Regulatory T cell adoptive transfer alters uterine immune populations, increasing a novel MHC-IIlow macrophage associated with healthy pregnancy.

Intrauterine fetal demise (IUFD) - fetal loss after 20 weeks - affects 6 pregnancies per 1,000 live births in the United States, and the majority are of unknown etiology. Maternal systemic regulatory T cell (Treg) deficits have been implicated in fetal loss, but whether mucosal immune cells at the maternal-fetal interface contribute to fetal loss is under-explored. We hypothesized that the immune cell composition and function of the uterine mucosa would contribute to the pathogenesis of IUFD. To investigate local immune mechanisms of IUFD, we used the CBA mouse strain, which naturally has mid-late gestation fetal loss. We performed a Treg adoptive transfer and interrogated both pregnancy outcomes and the impact of systemic maternal Tregs on mucosal immune populations at the maternal-fetal interface. Treg transfer prevented fetal loss and increased an MHC-IIlow population of uterine macrophages. Single-cell RNA-sequencing was utilized to precisely evaluate the impact of systemic Tregs on uterine myeloid populations. A population of C1q+, Trem2+, MHC-IIlow uterine macrophages were increased in Treg-recipient mice. The transcriptional signature of this novel uterine macrophage subtype is enriched in multiple studies of human healthy decidual macrophages, suggesting a conserved role for these macrophages in preventing fetal loss.

DICER1 RNase IIIb domain mutations trigger widespread miRNA dysregulation and MAPK activation in pediatric thyroid cancer.

DICER1 is a highly conserved RNase III endoribonuclease essential for the biogenesis of single-stranded mature microRNAs (miRNAs) from stem-loop precursor miRNAs. Somatic mutations in the RNase IIIb domain of DICER1 impair its ability to generate mature 5p miRNAs and are believed to drive tumorigenesis in DICER1 syndrome-associated and sporadic thyroid tumors. However, the DICER1-driven specific changes in miRNAs and resulting changes in gene expression are poorly understood in thyroid tissue. In this study, we profiled the miRNA (n=2,083) and mRNA (n=2,559) transcriptomes of 20 non-neoplastic, 8 adenomatous and 60 pediatric thyroid cancers (13 follicular thyroid cancers [FTC] and 47 papillary thyroid cancers [PTC]) of which 8 had DICER1 RNase IIIb mutations. All DICER1-mutant differentiated thyroid cancers (DTC) were follicular patterned (six follicular variant PTC and two FTC), none had lymph node metastasis. We demonstrate that DICER1 pathogenic somatic mutations were associated with a global reduction of 5p-derived miRNAs, including those particularly abundant in the non-neoplastic thyroid tissue such as let-7 and mir-30 families, known for their tumor suppressor function. There was also an unexpected increase of 3p miRNAs, possibly associated with DICER1 mRNA expression increase in tumors harboring RNase IIIb mutations. These abnormally expressed 3p miRNAs, which are otherwise low or absent in DICER1-wt DTC and non-neoplastic thyroid tissues, make up exceptional markers for malignant thyroid tumors harboring DICER1 RNase IIIb mutations. The extensive disarray in the miRNA transcriptome results in gene expression changes, which were indicative of positive regulation of cell-cycle. Moreover, differentially expressed genes point to increased MAPK signaling output and loss of thyroid differentiation comparable to the RAS-like subgroup of PTC (as coined by The Cancer Genome Atlas), which is reflective of the more indolent clinical behavior of these tumors.

Fusion Oncogenes Are Associated With Increased Metastatic Capacity and Persistent Disease in Pediatric Thyroid Cancers.

PURPOSE: In 2014, data from a comprehensive multiplatform analysis of 496 adult papillary thyroid cancer samples reported by The Cancer Genome Atlas project suggested that reclassification of thyroid cancer into molecular subtypes, RAS-like and BRAF-like, better reflects clinical behavior than sole reliance on pathologic classification. The aim of this study was to categorize the common oncogenic variants in pediatric differentiated thyroid cancer (DTC) and investigate whether mutation subtype classification correlated with the risk of metastasis and response to initial therapy in pediatric DTC. METHODS: Somatic cancer gene panel analysis was completed on DTC from 131 pediatric patients. DTC were categorized into RAS-mutant (H-K-NRAS), BRAF-mutant (BRAF p.V600E), and RET/NTRK fusion (RET, NTRK1, and NTRK3 fusions) to determine differences between subtype classification in regard to pathologic data (American Joint Committee on Cancer TNM) as well as response to therapy 1 year after initial treatment had been completed. RESULTS: Mutation-based subtype categories were significant in most variables, including age at diagnosis, metastatic behavior, and the likelihood of remission at 1 year. Patients with RET/NTRK fusions were significantly more likely to have advanced lymph node and distant metastasis and less likely to achieve remission at 1 year than patients within RAS- or BRAF-mut subgroups. CONCLUSION: Our data support that genetic subtyping of pediatric DTC more accurately reflects clinical behavior than sole reliance on pathologic classification with patients with RET/NTRK fusions having worse outcomes than those with BRAF-mutant disease. Future trials should consider inclusion of molecular subtype into risk stratification.

Scedar: A scalable Python package for single-cell RNA-seq exploratory data analysis.

In single-cell RNA-seq (scRNA-seq) experiments, the number of individual cells has increased exponentially, and the sequencing depth of each cell has decreased significantly. As a result, analyzing scRNA-seq data requires extensive considerations of program efficiency and method selection. In order to reduce the complexity of scRNA-seq data analysis, we present scedar, a scalable Python package for scRNA-seq exploratory data analysis. The package provides a convenient and reliable interface for performing visualization, imputation of gene dropouts, detection of rare transcriptomic profiles, and clustering on large-scale scRNA-seq datasets. The analytical methods are efficient, and they also do not assume that the data follow certain statistical distributions. The package is extensible and modular, which would facilitate the further development of functionalities for future requirements with the open-source development community. The scedar package is distributed under the terms of the MIT license at https://pypi.org/project/scedar.

Pch Genes Control Biofilm and Cell Adhesion in a Clinical Serotype O157:H7 Isolate.

In a previous study, induction of the Escherichia coli serotype O157:H7 SOS response decreased csgD expression in the clinical isolate PA20 at 30°C but strongly induced genes in the horizontally transferred-DNA regions (HTR), including many known virulence regulators. To determine the role of HTR regulators in the control of csgD and curli, specific regulators were plasmid-expressed in the wild-type and mutant strains of PA20 and its biofilm-forming derivative, 20R2R. At 30°C, plasmid over-expression of the O157:H7 group 3 perC homolog, pchE, strongly repressed PA20 csgD transcription (>7-fold) while the group 1 homologs, pchA and pchB, resulted in smaller reductions (<2.5-fold). However, SOS induction decreased rather than increased pchE expression (>6-fold) making group 1 pch, which are enhanced by the SOS response, the likely SOS-induced csgD repressors. Plasmid-based pchE over-expression also reduced 20R2R biofilm formation (>6-fold) and the curli-dependent, Congo red affinity of both PA20 and 20R2R. However, to properly appreciate the regulatory direction, expression patterns, and environmental consequences of these and other CsgD-controlled functions, a better understanding of natural pchE regulation will be required. The effects of HTR regulators on PA20 and 20R2R adhesion to HEp-2 cell at host temperature were also studied. Under conditions where prophage genes were not induced, curli, rather than espA, contributed to host cell adhesion in strain 20R2R. High levels of pchE expression in trans reduced curli-dependent cell adherence (>2-fold) to both 20R2R and the clinical isolate PA20, providing a host-adapting adhesion control mechanism. Expression of pchE was also repressed by induction of the SOS response at 37°C, providing a mechanism by which curli expression might complement EspA-dependent intimate adhesion initiated by the group1 pch homologs. This study has increased our understanding of the O157 pch genes at both host and environment temperatures, identifying pchE as a strong regulator of csgD and CsgD-dependent properties.

Sulfamethoxazole - Trimethoprim represses csgD but maintains virulence genes at 30°C in a clinical Escherichia coli O157:H7 isolate.

The high frequency of prophage insertions in the mlrA gene of clinical serotype O157:H7 isolates renders such strains deficient in csgD-dependent biofilm formation but prophage induction may restore certain mlrA properties. In this study we used transcriptomics to study the effect of high and low sulfamethoxazole-trimethoprim (SMX-TM) concentrations on prophage induction, biofilm regulation, and virulence gene expression in strain PA20 under environmental conditions following 5-hour and 12-hour exposures in broth or on agar. SMX-TM at a sub-lethal concentration induced strong RecA expression resulting in concentration- and time-dependent major transcriptional shifts with emphasis on up-regulation of genes within horizontally-transferred chromosomal regions (HTR). Neither high or low levels of SMX-TM stimulated csgD expression at either time point, but both levels resulted in slight repression. Full expression of Ler-dependent genes paralleled expression of group 1 pch homologues in the presence of high glrA. Finally, stx2 expression, which is strongly dependent on prophage induction, was enhanced at 12 hours but repressed at five hours, in spite of early SOS initiation by the high SMX-TM concentration. Our findings indicate that, similar to host conditions, exposure to environmental conditions increased the expression of virulence genes in a clinical isolate but genes involved in the protective biofilm response were repressed.

Whole-Genome Sequence of Escherichia coli Serotype O157:H7 Strain B6914-ARS.

Escherichia coli serotype O157:H7 strain B6914-MS1 is an isolate from the Centers for Disease Control and Prevention that is missing both Shiga toxin genes and has been used extensively in applied research studies. Here we report the genome sequence of strain B6914-ARS, a B6914-MS1 clone that has unique biofilm properties.

Genome Sequences of 34 Shiga Toxin-Producing Escherichia coli Isolates from Swine and Other Sources.

Shiga toxin-producing Escherichia coli (STEC) bacteria are foodborne pathogens that can be carried by various animals. The swine STEC population is partially composed of host-specific strains that are often not well characterized. In this work, the genome sequences of a number of swine STEC strains are presented.

Complete Genome Sequence of Campylobacter jejuni RM1246-ERRC, Which Exhibits Resistance to Quaternary Ammonium Compounds.

Campylobacter jejuni strain RM1246-ERRC is a clinical isolate. In laboratory experiments, RM1246-ERRC exhibited greater resistance to the antimicrobial effects of quaternary ammonium compounds than other C. jejuni strains. The chromosome of RM1246-ERRC is 1,659,694 bp with a G+C content of 30.56%. The strain also possesses a 45,197-bp plasmid.

Complete Genome Sequences of Escherichia coli O157:H7 Strains SRCC 1675 and 28RC, Which Vary in Acid Resistance.

The level of acid resistance among Escherichia coli O157:H7 strains varies, and strains with higher resistance to acid may have a lower infectious dose. The complete genome sequences belonging to two strains of Escherichia coli O157:H7 with different levels of acid resistance are presented here.

Complete Genome Sequence of UV-Resistant Campylobacter jejuni RM3194, Including an 81.08-Kilobase Plasmid.

Campylobacter jejuni strain RM3194 was originally isolated from a human with enteritis and contains a novel 81,079-bp plasmid. RM3194 has exhibited superior survival compared to other Campylobacter jejuni strains when challenged with UV light. The chromosome of RM3194 was determined to be 1,651,183 bp, with a G+C content of 30.5%.

Prokaryotic nucleotide composition is shaped by both phylogeny and the environment.

The causes of the great variation in nucleotide composition of prokaryotic genomes have long been disputed. Here, we use extensive metagenomic and whole-genome data to demonstrate that both phylogeny and the environment shape prokaryotic nucleotide content. We show that across environments, various phyla are characterized by different mean guanine and cytosine (GC) values as well as by the extent of variation on that mean value. At the same time, we show that GC-content varies greatly as a function of environment, in a manner that cannot be entirely explained by disparities in phylogenetic composition. We find environmentally driven differences in nucleotide content not only between highly diverged environments (e.g., soil, vs. aquatic vs. human gut) but also within a single type of environment. More specifically, we demonstrate that some human guts are associated with a microbiome that is consistently more GC-rich across phyla, whereas others are associated with a more AT-rich microbiome. These differences appear to be driven both by variations in phylogenetic composition and by environmental differences-which are independent of these phylogenetic composition differences. Combined, our results demonstrate that both phylogeny and the environment significantly affect nucleotide composition and that the environmental differences affecting nucleotide composition are far subtler than previously appreciated.

A toolkit for ARB to integrate custom databases and externally built phylogenies.

UNLABELLED: Researchers are perpetually amassing biological sequence data. The computational approaches employed by ecologists for organizing this data (e.g. alignment, phylogeny, etc.) typically scale nonlinearly in execution time with the size of the dataset. This often serves as a bottleneck for processing experimental data since many molecular studies are characterized by massive datasets. To keep up with experimental data demands, ecologists are forced to choose between continually upgrading expensive in-house computer hardware or outsourcing the most demanding computations to the cloud. Outsourcing is attractive since it is the least expensive option, but does not necessarily allow direct user interaction with the data for exploratory analysis. Desktop analytical tools such as ARB are indispensable for this purpose, but they do not necessarily offer a convenient solution for the coordination and integration of datasets between local and outsourced destinations. Therefore, researchers are currently left with an undesirable tradeoff between computational throughput and analytical capability. To mitigate this tradeoff we introduce a software package to leverage the utility of the interactive exploratory tools offered by ARB with the computational throughput of cloud-based resources. Our pipeline serves as middleware between the desktop and the cloud allowing researchers to form local custom databases containing sequences and metadata from multiple resources and a method for linking data outsourced for computation back to the local database. A tutorial implementation of the toolkit is provided in the supporting information, S1 Tutorial. AVAILABILITY: http://www.ece.drexel.edu/gailr/EESI/tutorial.php.

Plasma amino acids changes in complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is a severe chronic pain condition that most often develops following trauma. Blood samples were collected from 220 individuals, 160 CRPS subjects, and 60 healthy pain-free controls. Plasma amino acid levels were compared and contrasted between groups. L-Aspartate, L-glutamate, and L-ornithine were significantly increased, whereas L-tryptophan and L-arginine were significantly decreased in CRPS subjects as compared to controls. In addition, the L-kynurenine to L-tryptophan ratio demonstrated a significant increase, whereas the global arginine bioavailability ratio (GABR) was significantly decreased in the CRPS subjects. The CRPS subjects demonstrated a significant correlation between overall pain and the plasma levels of L-glutamate and the L-kynurenine to L-tryptophan ratio. CRPS subjects also showed a correlation between the decrease in plasma L-tryptophan and disease duration. This study shows that CRPS subjects exhibit significant changes in plasma levels of amino acids involved in glutamate receptor activation and in amino acids associated with immune function as compared to healthy pain-free controls. A better understanding of the role plasma amino acids play in the pathophysiology of CRPS may lead to novel treatments for this crippling condition.

Establishing a relationship between bacteria in the human gut and complex regional pain syndrome.

Complex Regional Pain Syndrome (CRPS) is a serious and painful condition involving the peripheral and central nervous systems. Full comprehension of the disorder's pathophysiology remains incomplete, but research implicates the immune system as a contributor to chronic pain. Because of the impact gastrointestinal bacteria have in the development and behavior of the immune system, this study compares the GI microbial communities of 16 participants with CRPS (5 of whom have intestinal discomforts) and 16 healthy controls using 454 sequencing technology. CRPS subjects were found to have significantly less diversity than their healthy counterparts. Statistical analysis of the phylogenetic classifications revealed significantly increased levels of Proteobacteria and decreased levels of Firmicutes in CRPS subjects. Clustering analysis showed significant separation between healthy controls and CRPS subjects. These results support the hypothesis that the GI microbial communities of CRPS participants differ from those of their healthy counterparts. These variations may hold the key to understanding how CRPS develops and provide information that could yield a potential treatment.

NBC: the Naive Bayes Classification tool webserver for taxonomic classification of metagenomic reads.

MOTIVATION: Datasets from high-throughput sequencing technologies have yielded a vast amount of data about organisms in environmental samples. Yet, it is still a challenge to assess the exact organism content in these samples because the task of taxonomic classification is too computationally complex to annotate all reads in a dataset. An easy-to-use webserver is needed to process these reads. While many methods exist, only a few are publicly available on webservers, and out of those, most do not annotate all reads. RESULTS: We introduce a webserver that implements the naïve Bayes classifier (NBC) to classify all metagenomic reads to their best taxonomic match. Results indicate that NBC can assign next-generation sequencing reads to their taxonomic classification and can find significant populations of genera that other classifiers may miss. AVAILABILITY: Publicly available at: http://nbc.ece.drexel.edu.

Outpatient intravenous ketamine for the treatment of complex regional pain syndrome: a double-blind placebo controlled study.

Complex regional pain syndrome (CRPS) is a severe chronic pain condition that most often develops following trauma. The pathophysiology of CRPS is not known but both clinical and experimental evidence demonstrate the important of the NMDA receptor and glial activation in its induction and maintenance. Ketamine is the most potent clinically available safe NMDA antagonist that has a well established role in the treatment of acute and chronic pain. This randomized double-blind placebo controlled trial was designed to evaluate the effectiveness of intravenous ketamine in the treatment of CRPS. Before treatment, after informed consent was obtained, each subject was randomized into a ketamine or a placebo infusion group. Study subjects were evaluated for at least 2 weeks prior to treatment and for 3 months following treatment. All subjects were infused intravenously with normal saline with or without ketamine for 4h (25ml/h) daily for 10 days. The maximum ketamine infusion rate was 0.35mg/kg/h, not to exceed 25mg/h over a 4h period. Subjects in both the ketamine and placebo groups were administered clonidine and versed. This study showed that intravenous ketamine administered in an outpatient setting resulted in statistically significant (p<0.05) reductions in many pain parameters. It also showed that subjects in our placebo group demonstrated no treatment effect in any parameter. The results of this study warrant a larger randomized placebo controlled trial using higher doses of ketamine and a longer follow-up period.

High-dose cyclophosphamide in the treatment of multiple sclerosis.

High dose cyclophosphamide (HDC) has been successfully used for the treatment of a variety of autoimmune diseases. In this study, we sought to determine whether the use of high dose cyclophosphamide provided stabilization of relapsing remitting MS (RRMS), secondary progressive MS (SPMS), or primary progressive MS (PPMS). The parameters evaluated were EDSS scores, lesion load and brain volumes by MRI and frequency of relapses. Twenty-three patients underwent immunoablative therapy with HDC and were followed for 3.5 years. Nine were relapsing remitting (RRMS), 11 secondary progressive (SPMS), and 3 primary progressive (PPMS). Four of 9 RRMS have had no clinical progression up to 3.5 years following treatment. Three of 9 patients maintained a normal neurologic examination with improved EDSS scores. Seven of the nine RRMS patients had reduction in flare frequency which was maintained for 3.5 years following treatment or no immunomodulating agents. Subgroup analysis in the RRMS patients of lesion load and brain parenchymal volume revealed a favorable trend in these parameters which did not reach statistical significance. The treatment was generally ineffective for SPMS and failed in the 2 PPMS patients. HDC was well tolerated, demonstrated a good safety profile and had minimal adverse effects. These results along with previous reports suggest that early use of HDC therapy in RRMS is promising.

Oligomerization state-dependent elevations of adiponectin in chronic daily headache.

OBJECTIVE: To evaluate serum adiponectin levels in female episodic migraineurs (EMs) and chronic daily headache (CDH) sufferers. BACKGROUND: Obesity is a risk factor for headache "chronification." Adiponectin (ADP) is an adipocytokine secreted primarily by adipose tissue. ADP and its oligomers (high-molecular-weight [HMW], middle-molecular-weight [MMW], and low-molecular-weight [LMW] ADP) have been shown to modulate several inflammatory pathways that have also been shown to be associated with migraine pathophysiology. METHODS: Age- and body mass index (BMI)-matched women participants were enrolled. Anthropometric measures (including waist-to-hip ratio [WHR] and BMI) were measured in all participants. Serum total ADP (T-ADP) levels and its oligomers were measured in EMs during headache-free periods and CDH sufferers at baseline level of pain, as compared with healthy control subjects using ELISA. RESULTS: Although total body obesity as estimated by BMI showed no significant association between participants, visceral obesity as estimated by WHR was significantly associated with CDH as compared with EMs and controls. WHR was also inversely related to both T-ADP (p = 0.008) and HMW-ADP (p = 0.002). After adjusting for WHR, serum T-ADP levels were higher in CDH sufferers (10.1 +/- 4.0) than in both EMs (8.6 +/- 3.5) and controls (7.5 +/- 2.4) (p = 0.024). In addition, HMW-ADP was higher in CDH (6.1 +/- 2.8) as compared with EMs (4.2 +/- 1.7) and controls (3.9 +/- 1.5) (p = 0.003). MMW-ADP was also higher in CDH (2.0 +/- 1.2) as compared with EMs (1.5 +/- 0.7) and controls (1.1 +/- 0.4) (p = 0.009). CONCLUSION: Serum adiponectin levels are increased in women chronic daily headache (CDH) sufferers. In addition, visceral obesity, as measured by waist-to-hip ratio, is a risk factor for CDH in women.

Changes in immune and glial markers in the CSF of patients with Complex Regional Pain Syndrome.

Complex Regional Pain Syndrome is a severe chronic pain condition characterized by sensory, autonomic, motor and dystrophic signs and symptoms. The pain in CRPS is continuous, it worsens over time, and it is usually disproportionate to the severity and duration of the inciting event. This study compares cerebrospinal fluid (CSF) levels of pro- and anti-inflammatory cytokines, chemokines and several biochemical factors (glial fibrillary acidic protein (GFAP), the nitric oxide metabolites (nitrate plus nitrite), the excitatory amino acid neurotransmitter glutamate, calcium, total protein and glucose) in patients afflicted with CRPS to levels found in patients suffering with other non-painful or painful conditions. The aim of the study is to determine the degree of involvement of glial cells and immune system mediators in the pathophysiology of CRPS. There was no elevation or reduction of a CSF marker that was specific for CRPS patients. However, there were several patterns of markers that could be helpful in both elucidating the mechanisms involved in the disease process and supporting the diagnosis of CRPS. The most common pattern was found in 50% (11 out of 22) of the CRPS patients and consisted of; elevated IL-6, low levels of IL-4 or IL-10, increased GFAP or MCP1 and increases in at least two of the following markers NO metabolites, calcium or glutamate. The results from this and other similar studies may aid in elucidating the mechanisms involved in the pathophysiology of CRPS. A better understanding of these mechanisms may lead to novel treatments for this very severe, life-altering illness.