae are powered by processes originating much further out in the magnetosphere of the dwarf star that couple energy into the lower atmosphere. The dissipated power is at least four orders of magnitude larger than what is produced in the Jovian magnetosphere, revealing aurorae to be a potentially ubiquitous signature of large-scale magnetospheres that can scale to luminosities far greater than those observed in our Solar System. These magnetospheric current systems may also play a part in powering some of the weather phenomena reported on brown dwarfs.

Economic burden of obesity and four obesity-related chronic diseases in rural Yunnan Province, China.

OBJECTIVE: To analyze the economic burden of obesity and four obesity-related chronic diseases in rural Yunnan Province, China. STUDY DESIGN: A community-based, cross-sectional study was conducted among rural residents in Yunnan Province. A multistage stratified random sampling approach was applied to collect a sample of the population aged ≥35 years in this region. METHODS: Questionnaires were conducted and measurements were taken from 5040 participants. A two-step model was used to measure direct economic burden of disease, whereas a human capital approach was applied to measure indirect economic burden. RESULTS: The prevalence of general obesity, central obesity, hypertension, diabetes, coronary heart disease, and stroke was 7.1%, 37.0%, 35.3%, 9.9%, 3.8%, and 1.7%, respectively, while obese participants as expected had a higher risk of the aforementioned four obesity-related illnesses than their counterparts (P < 0.01). The total, direct, and indirect costs of the four illnesses were $30,350.8 million, $28,642.5 million, and $1708.3 million, respectively, with 12.7% attributable to general obesity and 28.7% attributable to central obesity. CONCLUSIONS: The economic burden of the four studied chronic diseases attributable to obesity in rural Yunnan Province is substantial. Interventions for controlling obesity should be applied to prevent obesity-related diseases and reduce the economic burden of disease.

Structure and function of neonatal social communication in a genetic mouse model of autism.

A critical step toward understanding autism spectrum disorder (ASD) is to identify both genetic and environmental risk factors. A number of rare copy number variants (CNVs) have emerged as robust genetic risk factors for ASD, but not all CNV carriers exhibit ASD and the severity of ASD symptoms varies among CNV carriers. Although evidence exists that various environmental factors modulate symptomatic severity, the precise mechanisms by which these factors determine the ultimate severity of ASD are still poorly understood. Here, using a mouse heterozygous for Tbx1 (a gene encoded in 22q11.2 CNV), we demonstrate that a genetically triggered neonatal phenotype in vocalization generates a negative environmental loop in pup-mother social communication. Wild-type pups used individually diverse sequences of simple and complicated call types, but heterozygous pups used individually invariable call sequences with less complicated call types. When played back, representative wild-type call sequences elicited maternal approach, but heterozygous call sequences were ineffective. When the representative wild-type call sequences were randomized, they were ineffective in eliciting vigorous maternal approach behavior. These data demonstrate that an ASD risk gene alters the neonatal call sequence of its carriers and this pup phenotype in turn diminishes maternal care through atypical social communication. Thus, an ASD risk gene induces, through atypical neonatal call sequences, less than optimal maternal care as a negative neonatal environmental factor.

Economic burden and cost determinants of coronary heart disease in rural southwest China: a multilevel analysis.

OBJECTIVES: To estimate the economic burden of coronary heart disease (CHD) in a given year (2010), including direct and indirect costs, and examine the impact of contextual and individual socio-economic (SES) predictors on the costs of CHD among adults in rural southwest China. STUDY DESIGN: Cross-sectional community survey. METHODS: In total, 4595 adults (aged ≥18 years) participated in this study. A prevalence-based cost-of-illness approach was used to estimate the economic burden of CHD. Information on demographic characteristics of the study population and the economic consequences of CHD was obtained using a standard questionnaire. Multilevel linear regression was used to model the variation in costs of CHD. RESULTS: In the study population, the overall prevalence of CHD was 2.9% (3.5% for males, 2.3% for females). The total cost of CHD was estimated to be US$17 million. Inpatient hospitalizations represented the main component of direct costs of CHD, and direct costs accounted for the greatest proportion of the economic burden of CHD. Males were more likely to have a higher economic burden of CHD than females. A positive association was found between the individual's level of education and the economic burden of CHD. Residence in a higher-income community was associated with higher costs related to CHD. CONCLUSIONS: This study found that both contextual and individual SES were closely associated with the costs of CHD. Future strategies for CHD interventions and improved access to affordable medications to treat and control CHD should focus on less-educated individuals and communities with lower SES.

Trends in the Prevalence of Chronic Non-Communicable Diseases and Multimorbidity across Socioeconomic Gradients in Rural Southwest China.

OBJECTIVES: This study aimed to determine the changing prevalence of five chronic non-communicable diseases (NCDs)- hypertension, coronary heart disease (CHD), stroke, chronic obstructive pulmonary disease (COPD), and asthma-- and its multimorbidity (refers to the co-existence of two or more chronic diseases in an individual) across socioeconomic spectra in rural southwest China. MEASUREMENTS: Two cross-sectional health interviews and examination surveys were conducted among individuals aged ≥35 years in rural China. An individual socioeconomic position (SEP) index was constructed using principal component analysis. Anthropometric measurements, blood pressure, and post-bronchodilator spirometry tests were recorded for each participant. RESULTS: The mean age and proportion of men was 56.1 years and 48.4% in 2011, while was 56.6 years and 49.4% in 2021. From 2011 to 2021, the overall prevalence of hypertension, stroke and COPD increased from 26.1%, 1.1%, and 8.7% to 40.4%, 2.4%, and 12.8%, respectively (P < 0.01), while prevalence of CHD (2.1% vs. 2.2%) and asthma (1.4% vs. 1.5%) did not differ between the two study years (P > 0.05). The prevalence of NCDs multimorbidity increased from 2.3% to 9.7%, and was also observed among subgroups categorized by sex, age, ethnicity, level of education, income, and SEP (P < 0.01). In addition, the relative increases in the prevalence of multimorbidity were greater among men, old individuals, ethnic minorities, and those with low level of education and low SEP. Both in 2011 and 2021, ethnic minorities and individuals with lower level of education and low SEP had a higher prevalence of multimorbidity of the five studied chronic NCDs than their counterparts (P <0.01). CONCLUSIONS: The prevalence of NCDs multimorbidity increased substantially across all socioeconomic gradients in rural southwest China. Future interventions to further manage NCDs and their multimorbidity must be tailored to address socioeconomic factors.

Tooth Loss and Nutritional Status in Older Adults: A Systematic Review and Meta-analysis.

BACKGROUND/OBJECTIVE: Older adults are at risk for tooth loss and compromised nutritional status. Our objective was to conduct a systematic review and meta-analysis to answer the following question: Among adults aged ≥60 y living in developed countries, what are the associations between tooth loss and nutritional status as assessed by a validated nutrition screening or assessment tool? METHODS: PRISMA guidelines were followed. PubMed, Scopus, CINAHL, Web of Science, and MEDLINE were searched for studies published in English between 2009 and 2019 that met inclusion criteria. Data extracted included study and participant characteristics, dentition, and nutritional status. Risk of bias was assessed with a modified Newcastle-Ottawa Scale. Random effects meta-analysis was used. RESULTS: Of the 588 unduplicated articles identified, 78 were reviewed in full text, and 7 met inclusion criteria. Six studies were combined for a meta-analysis, which revealed that individuals who were completely edentulous or who lacked functional dentition had a 21% increased likelihood of being at risk of malnutrition or being malnourished, as compared with those who were dentulous or had functionally adequate dentition (risk ratio, 1.21; 95% CI, 1.11 to 1.32; I2 = 70%). Whether the article statistically adjusted for medical history explained most of the heterogeneity in the pooled effect. CONCLUSIONS AND IMPLICATIONS: Findings suggest that older adults with tooth loss are at greater risk of malnutrition than those with functionally adequate dentition. Use of validated tools to assess risk of malnutrition in older adults with tooth loss is important to promote early intervention and referral to optimize nutrition and oral health status. Findings were limited by heterogeneity, risk of bias, and overall quality of the studies reviewed. Cohort studies that adjust for known confounders and use consistent approaches to assess tooth loss and nutritional status are needed. KNOWLEDGE TRANSFER STATEMENT: The results of this study suggest that older adults with tooth loss are at greater risk of malnutrition than those with functionally adequate dentition. Screening of this population for malnutrition by health care professionals, including dentists and dietitians, may result in corresponding referrals to optimize nutrition and oral health status. Further research is needed with consistent approaches to assess tooth loss and nutritional status.

Differential predictions about future negative events in seasonal and non-seasonal depression.

BACKGROUND: Previous research indicates that individuals with seasonal depression (SD) do not exhibit the memory biases for negative self-referent information that characterize non-seasonal depression (NSD). The current study extended this work by examining processing of self-referent emotional information concerning potential future events in SD. METHOD: SD and NSD patients, along with never-depressed controls, completed a scenario-based measure of likelihood estimation for future positive and negative events happening either to the self or to another person. RESULTS: SD patients estimated future negative events as more likely to happen to both the self and others, relative to controls. In contrast, in the NSD sample this bias was specific to self-referred material. There were no group differences for positive events. CONCLUSIONS: These data provide further evidence that the self-referent bias for processing negative information that characterizes NSD can be absent in SD, this time in the domain of future event processing.

Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins.

Treatment of platelets with thrombin was shown previously to induce rapid changes in tyrosine phosphorylation of several platelet proteins. In this report, we demonstrate that a variety of agonists which induce platelet aggregation also stimulate tyrosine phosphorylation of three proteins with apparent molecular masses of 84, 95, and 97 kD. Since platelet aggregation requires the agonist-induced activation of an integrin receptor (GP IIb-IIIa) as well as the binding of fibrinogen to this receptor, we examined the relationship between tyrosine phosphorylation and the function of GP IIb-IIIa. When platelets were examined under conditions that either precluded the activation of GP IIb-IIIa (prior disruption of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (addition of RGDS or an inhibitory mAb), tyrosine phosphorylation of the 84-, 95-, and 97-kD proteins was not observed. However, although both GP IIb-IIIa activation and fibrinogen binding were necessary for tyrosine phosphorylation, they were not sufficient since phosphorylation was observed only under conditions in which the activated platelets were stirred and allowed to aggregate. In contrast, tyrosine phosphorylation was not dependent on another major platelet response, dense granule secretion. Furthermore, granule secretion did not require tyrosine phosphorylation of this set of proteins. These experiments demonstrate that agonist-induced tyrosine phosphorylation is linked to the process of GP IIb-IIIa-mediated platelet aggregation. Thus, tyrosine phosphorylation may be required for events associated with platelet aggregation or for events that follow aggregation.

AIR-2: An Aurora/Ipl1-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos.

An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms. Unlike other Aurora/Ipl1-related kinases, the Caenorhabditis elegans orthologue, AIR-2, is associated with meiotic and mitotic chromosomes. AIR-2 is initially localized to the chromosomes of the most mature prophase I-arrested oocyte residing next to the spermatheca. This localization is dependent on the presence of sperm in the spermatheca. After fertilization, AIR-2 remains associated with chromosomes during each meiotic division. However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes. AIR-2 also remains associated with both extruded polar bodies. In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant. Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis. The embryos accumulate large amounts of DNA and microtubule asters. Polar bodies are not extruded, but remain in the embryo where they continue to replicate. The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete. Additionally, staining with a marker of midbody microtubules revealed that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity. Our results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis.

Metaphase to anaphase (mat) transition-defective mutants in Caenorhabditis elegans.

The metaphase to anaphase transition is a critical stage of the eukaryotic cell cycle, and, thus, it is highly regulated. Errors during this transition can lead to chromosome segregation defects and death of the organism. In genetic screens for temperature-sensitive maternal effect embryonic lethal (Mel) mutants, we have identified 32 mutants in the nematode Caenorhabditis elegans in which fertilized embryos arrest as one-cell embryos. In these mutant embryos, the oocyte chromosomes arrest in metaphase of meiosis I without transitioning to anaphase or producing polar bodies. An additional block in M phase exit is evidenced by the failure to form pronuclei and the persistence of phosphohistone H3 and MPM-2 antibody staining. Spermatocyte meiosis is also perturbed; primary spermatocytes arrest in metaphase of meiosis I and fail to produce secondary spermatocytes. Analogous mitotic defects cause M phase delays in mitotic germline proliferation. We have named this class of mutants "mat" for metaphase to anaphase transition defective. These mutants, representing six different complementation groups, all map near genes that encode subunits of the anaphase promoting complex or cyclosome, and, here, we show that one of the genes, emb-27, encodes the C. elegans CDC16 ortholog.

Cytoplasmic flow and the establishment of polarity in C. elegans 1-cell embryos.

Early Caenorhabditis elegans embryos provide an excellent model for the study of developmental processes. Development can be studied by direct observation under the light microscope and can be perturbed using laser manipulations, drug inhibitor treatments, and genetic mutants. The first division of the C. elegans embryo is asymmetric, generating two daughter cells unequal in size and developmental fate. These distinct fates are generated by the partitioning of cytoplasmic determinants during the first mitotic cell cycle. Partitioning of these determinants is thought to be driven by cytoplasmic flow. Recent studies in C. elegans in the past year have identified a number of components necessary for this flow, giving us a clearer picture of the molecular mechanisms underlying developmental asymmetry.

Socioeconomic differences in prevalence, awareness, control and self-management of hypertension among four minority ethnic groups, Na Xi, Li Shu, Dai and Jing Po, in rural southwest China.

This study investigates socioeconomic differences in prevalence, awareness, control and self-management of hypertension in rural China. A cross-sectional survey was conducted among four ethnic minority groups in Yunnan Province: Na Xi, Li Shu, Dai and Jing Po. Approximately 5532 consenting individuals aged ⩾35 years (48.4% of whom were male) were selected to participate in the study using a stratified, multistage sampling technique. Information about participants' demographic characteristics and hypertension awareness, treatment, control and self-management practices was obtained using a standard questionnaire. The age-standardised prevalence of hypertension in the study population was 33.6%. In hypertensive subjects, the overall levels of awareness, treatment and control of hypertension were 42.1%, 28.5% and 6.7%, respectively. Approximately 58.7% of hypertensive patients regularly self-monitored blood pressure (BP), 64.7% adhered to their physician-prescribed anti-hypertensive drugs, and 88.0% took at least one measure to control BP. Hypertensive patients of Jing Po ethnicity had the lowest rates of awareness, treatment, control and self-management of hypertension among the four ethnic minority groups studied. Individuals with lower levels of education were more likely to be hypertensive. Further, individuals with lower levels of education had a lower probability of awareness of their hypertensive status and of treatment with antihypertensive medication. Access to medical services was positively associated with awareness of suffering from hypertension, being treated with antihypertensive medication, and compliance with antihypertensive drug treatment. This study suggests that effective strategies to enhance awareness, treatment and management of hypertension should focus on individuals with low levels of education and poor access to medical services.

A Caenorhabditis elegans wee1 homolog is expressed in a temporally and spatially restricted pattern during embryonic development.

A wee1 homolog, wee-1.1, is expressed in both a temporally and spatially restricted pattern during early Caenorhabditis elegans embryogenesis, and is undetectable throughout the remainder of embryogenesis. The wee-1.1 message appears to be zygotically expressed in the somatic founder cell E of the 12-cell embryo. This expression disappears when the E blastomere divides for the first time. The wee-1.1 message then appears transiently in the nuclei of the eight great-granddaughter cells of the AB somatic founder cell, just before these cells divide in the 16-cell embryo. Following this division, the wee-1.1 mRNA is no longer detectable throughout the remainder of embryogenesis. The expression of wee-1.1 in the E blastomere and in the AB progeny appears to be restricted to nuclei in prophase and metaphase of the cell cycle. Analysis of the wee-1.1 mRNA expression pattern in maternal-effect lethal mutants suggests that this expression pattern is restricted to cells of the E and AB fates in the early embryo. This mRNA expression pattern is restricted to a 10-15-min span of embryonic development and may be regulating the timing of crucial cell divisions at this early stage of development.

Molecular analyses of HIV-1 group O and HIV-2 variants from Africa.

Genetic variation among HIV isolates creates challenges for their detection by serologic and genetic techniques. To characterize the sequence variation and its correlation to serologic diversity of HIV-1 Group O and HIV-2 isolates, samples were identified by differential reactivity in selected commercial and research assays. Analysis of sera from Equatorial Guinea (EG) led to identification of 4 HIV-1 Group O variants. Viral RNA, extracted from these samples was used to PCR amplify overlapping sequences of the entire envelope gene using multiple primer pairs. Sequence analysis indicated that the V3 loop nucleotide and protein sequences aligned more closely with HIVANT70 compared to other Group O sequences. The amino acid sequences at the octameric tip of the V3 loop were RIGPLAWY, RIGPMAWY, or GLGPLAVY. The tetrameric tip GPLA is represented only once in the published 1994 HIV database (Los Alamos) but was present in 2 of 4 of EG samples. The immuno-dominant region (IDR) sequences derived from EG sera were unique in that none of the sequences were completely homologous to other HIV-1 group O variants. Further, the HIV-1 group O sequence variation could be correlated with differential serologic reactivity using IDR peptides. Compared to HIV-1, the sequence information on HIV-2 isolates is relatively limited, though the HIV-2 isolates also show genetic variation similar to HIV-1. To further establish a correlation between the genetic diversity and serologic detection of HIV-2, plasma samples from Western Africa were evaluated. Eight samples were selected based on weak serologic reactivity to env proteins. PCR amplification and sequence analysis of the gag, env V3 loop, and env IDR regions indicated that the samples could be classified as subtypes A (4 samples), B (3 samples) and D (1 sample). Across the subtypes, there was conservation in the IDR region of the sequence WGCAFRQVCHT. This region is absolutely conserved among the majority of currently known HIV-2 and related SIV viruses (1994 HIV database). One subtype B sample had a unique sequence immediately adjacent to the IDR, however, this did not change the serologic detection using a HIV-2 IDR specific monoclonal antibody.

Serologic and phylogenetic characterization of HIV-1 subtypes in Uganda.

OBJECTIVES: To determine the HIV genetic subtypes present in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate serologic detection of infection by commercial immunoassays; to evaluate samples for HIV-1 group O infections. METHODS: Sixty-four HIV-seropositive plasma samples were collected from the Nakasero Blood Bank, Kampala, Uganda. The plasma were evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. HIV-1 group M and O infections were identified on the basis of discordant seroreactivity in EIA and reactivity to group M and O antigens on the immunoblot. Regions of gag p24 and env gp41 were amplified using reverse transcriptase polymerase chain reaction, and genetic subtypes were determined by phylogenetic analysis. RESULTS: Serologic testing confirmed that 63 out of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of HIV-1 group O infections. Genetic subtyping determined that 25 samples were subtype A, three subtype C, 22 subtype D, and nine were heterogeneous for subtypes A and D. CONCLUSIONS: Despite the sequence variation observed in Uganda, commercial EIA based on HIV-1 subtype B proteins detected all the infections. In contrast, a peptide-based assay failed to detect three infections by subtype D viruses. This emphasizes the negative impact of HIV genetic variation on assays that rely on peptides to detect HIV infections. The number of infections with heterogeneous subtype (due to mixed infections or recombinant viruses) is high and reflects the growing complexity of the HIV epidemic in endemic regions where multiple subtypes are present in the population.

PIP: Extensive sequence heterogeneity between HIV-1 isolates has led to the classification of HIV-1 into group M (major) subtypes A-J, and group O (outlier). Some isolates have also been found to be the result of recombination between different group M subtypes. Findings are reported from a study conducted to determine the various HIV genetic subtypes in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate the serologic detection of infection by commercial immunoassays. 64 HIV-seropositive plasma samples were collected from the Nakasero Blood Bank in Kampala and evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. 63 of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of group O infections. According to phylogenetic analysis, 25 samples were subtype A, 3 subtype C, 22 subtype D, and 9 heterogenous for subtypes A and D. Despite the sequence variation observed in this study population, commercial EIA based upon HIV-1 subtype B proteins detected all of the infections. A peptide-based assay failed to detect 3 infections by subtype D viruses.

Epitope mapping of anti-HIV and anti-HCV monoclonal antibodies and characterization of epitope mimics using a filamentous phage peptide library.

A large filamentous phage library (1 x 10(9) clones) displaying random 30-amino-acid (aa) sequences on the N terminus of the pIII coat protein was constructed and characterized. Clones in the library were affinity selected for binding to monoclonal antibodies (mAb) against two viral antigens, the HIV gp120 protein and the HCV core protein. The obtained aa sequences precisely identified the epitopes recognized by the mAb. Binding of peptide-carrying phages to the Ab was demonstrated by ELISA, Western blot and the surface plasmon resonance (SPR) method. The mAb-specific peptides were transferred via genetic techniques onto the N terminus of Escherichia coli alkaline phosphatase (AP). When fused to the enzyme, the peptides maintained their ability to bind their respective mAb, indicating that the peptides contained the necessary contact residues for binding. The affinity of the peptides was estimated to be 100 nM by SPR. A comparison is presented of the relative affinities of phage-derived peptides to the native viral epitopes also displayed on the AP scaffold. The approach of transferring epitopes from phage to AP for further evaluation should be applicable to many other mAb or receptors.

The four cdc25 genes from the nematode Caenorhabditis elegans.

During eukaryotic evolution, multicellular organisms have evolved multiple members of gene families that may display unique, partially overlapping, or redundant functions during development. More than 75% of the C. elegans genome has been sequenced, which represents approximately 95% of the coding sequences. This provides a unique opportunity to identify most, if not all, of the members of a given gene family. We have searched the C. elegans genome database for members of a key family of cell cycle regulators, the CDC25 phosphatases, and have identified four genes. The four C. elegans genes represent a larger family within a single organism than has been reported so far in Drosophila, mice and humans. An amino acid comparison revealed a high degree of similarity and identity within the phosphatase domain. This analysis also identified an expanded consensus sequence that can be used to discover new members of the CDC25 phosphatase family. However, the four C. elegans sequences display a few novel amino acid substitutions in the residues surrounding the invariant catalytic motif CX5R. These data demonstrate the value of genome database searching for identifying new members of known gene families, understanding genetic diversity, and for studying gene structure.

High-level production of a secreted, heterodimeric alpha beta murine T-cell receptor in Escherichia coli.

For structural studies, high-level production of properly folded, disulfide-linked, unglycosylated protein in E. coli is an attractive alternative to production in eukaryotic systems. We describe here the production of heterodimeric, murine D10 T-cell receptor (sD10TCR) in E. coli as a secreted leucine zipper (LZ) fusion protein. Two genes, one (alpha-acid) encoding the alpha-chain variable and constant domains (V alpha and C alpha) of D10 TCR fused to an LZ 'acid' encoding sequence and the other (beta-base) encoding the beta-chain variable and constant domains (V beta and C beta) fused to an LZ 'base' encoding sequence, were co-expressed from a bacteriophage T7 promoter as a dicistronic message. Secreted alpha-acid and beta-base proteins formed proper inter- and intra-chain disulfide bonds in the periplasm, bypassing the need for in vitro protein refolding. Complementary LZ sequences facilitated the formation of alpha beta heterodimers. sD10TCR-LZ was purified by affinity chromotography using a D10 TCR clonotype-specific monoclonal antibody (mAb 3D3). Typical yields of purified protein were 4-5 mg/l of culture. Purified sD10TCR-LZ was reactive with a panel of conformationally sensitive TCR-specific monoclonal antibodies, consistent with its conformational integrity and appeared to be suitable for structural studies by X-ray crystallography or NMR spectroscopy.

Analysis of recombinant protein expression by MALDI-TOF mass spectrometry of bacterial colonies.

E. coli expressing soluble recombinant HIV antigens were analyzed directly by MALDI-TOF mass spectrometry (MS) from bacterial colonies picked from agar plates. An HIV envelope (ENV) antigen construct, penvA, was expressed in E. coli by transformation of the plasmid pPL/penvA-M. The plasmid was co-transformed into E. coli DH5 alpha cells with an equal quantity of the plasmid pKRR826, the parent vector without the penvA insert, and plated at medium density on L-agar plus ampicillin plates. A total of 24 colonies from four agar plates (six colonies per plate) were picked and transferred into 50% acetonitrile--0.1% trifluoroacetic acid aliquots for analysis by MALDI-TOF MS. The MS analysis detected 10 of 24 colonies expressing the recombinant protein; one colony expressed a mutant penvA protein; eleven of 24 colonies showed ions only from E. coli; and two of 24 colonies showed no detectable proteins. When E. coli transformed only with plasmid pPL/penvA-M were examined, all (10 of 10) colonies showed the penv insert by the MALDI-TOF MS method. The method is fast (less than 1.5 h for 24 colonies) and allows identification of colonies expressing intact or mutant proteins directly from culture plates without sample purification.