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PubMed2001

Identification of virulence mutants of the fungal pathogen Cryptococcus neoformans using signature-tagged mutagenesis.

Cryptococcus neoformans var. neoformans is an important opportunistic fungal pathogen of patients whose immune system has been compromised due to viral infection, antineoplastic chemotherapy, or tissue transplantation. As many as 13% of all AIDS patients suffer a life-threatening cryptococcal infection at some time during the course of their HIV disease. To begin to understand the molecular basis for virulence in Cryptococcus neoformans var. neoformans serotype A, we have employed signature-tagged mutagenesis (STM) to identify mutants with altered virulence in a mouse model. The critical parameters of signature-tagged mutagenesis in C. neoformans are explored. Data are presented showing that at least 100 different strains can be mixed together in a single animal with each participating in the infection and that there is no apparent interaction between a virulent strain and an avirulent strain in our animal model. Using signature-tagged mutagenesis, we identified 39 mutants with significantly altered growth in a competitive assay. Molecular analyses of these mutants indicated that 19 (49%) contained an insertion in the actin promoter by homologous recombination from a single crossover event, creating a duplication of the actin promoter and the integration of single or multiple copies of the vector. Analysis of the chromosomal insertion sites of those mutants that did not have an integration event in the actin promoter revealed an approximately random distribution among the chromosomes. Individual challenge of the putative mutants in a mouse model revealed five hypovirulent mutants and one hypervirulent mutant.

Actins↗
PubMed1990

1H NMR spectrum of the native human insulin monomer. Evidence for conformational differences between the monomer and aggregated forms.

The effects of high dilution on the 1H Fourier transform NMR spectrum of native human insulin at pH* 8.0 and 9.3 have been examined at 500 MHz resolution. The dependence of the spectrum on concentration and comparison with the spectrum of a biologically highly potent monomeric insulin mutant (SerB9----Asp) establish that at 36 microM (pH* 9.3) or 18 microM (pH* 8) and no added buffer or salts, human insulin is monomeric. Under these conditions of dilution, ionic strength, and pH*, human insulin and the SerB9----Asp mutant exhibit nearly identical 1H NMR spectra. At higher concentrations (i.e. greater than 36 microM to 0.91 mM), native human insulin dimerizes, and this aggregation causes a change in insulin conformation. Although there are many changes in the spectrum, the TyrB26 ring H3,5 proton signals located at 6.63 ppm and the methyl signal located at 0.105 ppm (characteristics of monomeric insulin) are particularly distinct signatures of the conformation change that accompanies dimerization. Magnetization transfer experiments show that the 0.105 ppm methyl signal shifts downfield to a new position at 0.45 ppm. We conclude that the 0.105 ppm methyl signal is due to a conformation in which a Leu methyl group is centered over and in van der Waals contact with the ring of an aromatic side chain. Dimerization causes a conformation change that alters this interaction, thereby causing the downfield shift. Nuclear Overhauser studies indicate that the methyl group involved is located within a cluster of aromatic side chains and that the closest ring-methyl group interaction is with the ring of PheB24.

Aspartic Acid↗
PubMed2006

Tumor metabolism: new opportunities for cancer therapy.

Mammalian cells depend on extracellular input for the regulation of growth, proliferation and survival. Cancer cells evade these requirements, and are able to take up nutrients in a cell-autonomous fashion, which allows continuous growth and proliferation. To fulfill the high bioenergetic demands imposed by transformation, tumors must develop alternative mechanisms of energy production. Accordingly, the biochemical signature of cancer cells involves a shift to aerobic glycolysis, also known as the "Warburg effect". This property of cancer cells has resulted of great utility in modern medicine for detection of early tumors by positron-emission scanning. Nonetheless, the underlying mechanisms and contribution of the Warburg effect to the malignant phenotype have remained obscure. Thanks to recent advances in cancer research, we are beginning to understand the link between cancer genetics and the abnormal use of glucose by tumors. A new scenario is thus emerging, in which bioenergetics would contribute to and sustain malignant transformation. These findings are not only important for a better understanding of tumorigenesis; tumor reliance on glycolysis can be exploited in the search for novel, more potent therapeutic approaches to cancer treatment.

Aerobiosis↗
PubMed1997

The involvement of serine 175 and alanine 185 of cytochrome b of Rhodobacter sphaeroides cytochrome bc1 complex in interaction with iron-sulfur protein.

An approach involving cysteine replacement of potentially noncritical amino acid residues, followed by chemical modification studies, was used to investigate structure-function of the "cd helix" of cytochrome b from Rhodobacter sphaeroides. Three amino acid residues, Ser-155, Ser-175, and Ala-185, which span this region of cytochrome b, were selected for this study. The S155C substitution yields cells unable to support photosynthetic growth, indicating that Ser-155 is a critical amino acid residue. Further mutational studies of Ser-155 indicate that the size of the amino acid side chain at this position is critical for photosynthetic growth of R. sphaeroides. On the other hand, the S175C and A185C substitutions yield cells with photosynthetic growth rates and enzyme kinetics of the bc1 complexes very similar to those of the unmutated complex, indicating that Ser-175 and Ala-185 are noncritical residues. Thus, engineered cysteines at these two positions of cytochrome b are suitable for membrane topology and domain/subunit interaction studies. Cys-175 does not react with a sulfhydryl-modifying reagent, N-ethylmaleimide (NEM), either in sealed, inside-out chromatophores or in detergent-disrupted chromatophores, indicating that position 175 of cytochrome b is inaccessible from both sides of the membrane and is probably buried within the protein complex. Cys-185 reacts with NEM only after detergent disruption of the sealed, inside-out chromatophores, indicating that this position of cytochrome b is accessible on the outer (periplasmic) surface of the membrane. These results place the cd helix of cytochrome b on the periplasmic side of the chromatophore membrane. When purified A185C-substituted bc1 complex was treated with NEM, about 87% of the activity was abolished due to NEM modification of Cys-185. The signature of the Rieske iron-sulfur center is broadened upon NEM modification of A185C, with the gx signal shifting from g = 1.80 to g = 1.75, suggesting that Ala-185 of cytochrome b interacts with the iron-sulfur protein. When purified S175C-substituted bc1 complex is treated with NEM, no change in the activity is observed, since Cys-175 is inaccessible to NEM. However, when the iron-sulfur protein is removed from the S175C-substituted bc1 complex, Cys-175 becomes accessible to NEM, indicating that Ser-175 of cytochrome b is shielded by the iron-sulfur protein in the bc1 complex.

Alanine↗
PubMed2002

Characterisation of BRH1, a brassinosteroid-responsive RING-H2 gene from Arabidopsis thaliana.

Although many important aspects of plant development are controlled by brassinosteroids (BRs), the early molecular events of their hormonal action are largely unknown. Using a differential-display RT-PCR screen designed to detect early response transcripts, those regulated by BR treatment in the absence of de novo protein synthesis, we identified an Arabidopsis thaliana (L.) Heynh. gene (designated BRH1) that encodes a novel RING finger protein. As deduced from a complete cDNA clone, the 170-amino-acid sequence of BRH1 forms an N-terminal hydrophobic domain and a C-terminal RING-H2 signature. In wild-type Arabidopsis, the level of the BRH1 transcript was rapidly down-regulated by brassinolide, but this effect was abolished in a BR-insensitive mutant deficient in the BRI1 receptor. BRH1 mRNA abundance was not influenced by other phytohormones, but the pathogen elicitor chitin induced a rapid and transient accumulation of the transcript. Antisense expression of BRH1 resulted in transgenic Arabidopsis plants with thicker inflorescence stems and altered leaf morphology, whereas in sense overexpression lines no phenotypic effect could be observed. Considering the potential of the RING proteins to participate in regulatory protein complexes, BR-dependent expression of BRH1 may suggest its involvement in later hormonal effects.

Amino Acid Sequence↗
PubMed2006

Genetic and phylogenetic analysis of multi-continent human influenza A(H1N2) reassortant viruses isolated in 2001 through 2003.

Genetic analyses were performed on 228 influenza A(H1) viruses derived from clinical subjects participating in an experimental vaccine trial conducted in 20 countries on four continents between 2001 and 2003. HA1 phylogenetic analysis of these viruses showed multiple clades circulated around the world with regional prevalence patterns. Sixty-five of the A(H1) viruses were identified as A(H1N2), 40 of which were isolated from South Africa. The A(H1) sequences of these viruses cluster with published H1N2 viruses phylogenetically and share with them diagnostic signature V169A and A193T changes. The results also showed for the first time that H1N2 viruses were prominent in South Africa during the 2001-2002 influenza season, accounting for over 90% of the A(H1) cases in our study, and infecting both children (29/31) and the elderly (11/13). Phylogenetic analysis of the 65 H1N2 viruses we identified, in conjunction with the 56 recent H1N2 viruses currently available in the database, provided a comprehensive view of the circulation and evolution of distinct clades of H1N2 viruses in a temporal manner between early 2001 and mid-2003, shortly after the appearance of these recent reassortant viruses in or near year 2000.

Age Factors↗
PubMed2004

The channel architecture of aquaporin 0 at a 2.2-A resolution.

We determined the x-ray structure of bovine aquaporin 0 (AQP0) to a resolution of 2.2 A. The structure of this eukaryotic, integral membrane protein suggests that the selectivity of AQP0 for water transport is based on the identity and location of signature amino acid residues that are hallmarks of the water-selective arm of the AQP family of proteins. Furthermore, the channel lumen is narrowed only by two, quasi-2-fold related tyrosine side chains that might account for reduced water conductance relative to other AQPs. The channel is functionally open to the passage of water because there are eight discreet water molecules within the channel. Comparison of this structure with the recent electron-diffraction structure of the junctional form of sheep AQP0 at pH 6.0 that was interpreted as closed shows no global change in the structure of AQP0 and only small changes in side-chain positions. We observed no structural change to the channel or the molecule as a whole at pH 10, which could be interpreted as the postulated pH-gating mechanism of AQP0-mediated water transport at pH >6.5. Contrary to the electron-diffraction structure, the comparison shows no evidence of channel gating induced by association of the extracellular domains of AQP0 at pH 6.0. Our structure aids the analysis of the interaction of the extracellular domains and the possibility of a cell-cell adhesion role for AQP0. In addition, our structure illustrates the basis for formation of certain types of cataracts that are the result of mutations.

Amino Acid Sequence↗
PubMed2006

Viral evolution in macaques coinfected with CCR5- and CXCR4-tropic SHIVs in the presence or absence of vaccine-elicited anti-CCR5 SHIV neutralizing antibodies.

Macaques were immunized with SF162 Env-based gp140 immunogens and challenged simultaneously with the CCR5-tropic homologous SHIV(SF162P4) and the CXCR4-tropic heterologous SHIV(SF33A) viruses. Both mock-immunized and immunized animals became dually infected. Prior immunization preferentially reduced the viral replication of the homologous virus during primary infection but the relative replication of the two coinfecting viruses during chronic infection was unaffected by prior immunization, despite the fact that five of six immunized animals maintained a significantly lower overall viral replication that the control animals. Neutralizing antibodies participated in controlling the replication of SHIV(SF162P4), but not that of SHIV(SF33A). Dual infection resulted in the emergence and predominance within the circulating CCR5 virus pool, of a variant with a distinct neutralization phenotype. The signature of this variant was the presence of three amino acid changes in gp120, two of which were located in the receptor and coreceptor binding sites. Also, a significant fraction of the viruses circulating in the blood, as early as two weeks post-infection, was recombinants and prior immunization did not prevent their emergence. These findings provide new insights into the dynamic interaction of CCR5- and CXCR4-tropic HIV isolates that are potentially relevant in better understanding HIV-mediated pathogenesis.

AIDS Vaccines↗
PubMed2004

Chromosome intrachanges and interchanges detected by multicolor banding in lymphocytes: searching for clastogen signatures in the human genome.

Genomic fingerprints of mutagenic agents would have wide applications in the field of cancer biology, epidemiology and prevention. The differential spectra of chromosomal aberrations induced by different clastogens suggest that ratios of specific aberrations can be exploited as biomarkers of carcinogen exposure. We have tested this hypothesis using the novel technique of multicolor banding in situ hybridization (mBAND) in human peripheral blood lymphocytes exposed in vitro to X rays, neutrons, heavy ions, or the restriction endonuclease AluI. In the heavy-ion-irradiated cells, we further analyzed aberrations in chromosome 5 using multicolor FISH (mFISH). Contrary to the expectations of biophysical models, our results do not support the use of the ratios of inter-/intrachromosomal exchanges or intra-/interarm intrachanges as fingerprints of exposure to densely ionizing radiation. However, our data point to measurable differences in the ratio of complex/simple interchanges after exposure to different clastogens. These data should be considered in current biophysical models of radiation action in living cells.

Biomarkers↗
PubMed2006

Effects of disrupting the polyketide synthase gene WdPKS1 in Wangiella [Exophiala] dermatitidis on melanin production and resistance to killing by antifungal compounds, enzymatic degradation, and extremes in temperature.

BACKGROUND: Wangiella dermatitidis is a human pathogenic fungus that is an etiologic agent of phaeohyphomycosis. W. dermatitidis produces a black pigment that has been identified as a dihydroxynaphthalene melanin and the production of this pigment is associated with its virulence. Cell wall pigmentation in W. dermatitidis depends on the WdPKS1 gene, which encodes a polyketide synthase required for generating the key precursor for dihydroxynaphthalene melanin biosynthesis. RESULTS: We analyzed the effects of disrupting WdPKS1 on dihydroxynaphthalene melanin production and resistance to antifungal compounds. Transmission electron microscopy revealed that wdpks1Delta-1 yeast had thinner cell walls that lacked an electron-opaque layer compared to wild-type cells. However, digestion of the wdpks1Delta-1 yeast revealed small black particles that were consistent with a melanin-like compound, because they were acid-resistant, reacted with melanin-binding antibody, and demonstrated a free radical signature by electron spin resonance analysis. Despite lacking the WdPKS1 gene, the mutant yeast were capable of catalyzing the formation of melanin from L-3,4-dihyroxyphenylalanine. The wdpks1Delta-1 cells were significantly more susceptible to killing by voriconazole, amphotericin B, NP-1 [a microbicidal peptide], heat and cold, and lysing enzymes than the heavily melanized parental or complemented strains. CONCLUSION: In summary, W. dermatitidis makes WdPKS-dependent and -independent melanins, and the WdPKS1-dependent deposition of melanin in the cell wall confers protection against antifungal agents and environmental stresses. The biological role of the WdPKS-independent melanin remains unclear.

Antifungal Agents↗
PubMed2006

Combined effects of the two reciprocal t(4;11) fusion proteins MLL.AF4 and AF4.MLL confer resistance to apoptosis, cell cycling capacity and growth transformation.

The reciprocal chromosomal translocation t(4;11) is correlated with infant, childhood, adult and therapy-related high-risk acute leukemia. Here, we investigated the biological effects of MLL.AF4, AF4.MLL or the combination of both reciprocal fusion proteins in a conditional in vitro cell culture model system. Several parameters like cell growth, cell cycling capacity, apoptotic behavior and growth transformation were investigated under physiological and stress conditions. Co-transfected cells displayed the highest resistance against apoptotic triggers, cell cycling capacity and loss-of-contact inhibition. These analyses were complemented by gene expression profiling experiments and specific gene signatures were established for each of the three cell lines. Interestingly, co-transfected cells strongly upregulate the homeobox gene Nanog. In combination with Oct4, the Nanog homeoprotein is steering maintenance of pluripotency and self-renewal in embryonic stem cells. Transcription of Nanog and other stem cell factors, like Oct4 and Bmi1, was verified in biopsy material of t(4;11) patient cells which express both reciprocal t(4;11) fusion genes. In conclusion, the presence of both reciprocal MLL fusion proteins confers biological properties known from t(4;11) leukemia, suggesting that each of the two fusion proteins contribute specific properties and, in combination, also synergistic effects to the leukemic phenotype.

Animals↗
PubMed2004

Differential regulation of gene expression following CD40 activation of leukemic compared to healthy B cells.

It is possible to differentiate malignant from healthy cells and to classify diseases based on identification of specific gene expression profiles. We hypothesized that gene expression profiling could also be used to identify differential activation of healthy and malignant cells, and as a model for this, we examined the molecular sequelae of CD40 activation of healthy and B-cell chronic lymphocytic leukemia (CLL) cells. Hierarchical clustering analysis of gene expression signatures grouped samples by CD40 activation status and further subclassified CD40-activated CLL cells from healthy B cells. Supervised analyses in healthy B cells compared to CLL cells identified differential regulation of genes governing cell cycle progression and apoptosis. CD40 signaling of CLL cells increases their susceptibility to immune recognition, but promotes survival and cell cycle arrest, making these cells potentially more resistant to chemotherapy. These results illustrate the utility of gene expression profiling to elucidate the molecular sequelae of signaling in healthy cells and altered signaling pathways in malignant cells. This type of approach should be useful to identify targets of therapy of malignant diseases.

Adult↗
PubMed1999

Effectors of a developmental mitogen-activated protein kinase cascade revealed by expression signatures of signaling mutants.

Despite the importance of mitogen-activated protein kinase (MAPK) signaling in eukaryotic biology, the mechanisms by which signaling yields phenotypic changes are poorly understood. We have combined transcriptional profiling with genetics to determine how the Kss1 MAPK signaling pathway controls dimorphic development in Saccharomyces cerevisiae. This analysis identified dozens of transcripts that are regulated by the pathway, whereas previous work had identified only a single downstream target, FLO11. One of the MAPK-regulated genes is PGU1, which encodes a secreted enzyme that hydrolyzes polygalacturonic acid, a structural barrier to microbial invasion present in the natural plant substrate of S. cerevisiae. A third key transcriptional target is the G(1) cyclin gene CLN1, a morphogenetic regulator that we show to be essential for pseudohyphal growth. In contrast, the homologous CLN2 cyclin gene is dispensable for development. Thus, the Kss1 MAPK cascade programs development by coordinately modulating a cell adhesion factor, a secreted host-destroying activity, and a specialized subunit of the Cdc28 cyclin-dependent kinase.

Cyclins↗
PubMed2026

Human IL-34 Deficiency Primes Microglia Toward Alzheimer's Disease-Associated States.

BACKGROUND: Genome-wide association studies (GWAS), with independent replication in large European consortia, have identified a common nonsense variant in IL-34 (Y213X) as a genetic risk factor for late-onset Alzheimer's disease (AD). However, the biological consequences of this IL-34 mutation in humans, its prevalence in the population, and the mechanisms by which IL-34-Y213X alters microglial homeostasis, cerebrospinal fluid (CSF) proteomic networks, and amyloid pathology remain poorly understood. METHODS: We combined human genetics, cerebrospinal fluid (CSF) and serum proteomics, transcriptomics, large-scale phenome-wide association analyses, and preclinical experimental models to define the impact of human IL-34 deficiency. IL-34 concentrations were first quantified in CSF and serum from deeply phenotyped AD cohorts stratified by the common IL-34-Y213X nonsense variant. IL-34 levels and IL-34-Y213X status were then integrated with unbiased CSF proteomic networks and AD biomarkers. Transcriptomic profiling of purified microglia from IL-34 knockout mice was performed to assess disease-associated microglial programs. Using APP/PS1 mice lacking IL-34, we examined the effects of IL-34 deficiency on microglial survival, tiling, and plaque encapsulation. Finally, we performed postmortem analyses of temporal cortex from AD patients carrying IL-34-Y213X to assess microglial density, spatial organization, and plaque-associated responses. FINDINGS: IL-34-Y213X was a strong, dose-dependent loss-of-function (LOF) allele that reduced IL-34 levels by up to 2.5 standard deviations in CSF and serum and was common in multiple populations. IL-34 deficiency reshaped CSF proteomic networks, downregulating axon guidance and microglial support modules while upregulating inflammatory and extracellular matrix signatures, and showed pleiotropic associations with neurological, inflammatory, and metabolic traits. Transcriptomic analysis of sorted microglia from healthy 9-month-old IL-34KO compare to wild-type mice revealed a profound pro-inflammatory and disease-associated microglial transcriptional program enriched for disease-associated microglia (DAM) signatures, inflammatory pathways, and AD risk genes including APOE, CLU, and CASS4. In APP/PS1 mice, genetic IL-34 deletion selectively depleted homeostatic gray-matter microglia, disrupted microglial tiling, and impaired plaque encapsulation, resulting in altered amyloid structure and enhancing neuritic injury. Concordantly, AD patients homozygous for IL-34-Y213X displayed markedly reduced cortical microglial density and increased microglial spatial dispersion, indicating a breakdown of the microglial network organization in the human brain. INTERPRETATION: A common human IL-34 LOF variant creates a naturally occurring model of IL-34 deficiency that links microglial survival, CSF network signatures, and amyloid pathology in both mice and humans. Importantly, IL-34 deficiency alone is sufficient to induce inflammatory, AD-associated microglial states beyond simply reducing microglial number. These findings identify IL-34/CSF1R signaling as a critical determinant of microglial resilience and a potential upstream pathway linking human genetic variation to AD susceptibility, highlighting IL-34-dependent pathways as promising targets for disease modification. FUNDING: This work was supported by grants from the Spanish Ministerio de Ciencia, Innovación y Universidades/FEDER/UE (PID2024-157400OB-I00) and FORTALECE program (FORT23/00008; Instituto de Salud Carlos III, Spain) to RRL and JLV, ISCIII of Spain co-financed by FEDER funds (European Union) through grants PI24/00308 (JV) and CIBERNED collaborative grant 2022/01 to JV, PID2023-147125OB-I00 and CEX2023-001386-S (Severo Ochoa Programme) to SMTBC. A.R. is supported by STAR Award. University of Texas System. Tx, United States, The South Texas ADRC. National Institute of Aging. National Institutes of Health. USA. (P30AG066546), the Keith M. Orme and Pat Vigeon Orme Endowed Chair in Alzheimer's and Neurodegenerative Diseases (2024-2025) and Patricia Ruth Frederick Distinguished Chair for Precision Therapeutics in Alzheimer's and Neurodegenerative Diseases (2025-2028). AR is also supported by the Agency for Innovation and Entrepreneurship (VLAIO) grant N° PR067/21 for the HARPONE project and the ADAPTED project the EU/EFPIA Innovative Medicines Initiative Joint Undertaking Grant N° 115975 and CIBERNED (ISCIII).

Journal Article↗
PubMed2005

A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program.

Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described.

Amino Acid Sequence↗
PubMed2006

A scoring system based on the expression of six surface molecules allows the identification of three prognostic risk groups in B-cell chronic lymphocytic leukemia.

We have previously identified 12 surface antigens whose differential expression represented the signature of B-cell chronic lymphocytic leukemia (B-CLL) subsets with different prognosis. In the present study, expression data for these antigens, as determined in 137 B-CLL cases, all with survivals, were utilized to devise a comprehensive immunophenotypic scoring system of prognostic relevance for B-CLL patients. In particular, univariate z score was employed to identify the markers with greater prognostic impact, while maximally selected log-rank statistics were chosen to define the optimal cut-off points capable to split patients into two groups with different survivals. A weighted immunophenotypic scoring system was developed by integrating results from these analyses. Six antigens were selected: three positive prognosticators (CD62L, CD54, CD49c) and three negative prognosticators (CD49d, CD38, CD79b), with cut-off values ranging from 30% to 50% of positive cells. By weighing the expression of each marker according to its statistical power, a complete scoring system, with point values comprised between 0 (complete absence of phenotypic conditions associated with good prognosis) and 9 (all the phenotypic conditions associated with good prognosis fulfilled), allowed to split the whole set of B-CLL patients, into three distinctive prognostic groups (P = 4.78 x 10(-11)) with high- (score 0-3), intermediate- (score 4-6), and low- (score 7-9) risk of death. The three risk groups showed different distribution of cases as for Rai's stages, IgVH mutations, and ZAP-70 expression. The proposed immunophenotypic scoring system may be an additional useful tool in routine diagnostic/prognostic procedures for B-CLL.

ADP-ribosyl Cyclase 1↗
PubMed2006

The molecular signature of selection underlying human adaptations.

In the last decade, advances in human population genetics and comparative genomics have resulted in important contributions to our understanding of human genetic diversity and genetic adaptation. For the first time, we are able to reliably detect the signature of natural selection from patterns of DNA polymorphism. Identifying the effects of natural selection in this way provides a crucial piece of evidence needed to support hypotheses of human adaptation. This review provides a detailed description of the theory and analytical approaches used to detect signatures of natural selection in the human genome. We discuss these methods in relation to four classic human traits--skin color, the Duffy blood group, bitter-taste sensation, and lactase persistence. By highlighting these four traits we are able to discuss the ways in which analyses of DNA polymorphism can lead to inferences regarding past histories of selection. Specifically, we can infer the importance of specific regimes of selection (i.e. directional selection, balancing selection, and purifying selection) in the evolution of a trait because these different types of selection leave different patterns of DNA polymorphism. In addition, we demonstrate how these types of data can be used to estimate the time frame in which selection operated on a trait. As the field has advanced, a general issue that has come to the forefront is how specific demographic events in human history, such as population expansions, bottlenecks, and subdivision of populations, have also left a signature across the genome that can interfere with our detection of the footprint of selection at particular genes. Therefore, we discuss this general problem with respect to the four traits reviewed here, and describe the ways in which the signature of selection can be teased from a background signature of demographic history. Finally, we move from a discussion of analyses of selection motivated by a "candidate-gene" approach, in which a priori information led to the analysis of specific gene, to discussion of "genome-scanning" approaches that are directed at discovering new genes that have been under positive selection. Such scans can be designed to detect those genes that have been positively selected in our divergence from chimpanzees, as well as those genes that have been under selection as human populations have migrated, differentiated, and adapted to specific geographic environments. We predict that both approaches will be applied in the future, enabling a greater insight into human species-wide adaptations, as well as the specific adaptations of human populations.

Animals↗
PubMed2005

Novel attenuated Salmonella enterica serovar Choleraesuis strains as live vaccine candidates generated by signature-tagged mutagenesis.

Salmonella enterica serovar Choleraesuis is a host-adapted pathogen that causes swine paratyphoid. Signature-tagged mutagenesis (STM) was used to understand the pathogenicity of S. enterica serovar Choleraesuis in its natural host and also to develop novel attenuated live vaccine candidates against this disease. A library of 960 signature-tagged mutants of S. enterica serovar Choleraesuis was constructed and screened for attenuation in pigs. Thirty-three mutants were identified by the STM screening, and these mutants were further screened for attenuation by in vivo and in vitro competitive growth. Of these, 20 mutants targeting the outer membrane, type III secretion, transporter, lipopolysaccharide biosynthesis, and other unknown proteins were confirmed for attenuation. Five highly attenuated mutants (SC2D2 [ssaV], SC4A9 [gifsy-1], SC6F9 [dgoT], SC12B12 [ssaJ], and SC10B1[spiA]) were selected and evaluated for safety and protective efficacy in pigs by comparison with a commercially available vaccine strain. STM-attenuated live vaccine strains SC4A9 (gifsy-1) and SC2D2 (ssaV) were superior to commercially available live vaccine because they provided both safety and a protective immune response against challenge in pigs.

Animals↗