Epigenetic networks driving T cell identity and plasticity during immunosenescence.

The aging process is associated with the accumulation of epigenetic alterations in immune cells, although the origin of these changes is not clear. Understanding this epigenetic drift in the immune system can provide essential information about the progression of the aging process and the immune history of each individual.

Exploring kidney allograft rejection: A proof-of-concept study using spatial transcriptomics.

In this proof-of-concept study, spatial transcriptomics combined with public single-cell ribonucleic acid-sequencing data were used to explore the potential of this technology to study kidney allograft rejection. We aimed to map gene expression patterns within diverse pathologic states by examining biopsies classified across nonrejection, T cell-mediated acute rejection, interstitial fibrosis, and tubular atrophy. Our results revealed distinct immune cell signatures, including those of T and B lymphocytes, monocytes, mast cells, and plasma cells, and their spatial organization within the renal interstitium. We also mapped chemokine receptors and ligands to study immune cell migration and recruitment. Finally, our analysis demonstrated differential spatial enrichment of transcription signatures associated with kidney allograft rejection across various biopsy regions. Interstitium regions displayed higher enrichment scores for rejection-associated gene expression patterns than tubular areas, which had negative scores. This implies that these signatures are primarily driven by processes unfolding in the renal interstitium. Overall, this study highlights the value of spatial transcriptomics for revealing cellular heterogeneity and immune signatures in renal transplant biopsies and demonstrates its potential for studying the molecular and cellular mechanisms associated with rejection. However, certain limitations must be borne in mind regarding the development and future applications of this technology.

Plasmids of the incompatibility group FIBK occur in Klebsiella variicola from diverse ecological niches.

Plasmids play a fundamental role in the evolution of bacteria by allowing them to adapt to different environments and acquire, through horizontal transfer, genes that confer resistance to different classes of antibiotics. Using the available in vitro and in silico plasmid typing systems, we analyzed a set of isolates and public genomes of K. variicola to study its plasmid diversity. The resistome, the plasmid multilocus sequence typing (pMLST), and molecular epidemiology using the MLST system were also studied. A high frequency of IncF plasmids from human isolates but lower frequency from plant isolates were found in our strain collection. In silico detection revealed 297 incompatibility (Inc) groups, but the IncFIBK (216/297) predominated in plasmids from human and environmental samples, followed by IncFIIK (89/297) and IncFIA/FIA(HI1) (75/297). These Inc groups were associated with clinically important ESBL (CTX-M-15), carbapenemases (KPC-2 and NDM-1), and colistin-resistant genes which were associated with major sequence types (ST): ST60, ST20, and ST10. In silico MOB typing showed 76% (311/404) of the genomes contained one or more of the six relaxase families with MOBF being most abundant. We identified untypeable plasmids carrying blaKPC-2, blaIMP-1, and blaSHV-187 but for which a relaxase was found; this may suggest that novel plasmid structures could be emerging in this bacterial species. The plasmid content in K. variicola has limited diversity, predominantly composed of IncFIBK plasmids dispersed in different STs. Plasmid detection using the replicon and MOB typing scheme provide a broader context of the plasmids in K. variicola. This study showed that whole-sequence-based typing provides current insights of the prevalence of plasmid types and their association with antimicrobial resistant genes in K. variicola obtained from humans and environmental niches.

Adaptive responses of yeast strains tolerant to acidic pH, acetate, and supraoptimal temperature.

Ethanol fermentations can be prematurely halted as Saccharomyces cerevisiae faces adverse conditions, such as acidic pH, presence of acetic acid, and supraoptimal temperatures. The knowledge on yeast responses to these conditions is essential to endowing a tolerant phenotype to another strain by targeted genetic manipulation. In this study, physiological and whole-genome analyses were conducted to obtain insights on molecular responses which potentially render yeast tolerant towards thermoacidic conditions. To this end, we used thermotolerant TTY23, acid tolerant AT22, and thermo-acid tolerant TAT12 strains previously generated by adaptive laboratory evolution (ALE) experiments. The results showed an increase in thermoacidic profiles in the tolerant strains. The whole-genome sequence revealed the importance of genes related to: H+, iron, and glycerol transport (i.e., PMA1, FRE1/2, JEN1, VMA2, VCX1, KHA1, AQY3, and ATO2); transcriptional regulation of stress responses to drugs, reactive oxygen species and heat-shock (i.e., HSF1, SKN7, BAS1, HFI1, and WAR1); and adjustments of fermentative growth and stress responses by glucose signaling pathways (i.e., ACS1, GPA1/2, RAS2, IRA2, and REG1). At 30 °C and pH 5.5, more than a thousand differentially expressed genes (DEGs) were identified in each strain. The integration of results revealed that evolved strains adjust their intracellular pH by H+ and acetic acid transport, modify their metabolism and stress responses via glucose signaling pathways, control of cellular ATP pools by regulating translation and de novo synthesis of nucleotides, and direct the synthesis, folding and rescue of proteins throughout the heat-shock stress response. Moreover, the motifs analysis in mutated transcription factors suggested a significant association of SFP1, YRR1, BAS1, HFI1, HSF1, and SKN7 TFs with DEGs found in thermoacidic tolerant yeast strains. KEY POINTS: • All the evolved strains overexpressed the plasma membrane H+ -ATPase PMA1 at optimal conditions • Tolerant strain TAT12 mutated genes encoding weak acid and heat response TFs HSF1, SKN7, and WAR1 • TFs HSF1 and SKN7 likely controlled the transcription of metabolic genes associated to heat and acid tolerance.

Insights behind the Relationship between Colorectal Cancer and Obesity: Is Visceral Adipose Tissue the Missing Link?

Colorectal cancer (CRC) is a major health problem worldwide, with an estimated 1.9 million new cases and 915,880 deaths in 2020 alone. The etiology of CRC is complex and involves both genetic and lifestyle factors. Obesity is a major risk factor for CRC, and the mechanisms underlying this link are still unclear. However, the generalized inflammatory state of adipose tissue in obesity is thought to play a role in the association between CRC risk and development. Visceral adipose tissue (VAT) is a major source of proinflammatory cytokines and other factors that contribute to the characteristic systemic low-grade inflammation associated with obesity. VAT is also closely associated with the tumor microenvironment (TME), and recent evidence suggests that adipocytes within the TME undergo phenotypic changes that contribute to tumor progression. In this review, we aim to summarize the current evidence linking obesity and CRC, with a focus on the role of VAT in tumor etiology and progression.

A Multi-center Genome-wide Association Study of Cervical Dystonia.

BACKGROUND: Several monogenic causes for isolated dystonia have been identified, but they collectively account for only a small proportion of cases. Two genome-wide association studies have reported a few potential dystonia risk loci; but conclusions have been limited by small sample sizes, partial coverage of genetic variants, or poor reproducibility. OBJECTIVE: To identify robust genetic variants and loci in a large multicenter cervical dystonia cohort using a genome-wide approach. METHODS: We performed a genome-wide association study using cervical dystonia samples from the Dystonia Coalition. Logistic and linear regressions, including age, sex, and population structure as covariates, were employed to assess variant- and gene-based genetic associations with disease status and age at onset. We also performed a replication study for an identified genome-wide significant signal. RESULTS: After quality control, 919 cervical dystonia patients compared with 1491 controls of European ancestry were included in the analyses. We identified one genome-wide significant variant (rs2219975, chromosome 3, upstream of COL8A1, P-value 3.04 × 10-8 ). The association was not replicated in a newly genotyped sample of 473 cervical dystonia cases and 481 controls. Gene-based analysis identified DENND1A to be significantly associated with cervical dystonia (P-value 1.23 × 10-6 ). One low-frequency variant was associated with lower age-at-onset (16.4 ± 2.9 years, P-value = 3.07 × 10-8 , minor allele frequency = 0.01), located within the GABBR2 gene on chromosome 9 (rs147331823). CONCLUSION: The genetic underpinnings of cervical dystonia are complex and likely consist of multiple distinct variants of small effect sizes. Larger sample sizes may be needed to provide sufficient statistical power to address the presumably multi-genic etiology of cervical dystonia. © 2021 International Parkinson and Movement Disorder Society.

Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment.

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.

New Bioconjugated Technetium and Rhenium Folates Synthesized by Transmetallation Reaction with Zinc Derivatives.

The zinc dithiocarbamates functionalized with folic acid 2Zn and 3Zn were synthesized with a simple straightforward method, using an appropriated folic acid derivative and a functionalized zinc dithiocarbamate (1Zn). Zinc complexes 2Zn and 3Zn show very low solubilities in water, making them useful for preparing Tc-99m radiopharmaceuticals with a potentially high molar activity. Thus, the transmetallation reaction in water medium between the zinc complexes 2Zn or 3Zn and the cation fac-[99mTc(H2O)3(CO)3]+, in the presence of the monodentate ligand TPPTS, leads to the formation of the 2 + 1 complexes fac-[99mTc(CO)3(SS)(P)] bioconjugated to folic acid (2Tc and 3Tc). In spite of the low solubility of 2Zn and 3Zn in water, the reaction yield is higher than 95%, and the excess zinc reagent is easily removed by centrifugation. The Tc-99m complexes were characterized by comparing their HPLC with those of the homologous rhenium complexes (2Re and 3Re) previously synthesized and characterized by standard methods. Preliminary in vivo studies with 2Tc and 3Tc indicate low specific binding to folate receptors. In summary, Tc-99m folates 2Tc and 3Tc were prepared in high yields, using a one-pot transmetallation reaction with low soluble zinc dithiocarbamates (>1 ppm), at moderate temperature, without needing a subsequent purification step.

Direct Synthesis of Rhenium and Technetium-99m Metallosurfactants by a Transmetallation Reaction of Lipophilic Groups: Potential Applications in the Radiolabeling of Liposomes.

A new zinc dithiocarbamate functionalized with palmitoyl groups is described as a useful tool for the preparation of metallosurfactants through a transmetallation reaction with the transition metals rhenium and technetium. An amphiphilic rhenium complex is synthesized by a transmetallation reaction with the zinc complex in presence of the polar phosphine sodium triphenylphosphine trisulfonate, which leads to a rhenium complex with a lipophilic dithiocarbamate and a polar phosphine ligand. The study of this rhenium complex has shown that it self-aggregates, leading to the formation of aggregates that have been analyzed by dynamic light scattering and cryotransmission electron microscopy (cryo-TEM). In addition, this amphiphilic rhenium complex is incorporated into soy phosphatidylcholine liposomes, whether liposomes are prepared by mixing phospholipid and the rhenium complex or by the incorporation of the rhenium complex into preformed liposomes. The one-pot reaction of the radiocompound [99mTc(H2O)3(CO)3]+ with the above-mentioned zinc dithiocarbamate, the phosphine sodium triphenylphosphine trisulfonate and the phospholipid soy phosphatidylcholine, leads to liposomes labeled with a Tc-99m homologous complex of the rhenium complex, in accordance with the high-performance liquid chromatography (HPLC) data.

Immune Landscape in Tumor Microenvironment: Implications for Biomarker Development and Immunotherapy.

Integration of the tumor microenvironment as a fundamental part of the tumorigenic process has undoubtedly revolutionized our understanding of cancer biology. Increasing evidence indicates that neoplastic cells establish a dependency relationship with normal resident cells in the affected tissue and, furthermore, develop the ability to recruit new accessory cells that aid tumor development. In addition to normal stromal and tumor cells, this tumor ecosystem includes an infiltrated immune component that establishes complex interactions that have a critical effect during the natural history of the tumor. The process by which immune cells modulate tumor progression is known as immunoediting, a dynamic process that creates a selective pressure that finally leads to the generation of immune-resistant cells and the inability of the immune system to eradicate the tumor. In this context, the cellular and functional characterization of the immune compartment within the tumor microenvironment will help to understand tumor progression and, ultimately, will serve to create novel prognostic tools and improve patient stratification for cancer treatment. Here we review the impact of the immune system on tumor development, focusing particularly on its clinical implications and the current technologies used to analyze immune cell diversity within the tumor.

Oscheius myriophila (Nematoda: Rhabditida) isolated in sugar cane soils in Mexico with potential to be used as entomopathogenic nematode.

A survey of entomopathogenic nematodes was conducted in sugar cane crops in a total of 14 soils, and positive results were obtained for strain MC5-2014 in the municipality of Tepalcingo, Morelos, in soil with a sandy loam texture and a pH of 6.4. Species determination was performed via morphological and morphometric techniques by searching for a tubular stoma with a swollen cylindrical pharyngeal body and a metacorpus in the basal part. The range of body length (L) was 750 to 1200 µm in females and 720 to 910 µm in males, while the corresponding maximum widths (W) of the body were 30 to 60 µm and 20 to 30 µm, respectively. Males exhibited bursa with a 1 + 1 + 3 + 3 distribution of papillae, and females exhibited a vulva located at the mid-body. For molecular identification, the ITS region of ribosomal DNA was used. Virulence tests (LC50) were conducted with Galleria mellonella, and a value of 4.732 was obtained for infective juveniles (IJs). Taking taxonomic and molecular characteristics into account, the isolate was determined to be Oscheius myriophila. The isolation of this strain represents the first geographic report of O. myriophila in Mexico, and it should be noted that the cultivation of sugar cane occurs with constant application of insecticides, herbicides, fungicides, and fertilizers as well as harvesting activities such as burning of the crop for harvest. The O. myriophila isolate has the potential to be used in the future as a method of biological control in our country.A survey of entomopathogenic nematodes was conducted in sugar cane crops in a total of 14 soils, and positive results were obtained for strain MC5-2014 in the municipality of Tepalcingo, Morelos, in soil with a sandy loam texture and a pH of 6.4. Species determination was performed via morphological and morphometric techniques by searching for a tubular stoma with a swollen cylindrical pharyngeal body and a metacorpus in the basal part. The range of body length (L) was 750 to 1200 µm in females and 720 to 910 µm in males, while the corresponding maximum widths (W) of the body were 30 to 60 µm and 20 to 30 µm, respectively. Males exhibited bursa with a 1 + 1 + 3 + 3 distribution of papillae, and females exhibited a vulva located at the mid-body. For molecular identification, the ITS region of ribosomal DNA was used. Virulence tests (LC50) were conducted with Galleria mellonella, and a value of 4.732 was obtained for infective juveniles (IJs). Taking taxonomic and molecular characteristics into account, the isolate was determined to be Oscheius myriophila. The isolation of this strain represents the first geographic report of O. myriophila in Mexico, and it should be noted that the cultivation of sugar cane occurs with constant application of insecticides, herbicides, fungicides, and fertilizers as well as harvesting activities such as burning of the crop for harvest. The O. myriophila isolate has the potential to be used in the future as a method of biological control in our country.

Epigenetic Networks Regulate the Transcriptional Program in Memory and Terminally Differentiated CD8+ T Cells.

Epigenetic mechanisms play a critical role during differentiation of T cells by contributing to the formation of stable and heritable transcriptional patterns. To better understand the mechanisms of memory maintenance in CD8+ T cells, we performed genome-wide analysis of DNA methylation, histone marking (acetylated lysine 9 in histone H3 and trimethylated lysine 9 in histone), and gene-expression profiles in naive, effector memory (EM), and terminally differentiated EM (TEMRA) cells. Our results indicate that DNA demethylation and histone acetylation are coordinated to generate the transcriptional program associated with memory cells. Conversely, EM and TEMRA cells share a very similar epigenetic landscape. Nonetheless, the TEMRA transcriptional program predicts an innate immunity phenotype associated with genes never reported in these cells, including several mediators of NK cell activation (VAV3 and LYN) and a large array of NK receptors (e.g., KIR2DL3, KIR2DL4, KIR2DL1, KIR3DL1, KIR2DS5). In addition, we identified up to 161 genes that encode transcriptional regulators, some of unknown function in CD8+ T cells, and that were differentially expressed in the course of differentiation. Overall, these results provide new insights into the regulatory networks involved in memory CD8+ T cell maintenance and T cell terminal differentiation.

Enhanced Tolerance against a Fungal Pathogen and Insect Resistance in Transgenic Tobacco Plants Overexpressing an Endochitinase Gene from Serratia marcescens.

In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the corpse of a Diatraea magnifactella lepidopteran, which is an important sugarcane pest. The chitinase gene SmchiC amplified from the S. marcescens genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco (Nicotiana tabacum L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus Botrytis cinerea and to the pest Spodoptera frugiperda was evaluated: both the activity of chitinase as well as the resistance against B. cinerea and S. frugiperda was significantly higher in transgenic plants compared to the wild-type.

Heat shock protein 104 (Hsp104)-mediated curing of [PSI+] yeast prions depends on both [PSI+] conformation and the properties of the Hsp104 homologs.

Prions arise from proteins that have two possible conformations: properly folded and non-infectious or misfolded and infectious. The [PSI+] yeast prion, which is the misfolded and self-propagating form of the translation termination factor eRF3 (Sup35), can be cured of its infectious conformation by overexpression of Hsp104, which helps dissolve the prion seeds. This dissolution depends on the trimming activity of Hsp104, which reduces the size of the prion seeds without increasing their number. To further understand the relationship between trimming and curing, trimming was followed by measuring the loss of GFP-labeled Sup35 foci from both strong and weak [PSI+] variants; the former variant has more seeds and less soluble Sup35 than the latter. Overexpression of Saccharomyces cerevisiae Hsp104 (Sc-Hsp104) trimmed the weak [PSI+] variants much faster than the strong variants and cured the weak variants an order of magnitude faster than the strong variants. Overexpression of the fungal Hsp104 homologs from Schizosaccharomyces pombe (Sp-Hsp104) or Candida albicans (Ca-Hsp104) also trimmed and cured the weak variants, but interestingly, it neither trimmed nor cured the strong variants. These results show that, because Sc-Hsp104 has greater trimming activity than either Ca-Hsp104 or Sp-Hsp104, it cures both the weak and strong variants, whereas Ca-Hsp104 and Sp-Hsp104 only cure the weak variants. Therefore, curing by Hsp104 overexpression depends on both the trimming ability of the fungal Hsp104 homolog and the strength of the [PSI+] variant: the greater the trimming activity of the Hsp104 homolog and the weaker the variant, the greater the curing.

Adsorption of Pharmaceutical Pollutants from Water Using Covalent Organic Frameworks.

Capture of pharmaceutical pollutants from water was studied using a novel fluorine-bearing covalent organic framework TpBD-(CF3 )2 , which showed ibuprofen adsorption capacity of 119 mg g-1 at neutral pH. This value is further enhanced at pH 2, highlighting the potential of this class of materials to serve as adsorbents even under harsh conditions. The adsorbed pharmaceutical can be recovered from TpBD-(CF3 )2 in high yield, offering the option of recycling both the adsorbent and the pharmaceutical. The high efficiency of ibuprofen capture as compared to other less lipophilic pharmaceuticals suggests that COFs can be pre-designed for selective capture of contaminants.

Long-term safety and efficacy of levodopa-carbidopa intestinal gel in advanced Parkinson's disease.

BACKGROUND: Levodopa-carbidopa intestinal gel (designated as carbidopa-levodopa enteral suspension in the United States) provides stable plasma levodopa concentrations and reduces motor fluctuations in advanced Parkinson's disease patients through continuous delivery of levodopa via percutaneous endoscopic gastrojejunostomy. We report long-term safety and efficacy outcomes from an open-label phase 3 treatment program. METHODS: PD patients (n = 262) who completed a 12-week double-blind study and its 52-week open-label extension or a separate 54-week open-label study were enrolled in this ongoing phase 3 open-label, multinational study (NCT00660673). Safety and efficacy assessments were collected every 6 months. RESULTS: Mean total duration of exposure to levodopa-carbidopa intestinal gel was 4.1 years (range, 1.2 to 6.9 years). The overall discontinuation rate was 34% (average annual discontinuation rate, 10%). Although most patients (94%) reported an adverse event, the rate of adverse events decreased over time; 53% experienced a serious adverse event. Of patients in this extension study, 54% required jejunal tube replacement during the study, and 37% required percutaneous endoscopic gastrostomy tube replacement. Most patients were on levodopa monotherapy. Patients maintained reductions in "off" time and increases in mean "on" time without dyskinesia from initial levodopa-carbidopa intestinal gel infusion to he study end point (P < 0.001; n = 81). Activities of daily living and quality-of-life assessments demonstrated significant improvements that persisted through the study. CONCLUSIONS: This long-term study demonstrates sustained and clinically meaningful benefits from levodopa-carbidopa intestinal gel in advanced PD patients. Although adverse event rates decreased over time, vigilance is required for device-related complications and adverse events. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Inhibition of Bromodomain and Extraterminal Domain Family Proteins Ameliorates Experimental Renal Damage.

Renal inflammation has a key role in the onset and progression of immune- and nonimmune-mediated renal diseases. Therefore, the search for novel anti-inflammatory pharmacologic targets is of great interest in renal pathology. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) proteins, was previously found to preserve renal function in experimental polycystic kidney disease. We report here that JQ1-induced BET inhibition modulated the in vitro expression of genes involved in several biologic processes, including inflammation and immune responses. Gene silencing of BRD4, an important BET protein, and chromatin immunoprecipitation assays showed that JQ1 alters the direct association of BRD4 with acetylated histone-packaged promoters and reduces the transcription of proinflammatory genes (IL-6, CCL-2, and CCL-5). In vivo, JQ1 abrogated experimental renal inflammation in murine models of unilateral ureteral obstruction, antimembrane basal GN, and infusion of Angiotensin II. Notably, JQ1 downregulated the expression of several genes controlled by the NF-κB pathway, a key inflammatory signaling pathway. The RelA NF-κB subunit is activated by acetylation of lysine 310. In damaged kidneys and cytokine-stimulated renal cells, JQ1 reduced the nuclear levels of RelA NF-κB. Additionally, JQ1 dampened the activation of the Th17 immune response in experimental renal damage. Our results show that inhibition of BET proteins reduces renal inflammation by several mechanisms: chromatin remodeling in promoter regions of specific genes, blockade of NF-κB pathway activation, and modulation of the Th17 immune response. These results suggest that inhibitors of BET proteins could have important therapeutic applications in inflammatory renal diseases.

Psychiatric associations of adult-onset focal dystonia phenotypes.

BACKGROUND: Depression and anxiety frequently accompany the motor manifestations of isolated adult-onset focal dystonias. Whether the body region affected when this type of dystonia first presents is associated with the severity of these neuropsychiatric symptoms is unknown. OBJECTIVES: The aim of this study was to determine whether depression, anxiety and social anxiety vary by dystonia onset site and evaluate whether pain and dystonia severity account for any differences. METHODS: Patients with isolated focal dystonia evaluated within 5 years from symptom onset, enrolled in the Natural History Project of the Dystonia Coalition, were included in the analysis. Individual onset sites were grouped into five body regions: cervical, laryngeal, limb, lower cranial and upper cranial. Neuropsychiatric symptoms were rated using the Beck Depression Inventory, Hospital Anxiety and Depression Scale and Liebowitz Social Anxiety Scale. Pain was estimated using the 36-Item Short Form Survey. RESULTS: Four hundred and seventy-eight subjects met our inclusion criteria. High levels of depression, anxiety and social anxiety occurred in all groups; however, the severity of anxiety and social anxiety symptoms varied by onset site group. The most pronounced differences were higher anxiety in cervical and laryngeal, lower anxiety in upper cranial and higher social anxiety in laryngeal. Increases in pain were associated with worse neuropsychiatric symptom scores within all groups. Higher anxiety and social anxiety in laryngeal and lower anxiety in upper cranial persisted after correcting for pain and dystonia severity. CONCLUSION: Anxiety and social anxiety severity vary by onset site of focal dystonia, and this variation is not explained by differences in pain and dystonia severity.

Regulation of the transcriptional program by DNA methylation during human αß T-cell development.

Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis.

Effects of Bacillus thuringiensis strains virulent to Varroa destructor on larvae and adults of Apis mellifera.

The sublethal effects of two strains of Bacillus thuringiensis, which were virulent in vitro to Varroa destructor, were measured on Apis mellifera. The effects of five concentrations of total protein (1, 5, 25, 50 and 100µg/mL) from the EA3 and EA26.1 strains on larval and adult honey bees were evaluated for two and seven days under laboratory conditions. Based on the concentrations evaluated, total protein from the two strains did not affect the development of larvae, the syrup consumption, locomotor activity or proboscis extension response of adults. These same parameters were also tested for the effects of three concentrations (1, 10 and 15µg/kg) of cypermethrin as a positive control. Although no significant differences were observed after two days of treatment with cypermethrin, a dose-response relationship in syrup consumption and locomotor activity was observed. A significant reduction in the proboscis extension response of the bees treated with cypermethrin was also observed. Therefore, in contrast to cypermethrin, our results indicate that the EA3 and EA26.1 strains of B. thuringiensis can be used in beehives to control V. destructor and reduce the negative effects of this mite on colonies without adverse effects on the larvae and adults of A. mellifera. Additionally, the overuse of synthetic miticides, which produce both lethal and sublethal effects on bees, can be reduced.