In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera ligustica)

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (<24 h) stronger expression of proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24–48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48–72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological pathways and key node proteins allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects.Embryogenesis is an important period during which the body plan of adult honey bees (Apis mellifera L.) is formed. This life stage, lasting 72 h, occurs during the egg laid by the queen before bees hatch as young larva. Worker bees are derived from fertilized eggs and develop through four distinct stages until the imago eventually emerges: egg, larva, pupa, and emerging adult (1–4). The worker is the dominate caste and engages in almost all aspects of social life: taking care of larvae, cleaning the hive, guarding the nest, and foraging for nectar and pollen for the colony. Understanding the developmental mechanism of embryogenesis of honey bee workers at the protein level is conducive to gaining a new insight into honey bee embryology, but information about the mechanisms of honey bee embryos at molecular level is still very limited.The embryo is recognized as an ideal model for genetic modification as compared with larva, pupa, and emerged adults (5). The environment for embryonic development requires a constant temperature of 34 °C and 80% relative humidity, which can easily be simulated under laboratory conditions. In contrast, rearing larvae or pupae is more challenging because they demand a specific temperature, humidity, and nutrition in the colony environment (1, 5). Furthermore, the honey bee has adapted an evolutionary strategy for better colony survival that makes it difficult to rear experimentally modified larvae and pupae within the colony (6, 7), nurse bees use acute judgment to identify and remove abnormal eggs or larvae (8). This adaptation makes raising experimentally treated bees, such as genetically manipulated eggs and larvae, very difficult in the honey bee colony (9–11). Because of totipotency and multiple differentiation potential, modified eggs could be hatched out normally and eventually some of them could be induced to morphologically and physiologically normal adult queens (12), increasing their usefulness as a model system. Moreover, the chorion of honey bee egg is more suitable for puncturing a hole for microinjection as it is much thinner than that of the fruit fly (Drosophila melonogastero) or the silk worm (Bombyx mori) (0.1–0.25 μm for honey bees compared with ∼17 μm for silk worm) (13, 14). These superiorities are quite promising for in vivo transgenic research on honey bee embryos.Until now, a number of genetic manipulations of the honey bee embryo have been developed. For example, embryonic cells in the pre-gastrula stage that have been transplanted with nuclear materials have developed into chimeric honey bee larvae (15). RNA interference (RNAi) has been used for honey bee embryos in vivo to characterize the functioning of specific genes (16) and for genetic effects on morphological differentiation (17, 18). Moreover, the cultivation of short-term (19–21), long-term (22), and immortalized cell lines (23), and the expression of non-Apis genes in cultured embryonic cells (24) have opened up a new era for genetic manipulation of honey bee embryos.Like Drosophila, Apis is a long germ insect in which segmentation occurs across the whole body (25). To date, although several studies have examined morphological change (2, 26, 27) and gene expression (25, 28, 29) during the period of embryogenesis in the honey bee, only a few works report on the preliminary results of the unraveling molecular underpinnings of worker (30) and drone (31) embryogenesis at the proteomic level, identifying only 107 proteins. MS-based proteomics is the primary technology that enables a system-wide view of proteomes and their changes. The development of MS with high resolution, high mass accuracy, and high sequencing speed now allows routine identification and quantification of proteins in a comprehensive and unbiased manner in biological samples with high confidence (32). These technological advances in LC-MS now allow the study of protein expression on a system-wide level (33). Therefore, an in-depth characterization of the proteome changes during the honey bee embryogenesis will provide greater understanding of the molecular mechanisms that underlie the process of embryogenesis in honey bee workers, and offers new insights into the embryology of other social insects.     

Proteome Analysis of Cerebrospinal Fluid in Amyotrophic Lateral Sclerosis (ALS)

Cerebrospinal fluid (CSF) is a promising source of biomarkers in amyotrophic lateral sclerosis (ALS). Using the two-dimensional difference in gel electrophoresis (2-D-DIGE), we compared CSF samples from patients with ALS (n = 14) with those from normal controls (n = 14). Protein spots that showed significant differences between patients and controls were selected for further analysis by MALDI-TOF mass spectrometry. For validation of identified spots western blot analysis and ELISA was performed. We identified 2 proteins that were upregulated and 3 proteins that were down-regulated in CSF in ALS. Of these, two proteins (Zn-alpha-2-glycoprotein and ceruloplasmin precursor protein) have not been reported in CSF of patients with ALS so far. In contrast, several other proteins (transferrin, alpha-1-antitrypsin precursor and beta-2-microglobulin) seem to be unspecifically affected in different neurological diseases and may therefore be of limited value as disease-related biochemical markers in ALS. Further evaluation of the candidate proteins identified here is necessary.     

Establishing the Proteome of Normal Human Cerebrospinal Fluid

Background

Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and access to true normal samples hindered attaining this goal.

Methods and Principal Findings

We applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject.

Conclusions

Our results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features.     

Analysis of the Cerebrospinal Fluid Proteome in Alzheimer's Disease

Alzheimer’s disease is a neurodegenerative disorder accounting for more than 50% of cases of dementia. Diagnosis of Alzheimer’s disease relies on cognitive tests and analysis of amyloid beta, protein tau, and hyperphosphorylated tau in cerebrospinal fluid. Although these markers provide relatively high sensitivity and specificity for early disease detection, they are not suitable for monitor of disease progression. In the present study, we used label-free shotgun mass spectrometry to analyse the cerebrospinal fluid proteome of Alzheimer’s disease patients and non-demented controls to identify potential biomarkers for Alzheimer’s disease. We processed the data using five programs (DecyderMS, Maxquant, OpenMS, PEAKS, and Sieve) and compared their results by means of reproducibility and peptide identification, including three different normalization methods. After depletion of high abundant proteins we found that Alzheimer’s disease patients had lower fraction of low-abundance proteins in cerebrospinal fluid compared to healthy controls (p<0.05). Consequently, global normalization was found to be less accurate compared to using spiked-in chicken ovalbumin for normalization. In addition, we determined that Sieve and OpenMS resulted in the highest reproducibility and PEAKS was the programs with the highest identification performance. Finally, we successfully verified significantly lower levels (p<0.05) of eight proteins (A2GL, APOM, C1QB, C1QC, C1S, FBLN3, PTPRZ, and SEZ6) in Alzheimer’s disease compared to controls using an antibody-based detection method. These proteins are involved in different biological roles spanning from cell adhesion and migration, to regulation of the synapse and the immune system.     

Nucleotide Degradation Products in Cerebrospinal Fluid (CSF) in Inherited and Acquired Pathologies

CSF purines were grossly elevated compared with controls only in adenylosuccinate lyase (ADSL) deficiency and TB meningitis. The former representing low permeability, the latter severe damage to the normal blood/brain barrier. By contrast, the similarity to controls, with no difference between Lesch–Nyhan disease (LND) or LND variants, would exclude hypoxia as a factor in the severe neurological deficits in LND. Similar findings in purine nucleoside phosphorylase (PNP) deficiency (although nucleosides replace the normal bases) likewise exclude hypoxia in the aetiology of the albeit milder neurological deficits.     

Cerebrospinal Fluid (CSF) 25-Hydroxyvitamin D Concentration and CSF Acetylcholinesterase Activity Are Reduced in Patients with Alzheimer's Disease

Background

Little is known of vitamin D concentration in cerebrospinal fluid (CSF) in Alzheimer´s disease (AD) and its relation with CSF acetylcholinesterase (AChE) activity, a marker of cholinergic function.

Methods

A cross-sectional study of 52 consecutive patients under primary evaluation of cognitive impairment and 17 healthy controls. The patients had AD dementia or mild cognitive impairment (MCI) diagnosed with AD dementia upon follow-up (n = 28), other dementias (n = 12), and stable MCI (SMCI, n = 12). We determined serum and CSF concentrations of calcium, parathyroid hormone (PTH), 25-hydroxyvitamin D (25OHD), and CSF activities of AChE and butyrylcholinesterase (BuChE).

Findings

CSF 25OHD level was reduced in AD patients (P < 0.05), and CSF AChE activity was decreased both in patients with AD (P < 0.05) and other dementias (P < 0.01) compared to healthy controls. None of the measured variables differed between BuChE K-variant genotypes whereas the participants that were homozygous in terms of the apolipoprotein E (APOE) ε4 allele had decreased CSF AChE activity compared to subjects lacking the APOE ε4 allele (P = 0.01). In AD patients (n=28), CSF AChE activity correlated positively with CSF levels of total tau (T-tau) (r = 0.44, P < 0.05) and phosphorylated tau protein (P-tau) (r = 0.50, P < 0.01), but CSF activities of AChE or BuChE did not correlate with serum or CSF levels of 25OHD.

Conclusions

In this pilot study, both CSF 25OHD level and CSF AChE activity were reduced in AD patients. However, the lack of correlations between 25OHD levels and CSF activities of AChE or BuChE might suggest different mechanisms of action, which could have implications for treatment trials.     

Absolute Proteome and Phosphoproteome Dynamics during the Cell Cycle of Schizosaccharomyces pombe (Fission Yeast)

To quantify cell cycle-dependent fluctuations on a proteome-wide scale, we performed integrative analysis of the proteome and phosphoproteome during the four major phases of the cell cycle in Schizosaccharomyces pombe. In highly synchronized cells, we identified 3753 proteins and 3682 phosphorylation events and relatively quantified 65% of the data across all phases. Quantitative changes during the cell cycle were infrequent and weak in the proteome but prominent in the phosphoproteome. Protein phosphorylation peaked in mitosis, where the median phosphorylation site occupancy was 44%, about 2-fold higher than in other phases. We measured copy numbers of 3178 proteins, which together with phosphorylation site stoichiometry enabled us to estimate the absolute amount of protein-bound phosphate, as well as its change across the cell cycle. Our results indicate that 23% of the average intracellular ATP is utilized by protein kinases to phosphorylate their substrates to drive regulatory processes during cell division. Accordingly, we observe that phosphate transporters and phosphate-metabolizing enzymes are phosphorylated and therefore likely to be regulated in mitosis.Cell replication involves a complex series of highly regulated and evolutionary conserved events, called the “cell cycle.” Aberrations in the cell cycle have severe implications and can cause cancerous growth. A detailed understanding of the cell cycle and its regulation may identify additional targets for cancer therapy (1–3). The cell cycle has been the subject of previous proteomics studies. Olsen et al. (4) measured the dynamics of thousands of proteins and phosphorylation events across cell cycle phases of HeLa cells, providing insights into the underlying regulatory mechanisms and pointing to a general increase in phosphorylation site occupancy during M phase. In a targeted study, Pagliuca et al. (5) investigated interactors of cyclins E1, A2, and B1 in HeLa cells, revealing key mechanistic links between DNA replication and mitosis.Schizosaccharomyces pombe (fission yeast) is a unicellular organism, which can easily be genetically manipulated and carries many cell cycle features similar to metazoan cells. It is an important model organism to study the cell cycle and its checkpoint controls (6). Recent global proteomics studies of yeasts and their cell cycle (7–13) have mainly focused on Saccharomyces cerevisiae (budding yeast), with only a few studies of fission yeast (14, 15), although the fission yeast cell cycle may be more representative of eukaryotic cell cycles (16). However, attention of the proteomics community toward S. pombe is increasing. Recent proteomics studies covered up to 4087 S. pombe proteins (71% of the predicted proteome) and 1544 phosphoproteins in both asynchronous and synchronized cell cultures (17–22); however, a comprehensive analysis of the S. pombe cell cycle is so far missing.Here, we use high resolution mass spectrometry in combination with stable isotope labeling by amino acids in the cell culture (SILAC)¹ method, termed super-SILAC (23), and intensity-based absolute quantification (iBAQ) (24) to measure relative and absolute dynamics of the proteome and phosphoproteome during the cell cycle of fission yeast. We estimate copy numbers for 3178 S. pombe proteins, and we combine these data with calculated phosphorylation site stoichiometry to estimate the total amount of protein-bound phosphate and its dynamics across the cell cycle. Providing the global absolute dynamics and stoichiometry of proteins and their modifications will be a valuable resource for classical and systems biologists alike.     

Cerebrospinal Fluid (CSF) Neuronal Biomarkers across the Spectrum of HIV Infection: Hierarchy of Injury and Detection

The character of central nervous system (CNS) HIV infection and its effects on neuronal integrity vary with evolving systemic infection. Using a cross-sectional design and archived samples, we compared concentrations of cerebrospinal fluid (CSF) neuronal biomarkers in 143 samples from 8 HIV-infected subject groups representing a spectrum of untreated systemic HIV progression and viral suppression: primary infection; four groups of chronic HIV infection neuroasymptomatic (NA) subjects defined by blood CD4+ T cells of >350, 200–349, 50–199, and <50 cells/µL; HAD; treatment-induced viral suppression; and ‘elite’ controllers. Samples from 20 HIV-uninfected controls were also examined. The neuronal biomarkers included neurofilament light chain protein (NFL), total and phosphorylated tau (t-tau, p-tau), soluble amyloid precursor proteins alpha and beta (sAPPα, sAPPβ) and amyloid beta (Aβ) fragments 1–42, 1–40 and 1–38. Comparison of the biomarker changes showed a hierarchy of sensitivity in detection and suggested evolving mechanisms with progressive injury. NFL was the most sensitive neuronal biomarker. Its CSF concentration exceeded age-adjusted norms in all HAD patients, 75% of NA CD4<50, 40% of NA CD4 50–199, and 42% of primary infection, indicating common neuronal injury with untreated systemic HIV disease progression as well as transiently during early infection. By contrast, only 75% of HAD subjects had abnormal CSF t-tau levels, and there were no significant differences in t-tau levels among the remaining groups. sAPPα and β were also abnormal (decreased) in HAD, showed less marked change than NFL with CD4 decline in the absence of HAD, and were not decreased in PHI. The CSF Aβ peptides and p-tau concentrations did not differ among the groups, distinguishing the HIV CNS injury profile from Alzheimer''s disease. These CSF biomarkers can serve as useful tools in selected research and clinical settings for patient classification, pathogenetic analysis, diagnosis and management.     

Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis

Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a manganese transporter called SsaB for virulence and O₂ tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper (Makhlynets, O., Boal, A. K., Rhodes, D. V., Kitten, T., Rosenzweig, A. C., and Stubbe, J. (2014) J. Biol. Chem. 289, 6259–6272) indicates that in the presence of O₂, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (Fe^III₂-Y^•) in vitro, whereas assembly of a dimanganese-tyrosyl radical (Mn^III₂-Y^•) cofactor requires NrdI, and Mn^III₂-Y^• is more active than Fe^III₂-Y^• with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically, whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O₂-independent adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O₂ was partially restored. The combined results suggest that Mn^III₂-Y^•-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why manganese is necessary for virulence and O₂ tolerance in many bacterial pathogens possessing a class Ib RNR and suggests NrdF and NrdI may serve as promising new antimicrobial targets.     

Characterization of the Serpin, α1-Antichymotrypsin, in Normal Human Cerebrospinal Fluid

Cerebrospinal fluid (CSF) from 20 male patients with nonneurologic disease (age 64.5 +/- 2.8 SEM) was analyzed for the presence of the serpin alpha 1-antichymotrypsin (alpha 1-ACT). A chymotrypsin-specific chromogenic substrate (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) was used to examine the CSF samples. All CSF samples showed inhibitory activity ranging from 45 to 80% inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the samples revealed the presence of a 68-kDa protein migrating identical to authentic human plasma alpha 1-ACT. Complex formation was performed with iodinated bovine chymotrypsin for several representative CSF samples having the highest chymotrypsin inhibitory activity. Comparison was made with complex formation performed with commercially available authentic human plasma alpha 1-ACT. These studies showed the formation of complexes at 37 degrees C, regardless of whether the sample was subsequently boiled or not. In the case of CSF, two complex bands, mass smaller than with plasma alpha 1-ACT, were formed at the lower temperature whereas a single higher Mr band was formed when the samples were boiled. To determine whether cleavage of the serpin occurred, these studies were repeated using human neutrophil cathepsin G as target protease. A complex of approximately 90 kDa was formed with human alpha 1-ACT under these same conditions. alpha 1-ACT has been detected in senile amyloid plaques in brains of Alzheimer's disease patients, the only plasma serine protease inhibitor localized to these structures. Another serpin, protease nexin I, is also found in these plaques, but this inhibitor does not circulate in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Drosophila Evolution over Space and Time (DEST): A New Population Genomics Resource

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome data sets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate data sets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in >20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This data set, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental metadata. A web-based genome browser and web portal provide easy access to the SNP data set. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan data set. Our resource will enable population geneticists to analyze spatiotemporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.     

Angiotensinogen in Cerebrospinal Fluid Corresponds Chromatographically to the γ-Form of Plasma Angiotensinogen

Angiotensinogen (Aogen) (CA 11002-13-14), the prohormone of the neuro- and vasoactive peptide angiotensin II (Ang II) (CA 11128-99-7), is found in dog brain as well as in dog plasma. At 2-4 micrograms/ml CSF, Aogen comprises 1-2% of the total protein in dog CSF. Immunopurified CSF and plasma Aogen were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anion-exchange HPLC. Two major (alpha- and beta-) forms and one minor (gamma-) form of Aogen were observed in dog plasma. The majority of Aogen in dog CSF was chromatographically identical to the gamma-form of plasma Aogen; alpha- and beta-Aogen forms comprised less than 5% of the total CSF Aogen. The N-terminal amino acid sequences of alpha-, beta-, and gamma-Aogen identified these proteins as members of the Aogen family. The N-terminal amino acid sequence of CSF gamma-Aogen was Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-Leu-Leu-Val-Tyr-Ser-Lys-Ser-Ser-(X)-Glu- . More basic than either alpha- or beta-Aogen, gamma-Aogen was shown to be a glycoprotein with an apparent molecular weight (Mr) of 58,000. CSF [des Ang I]-Aogen exhibited a greater anion-exchange HPLC retention. CSF, however, contained only minor amounts of [des Ang I]-Aogen. These analyses have demonstrated that brain overwhelmingly releases one particular Aogen into the CSF; however, very little of this brain Aogen is utilized for the production of Ang I.     

Cerebrospinal Fluid (CSF) CD8+ T-Cells That Express Interferon-Gamma Contribute to HIV Associated Neurocognitive Disorders (HAND)

BackgroundHIV associated neurocognitive disorders (HAND) continue to affect cognition and everyday functioning despite anti-retroviral treatment (ART). Previous studies focused on mechanisms related to monocyte/macrophage mediated inflammation. However, in the ART era, there is increasing evidence for the involvement of CD8+ T-cells in CNS pathogenesis.MethodsTo investigate the relationship between T-cell responses and neurocognitive impairment (NCI), cerebrospinal fluid (CSF) and peripheral blood CD4+ and CD8+ T-cell intracellular cytokine (IFNγ, IL-2, TNFα) and lytic marker (CD107a) expression were assessed in HIV infected subjects who underwent comprehensive neurocognitive (NC) evaluation and either initiated or changed ART.ResultsData were collected from 31 participants at 70 visits. The frequency of cytokine expressing T-cells in CSF was significantly higher than in peripheral blood for CD4+T-cells: TNFα, IL-2, IFNγ and CD8+T-cells: IL-2 and IFNγ. Analysis of T-cell activity and NCI as a function of CSF HIV RNA levels suggested a general association between NCI, high CSF CD8+ (but not CD4+T-cell) cytokine expression and CSF HIV RNA <10³ copies/ml (p<0.0001). Specifically, CSF CD8+ T-cell IFNγ expression correlated with severity of NCI (r = 0.57, p = 0.004). Multivariable analyses indicated that CSF CD8+T-cell IFNγ and myeloid activation (CD163) contributed equally and independently to cognitive status and a composite variable produced the strongest correlation with NCI (r = 0.83, p = 0.0001). In contrast, CD8+ cytolytic activity (CD107a expression) was negatively correlated with NCI (p = 0.05) but was dependent on CD4 levels >400/μl and low CSF HIV RNA levels (<10³ copies/ml). In our longitudinal analysis of 16 subjects, higher CSF CD8+IFNγ expression at baseline predicted NC decline at follow-up (p = 0.02). Severity of NCI at follow-up correlated with level of residual HIV RNA in CSF.ConclusionsPresence of IFNγ expressing CD8+ T-cells, absence of cytolytic CD8+ T-cells, high myeloid activation, and failure of ART to suppress HIV replication in CSF contribute to increased risk of HAND.     

Effect of Freezing on γ-Aminobutyric Acid Levels in Human Cerebrospinal Fluid

Abstract Using a radioreceptor assay, the concentration of γ -aminobutyric acid (GABA) in human cerebrospinal fluid (CSF) was found to be elevated significantly following a single deep-freeze to –70°C and thaw. Mean CSF GABA (± SD) in unfrozen CSF was 173 ± 73 pmol/ml ( n = 24). After a single deep-freeze, the mean level was 243 ± 106 pmol/ml ( p < 0.02). Subsequent freeze-thaw cycles resulted in further irregular and unpredictable elevations in CSF GABA. Mean level after two freezes was 379 ± 125 pmol/ml and after three freezes 654 ± 411 pmol/ml. These changes could result in the incorrect interpretation of results in patients suffering from neurological diseases.     

Paridris Kieffer of the New World (Hymenoptera,Platygastroidea, Platygastridae)

Paridris in the New World is revised (Hymenoptera: Platygastridae). Fifteen species are described, of which 13 are new. Paridris aenea (Ashmead)(Mexico (Tamaulipas) and West Indies south to Bolivia and southern Brazil (Rio de Janeiro state)), Paridris armata Talamas, sp. n. (Venezuela), Paridris convexa Talamas, sp. n. (Costa Rica, Panama), Paridris dnophos Talamas, sp. n. (Mexico (Vera Cruz) south to Bolivia and central Brazil (Goiás)), Paridris gongylos Talamas & Masner, sp. n. (United States: Appalachian Mountains of Virginia, Tennessee, South Carolina), Paridris gorn Talamas & Masner, sp. n. (United States: Ohio south to Alabama, Georgia), Paridris invicta Talamas & Masner, sp. n. (Brazil: São Paulo), Paridris isabelicae Talamas & Masner, sp. n. (Cuba, Dominican Republic), Paridris lemete Talamas & Masner, sp. n. (Puerto Rico), Paridris minor Talamas, sp. n. (Cuba), Paridris nayakorum Talamas, sp. n. (Costa Rica), Paridris pallipes (Ashmead)(southeastern Canada, United States south to Costa Rica, also Brazil (São Paulo), Paridris psydrax Talamas & Masner, sp. n. (Argentina, Mexico, Paraguay, United States, Venezuela), Paridris saurotos Talamas, sp. n. (Jamaica), Paridris soucouyant Talamas & Masner, sp. n. (Colombia, Trinidad and Tobago, Venezuela). Paridris brevipennis Fouts, Paridris laeviceps (Ashmead), and Paridris nigricornis (Fouts) are treated as junior synonyms of Paridris pallipes; Paridris opaca is transferred to Probaryconus. Lectotypes are designated for Idris aenea Ashmead and Caloteleia aenea Ashmead.