Several neurologic disorders have been treated with therapeutic plasma exchange (TPE). Case reports, case series, and clinical trials have published results regarding the outcomes in such patients. The data gathered have been used to formulate evidence-based guidelines, which can be used to guide therapy in patients with these neurological disorders. Adequately designed and powered randomized controlled trials have proven the efficacy of TPE in some disease entities, while other diseases are lacking such data. In the latter, decisions for the use of TPE must be made using the limited published data available. In this review, we discuss the published evidence regarding the use of TPE in neurological disorders, focusing on the most recent guidelines published by the American Society of Apheresis in 2010 and the American Academy of Neurology in 2011.
Sirolimus is an immunosuppressant used to prevent graft versus host disease in allogeneic hematopoietic stem cell transplant recipients. It has a large volume of distribution (12 +/- 7.5 l/kg) and within the intravascular space approximately 95% of it is bound to red blood cells. Because of potential toxic effects at high trough levels, therapeutic drug monitoring is recommended for sirolimus. We present a case of severe hepatic dysfunction due to Hepatitis B and sirolimus toxicity, in a 51-year-old male stem cell transplant recipient. An automated red cell exchange decreased his blood sirolimus level from 22.6 to 10.3 ng/ml (55% reduction) and improved his liver enzymes. Re-equilibration of sirolimus from other compartments to the blood necessitated a series of four red cell exchanges, after which the sirolimus level was 4.7 ng/ml. Although the patient ultimately succumbed to multiorgan failure, red cell exchange may be considered for acute removal of sirolimus in selected patients.
Prospective surveillance of D- recipients of D+ apheresis platelets: alloimmunization against D is not detected
BACKGROUND: Recent retrospective studies indicate that D- recipients of D+ apheresis platelets (PLTs) are not alloimmunized to D. Our hospital policy is to offer RhIG to D- women of childbearing age who received D+ apheresis PLTs but not to other D- recipients of D+ apheresis PLTs. We instituted prospective surveillance of the D- recipients who were not given RhIG.
STUDY DESIGN AND METHODS: All apheresis PLT recipients were prospectively entered into a database that recorded the patient's age, sex, diagnosis, D status, apheresis PLT transfusions, and antibody screen results from before and after PLT transfusions. Data are reported for PLTs transfused between October 16, 2012, and April 16, 2014, and antibody screens obtained through June 16, 2014. The analysis excludes neonates; women not more than 50 years of age; and patients who also received D+ red blood cells, received only D- PLTs, received RhIG, were previously alloimmunized to D, and did not have a follow-up antibody screen after the first D-incompatible apheresis PLT transfusion.
RESULTS: A total of 158 of 1107 apheresis PLT recipients were D-. Seventy-nine were eligible for analysis based on the exclusion criteria listed above. None became alloimmunized to D during the observation interval. In 45 (57%) cases the last follow-up antibody screen was obtained at least 4 weeks after the first D-incompatible apheresis PLT transfusion.
CONCLUSION: Prospective surveillance confirms prior retrospective observations that D- patients do not appear to risk D alloimmunization after receiving D+ apheresis PLTs.
Physiological measurements corroborate symptomatic improvement after therapeutic leukapheresis in a pregnant woman with chronic myelogenous leukemia
Therapeutic leukapheresis can control the white blood cell count (WBC) of pregnant women with chronic myelogenous leukemia (CML) who have hyperleukocytosis without leukostasis. The medical justification for this treatment has not been objectively documented. We report a 27-year-old woman, diagnosed with CML at 10-week gestation, who developed severe dyspnea on exertion. A workup that included chest CT and echocardiography with a bubble study detected no cardiopulmonary pathology to explain her symptoms, and thus she was referred for leukapheresis. Prior to her first leukapheresis, which lowered her WBC from 154 x 103 /muL to 133 x 103 /muL, her oxygen saturation (SpO2 ) on room air decreased from 98 to 93% during 100 feet of slow ambulation and she was dyspneic. Just after the leukapheresis, her dyspnea on exertion was much improved and her SpO2 remained at 98% with repeat ambulation. Spirometry and lung volume studies obtained before and after her first leukapheresis demonstrated 32 and 31% improvements in forced vital capacity and forced expiratory volume in 1 s respectively, a 25% increase in functional residual capacity, and a 142% improvement in expiratory reserve volume. Residual volume decreased by almost 20%. Three times in a week, leukapheresis was continued until her WBC was controlled with interferon alpha-2b approximately 4 weeks later. Her dyspnea had completely resolved. She gave birth by elective caesarean section to a healthy boy at 32 weeks. Corroboration of symptom relief by leukapheresis with physiological data may justify such treatment in pregnant patients with CML.
Therapeutic leukocytapheresis for improvement in respiratory function in a woman with hyperleukocytosis and mantle cell lymphoma with a circulating small lymphocyte phenotype
Mantle cell lymphoma is an aggressive malignant B-cell disorder that often presents with a leukemic picture. Circulating lymphoma cell morphology may vary from small round mature-appearing lymphocytes resembling the lymphocytes of chronic lymphocytic leukemia to large prolymphocytoid or blastoid cells. Rare reports of hyperleukocytosis with leukostasis, treated with leukocytapheresis, are described in patients with prolymphocytoid or blastoid morphology. We report an 88 year old woman with mantle cell lymphoma, hyperleukocytosis (WBC > 400 x 103 /microL) with severe respiratory compromise but without interstitial or alveolar infiltrates on radiograph or computerized tomography of the chest. She was afebrile and had no central nervous system signs. Circulating lymphoma cell morphology was predominantly of the small lymphocyte type. A two-whole-blood-volume leukocytapheresis reduced her WBC from 465 to 221 x 103 /microL in 150 min. Her respiratory rate decreased from 28/min to 18/min and her arterial oxygen saturation (SpO2 ) rose from 91% to 97% on 6 L/min of oxygen by nasal cannula. Severe breathlessness before the procedure abated completely by the end of the procedure. Respiratory compromise may occur in mantle cell lymphoma with hyperleukocytosis with a mature lymphoma cell phenotype, even without a clear picture of leukostasis. Although the ultimate survival of the patient depends on treatment with chemotherapy, leukocytapheresis for alleviation of symptoms may be warranted and should be considered. Respiratory status and response to leukocytapheresis should be documented with physiological measurements.
Although RNA-seq is a powerful tool, the considerable time and cost associated with library construction has limited its utilization for various applications. RNAtag-Seq, an approach to generate multiple RNA-seq libraries in a single reaction, lowers time and cost per sample, and it produces data on prokaryotic and eukaryotic samples that are comparable to those generated by traditional strand-specific RNA-seq approaches.
Understanding of mammalian enhancers is limited by the lack of a technology to rapidly and thoroughly test the cell type-specific function. Here, we use a nuclease-deficient Cas9 (dCas9)-histone demethylase fusion to functionally characterize previously described and new enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors.
Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses
CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes-derived Cas9 system (SpCas9) targeting Pten, a gene involved in NASH and a negative regulator of the PI3K-AKT pathway, in mouse liver. We found that the Ad vector mediated efficient Pten gene editing even in the presence of typical Ad vector-associated immunotoxicity in the liver. Four months after vector infusion, mice receiving the Pten gene-editing Ad vector showed massive hepatomegaly and features of NASH, consistent with the phenotypes following Cre-loxP-induced Pten deficiency in mouse liver. We also detected induction of humoral immunity against SpCas9 and the potential presence of an SpCas9-specific cellular immune response. Our findings provide a strategy to model human liver diseases in mice and highlight the importance considering Cas9-specific immune responses in future translational studies involving in vivo delivery of CRISPR/Cas9.
Evolution of proteins in bacteria and archaea living in different conditions leads to significant correlations between amino acid usage and environmental temperature. The origins of these correlations are poorly understood, and an important question of protein theory, physics-based prediction of types of amino acids overrepresented in highly thermostable proteins, remains largely unsolved. Here, we extend the random energy model of protein folding by weighting the interaction energies of amino acids by their frequencies in protein sequences and predict the energy gap of proteins designed to fold well at elevated temperatures. To test the model, we present a novel scalable algorithm for simultaneous energy calculation for many sequences in many structures, targeting massively parallel computing architectures such as graphics processing unit. The energy calculation is performed by multiplying two matrices, one representing the complete set of sequences, and the other describing the contact maps of all structural templates. An implementation of the algorithm for the CUDA platform is available at http://www.github.com/kzeldovich/galeprot and calculates protein folding energies over 250 times faster than a single central processing unit. Analysis of amino acid usage in 64-mer cubic lattice proteins designed to fold well at different temperatures demonstrates an excellent agreement between theoretical and simulated values of energy gap. The theoretical predictions of temperature trends of amino acid frequencies are significantly correlated with bioinformatics data on 191 bacteria and archaea, and highlight protein folding constraints as a fundamental selection pressure during thermal adaptation in biological evolution.
Novel Observations From Next-Generation RNA Sequencing of Highly Purified Human Adult and Fetal Islet Cell Subsets
Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic alpha- and beta-cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase beta-cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify alpha-, beta-, and delta-cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the subpopulations by flow cytometry, and, using next-generation RNA sequencing, we report the detailed transcriptomes of fetal and adult alpha- and beta-cells. We observed that human islet composition was not influenced by age, sex, or BMI, and transcripts for inflammatory gene products were noted in fetal beta-cells. In addition, within highly purified adult glucagon-expressing alpha-cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet alpha- and beta-cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes. long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
Ixodes scapularis is the principal vector of Lyme disease on the East Coast and in the upper Midwest regions of the United States, yet the tick is also present in the Southeast, where Lyme disease is absent or rare. A closely related species, I. affinis, also carries the pathogen in the South but does not seem to transmit it to humans. In order to better understand the geographic diversity of the tick, we analyzed the microbiota of 104 adult I. scapularis and 13 adult I. affinis ticks captured in 19 locations in South Carolina, North Carolina, Virginia, Connecticut, and New York. Initially, ticks from 4 sites were analyzed by 454 pyrosequencing. Subsequently, ticks from these sites plus 15 others were analyzed by sequencing with an Illumina MiSeq machine. By both analyses, the microbiomes of female ticks were significantly less diverse than those of male ticks. The dissimilarity between tick microbiomes increased with distance between sites, and the state in which a tick was collected could be inferred from its microbiota. The genus Rickettsia was prominent in all locations. Borrelia was also present in most locations and was present at especially high levels in one site in western Virginia. In contrast, members of the family Enterobacteriaceae were very common in North Carolina I. scapularis ticks but uncommon in I. scapularis ticks from other sites and in North Carolina I. affinis ticks. These data suggest substantial variations in the Ixodes microbiota in association with geography, species, and sex.
In Drosophila ovarian germ cells, PIWI-interacting RNAs (piRNAs) direct Aubergine and Argonaute3 to cleave transposon transcripts and instruct Piwi to repress transposon transcription, thereby safeguarding the germline genome. Here, we report that RNA cleavage by Argonaute3 initiates production of most Piwi-bound piRNAs. We find that the cardinal function of Argonaute3, whose piRNA guides predominantly correspond to sense transposon sequences, is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather than to make piRNAs that guide post-transcriptional silencing by Aubergine. We also find that the Tudor domain protein Qin prevents Aubergine's cleavage products from becoming Piwi-bound piRNAs, ensuring that antisense piRNAs guide Piwi. Although Argonaute3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. This alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs.
Plasmodium vivax infections often recur due to relapse of hypnozoites from the liver. In malaria-endemic areas, tools to distinguish relapse from reinfection are needed. We applied amplicon deep sequencing to P. vivax isolates from 78 Cambodian volunteers, nearly one-third of whom suffered recurrence at a median of 68 days. Deep sequencing at a highly variable region of the P. vivax merozoite surface protein 1 gene revealed impressive diversity-generating 67 unique haplotypes and detecting on average 3.6 cocirculating parasite clones within individuals, compared to 2.1 clones detected by a combination of 3 microsatellite markers. This diversity enabled a scheme to classify over half of recurrences as probable relapses based on the low probability of reinfection by multiple recurring variants. In areas of high P. vivax diversity, targeted deep sequencing can help detect genetic signatures of relapse, key to evaluating antivivax interventions and achieving a better understanding of relapse-reinfection epidemiology.
Differential Toxicity of Nuclear RNA Foci versus Dipeptide Repeat Proteins in a Drosophila Model of C9ORF72 FTD/ALS
Dipeptide repeat (DPR) proteins are toxic in various models of FTD/ALS with GGGGCC (G4C2) repeat expansion. However, it is unclear whether nuclear G4C2 RNA foci also induce neurotoxicity. Here, we describe a Drosophila model expressing 160 G4C2 repeats (160R) flanked by human intronic and exonic sequences. Spliced intronic 160R formed nuclear G4C2 sense RNA foci in glia and neurons about ten times more abundantly than in human neurons; however, they had little effect on global RNA processing and neuronal survival. In contrast, highly toxic 36R in the context of poly(A)(+) mRNA were exported to the cytoplasm, where DPR proteins were produced at >100-fold higher level than in 160R flies. Moreover, the modest toxicity of intronic 160R expressed at higher temperature correlated with increased DPR production, but not RNA foci. Thus, nuclear RNA foci are neutral intermediates or possibly neuroprotective through preventing G4C2 RNA export and subsequent DPR production.
Updates to the Integrated Protein-Protein Interaction Benchmarks: Docking Benchmark Version 5 and Affinity Benchmark Version 2
We present an updated and integrated version of our widely used protein-protein docking and binding affinity benchmarks. The benchmarks consist of non-redundant, high-quality structures of protein-protein complexes along with the unbound structures of their components. Fifty-five new complexes were added to the docking benchmark, 35 of which have experimentally measured binding affinities. These updated docking and affinity benchmarks now contain 230 and 179 entries, respectively. In particular, the number of antibody-antigen complexes has increased significantly, by 67% and 74% in the docking and affinity benchmarks, respectively. We tested previously developed docking and affinity prediction algorithms on the new cases. Considering only the top 10 docking predictions per benchmark case, a prediction accuracy of 38% is achieved on all 55 cases and up to 50% for the 32 rigid-body cases only. Predicted affinity scores are found to correlate with experimental binding energies up to r=0.52 overall and r=0.72 for the rigid complexes.
Duchenne muscular dystrophy (DMD), caused by mutations at the dystrophin gene, is the most common form of muscular dystrophy. There is no cure for DMD and current therapeutic approaches to restore dystrophin expression are only partially effective. The absence of dystrophin in muscle results in dysregulation of signaling pathways, which could be targets for disease therapy and drug discovery. Previously, we identified two exceptional Golden Retriever muscular dystrophy (GRMD) dogs that are mildly affected, have functional muscle, and normal lifespan despite the complete absence of dystrophin. Now, our data on linkage, whole-genome sequencing, and transcriptome analyses of these dogs compared to severely affected GRMD and control animals reveals that increased expression of Jagged1 gene, a known regulator of the Notch signaling pathway, is a hallmark of the mild phenotype. Functional analyses demonstrate that Jagged1 overexpression ameliorates the dystrophic phenotype, suggesting that Jagged1 may represent a target for DMD therapy in a dystrophin-independent manner.
Evaluation of preprocessing, mapping and postprocessing algorithms for analyzing whole genome bisulfite sequencing data
Cytosine methylation regulates many biological processes such as gene expression, chromatin structure and chromosome stability. The whole genome bisulfite sequencing (WGBS) technique measures the methylation level at each cytosine throughout the genome. There are an increasing number of publicly available pipelines for analyzing WGBS data, reflecting many choices of read mapping algorithms as well as preprocessing and postprocessing methods. We simulated single-end and paired-end reads based on three experimental data sets, and comprehensively evaluated 192 combinations of three preprocessing, five postprocessing and five widely used read mapping algorithms. We also compared paired-end data with single-end data at the same sequencing depth for performance of read mapping and methylation level estimation. Bismark and LAST were the most robust mapping algorithms. We found that Mott trimming and quality filtering individually improved the performance of both read mapping and methylation level estimation, but combining them did not lead to further improvement. Furthermore, we confirmed that paired-end sequencing reduced error rate and enhanced sensitivity for both read mapping and methylation level estimation, especially for short reads and in repetitive regions of the human genome.
Background: The effect of adiposity on bone mass in the early phases of inflammatory bowel disease (IBD) in children and adolescents is unclear.
Aims: To determine the role of adiposity on bone mass in the first 3 years of diagnosis of IBD.
Hypothesis: Increased adiposity will be associated with increased bone mass in both the controls and IBD subjects.
Setting: University tertiary institution.
Methods: Height-adjusted bone mineral density (BMD) z-scores of 25 subjects, age 13.97 ± 2.70y, diagnosed with IBD for < 4 years were compared to 24 controls, age 13.65 ± 2.60y. Overweight was defined as BMI of ≥85th but <95th percentile, and obesity as BMI ≥95thpercentile. Severity of IBD was determined by the Pediatric Crohn’s Disease Activity Index and Lichtiger Colitis Activity Index.
Results: Prior to stratification by BMI criterion, height-adjusted BMD z-scores were non-significantly lower in IBD subjects vs. controls for both the femoral neck (-0.8 ± 1.1 vs. -0.06 ± 1.1, p=0.070) and lumbar vertebrae (-0.4 ± 1.2 vs. 0.2 ± 1.2, p=0.086). Following stratification, height-adjusted BMD z-scores were significantly lower in the overweight/obese IBD subjects vs. overweight/obese controls for femoral neck (-0.9 ± 0.9 vs. 0.3 ± 1.3, p=0.032); and non-significantly lower for the lumbar spine z-score (-0.4 ± 1.6 vs. 0.5 ± 1.3, p=0.197). BMD z-score had no relationship with the duration of disease, steroid therapy, and the severity of disease.
Conclusion: Adiposity was associated with reduced bone mass in the early phases of IBD, but with increased bone mass in the controls.
High-throughput crystallographic approaches require integrated software solutions to minimize the need for manual effort. REdiii is a system that allows fully automated crystallographic structure solution by integrating existing crystallographic software into an adaptive and partly autonomous workflow engine. The program can be initiated after collecting the first frame of diffraction data and is able to perform processing, molecular-replacement phasing, chain tracing, ligand fitting and refinement without further user intervention. Preset values for each software component allow efficient progress with high-quality data and known parameters. The adaptive workflow engine can determine whether some parameters require modifications and choose alternative software strategies in case the preconfigured solution is inadequate. This integrated pipeline is targeted at providing a comprehensive and efficient approach to screening for ligand-bound co-crystal structures while minimizing repetitiveness and allowing a high-throughput scientific discovery process.
Deaminase activity mediated by the human APOBEC3 family of proteins contributes to genomic instability and cancer. APOBEC3A is by far the most active in this family and can cause rapid cell death when overexpressed, but in general how the activity of APOBEC3s is regulated on a molecular level is unclear. In this study, the biochemical and structural basis of APOBEC3A substrate binding and specificity is elucidated. We find that specific binding of single-stranded DNA is regulated by the cooperative dimerization of APOBEC3A. The crystal structure elucidates this homodimer as a symmetric domain swap of the N-terminal residues. This dimer interface provides insights into how cooperative protein-protein interactions may affect function in the APOBEC3 enzymes and provides a potential scaffold for strategies aimed at reducing their mutation load.