Feasibility of fine needle aspiration for diagnosis of b-cell lymphoma of the thyroid: a case series and review of the literature.

BACKGROUND: Primary thyroid lymphoma (PTL) is a rare cancer accounting for approximately 5% of thyroid malignancies. Historically, incisional biopsy has been the gold standard for definitive diagnosis of PTL, however, the use of cell block as an adjunct to fine needle aspiration (FNA) provides a high sensitivity and specificity for diagnosis and classification. METHODS: Three patients presented with a symptomatic enlarging thyroid mass. Patient 1 underwent incisional biopsy under general anesthesia, Patient 2 underwent core needle biopsy to avoid high risk intubation, and Patient 3 underwent fine needle aspiration alone with the use of cell block. RESULTS: All patients were diagnosed with a fully classified non-Hodgkin's lymphoma using immunohistochemistry, flow cytometry, and fluorescence in situ hybridization (FISH) analysis. CONCLUSIONS: FNA for diagnosis of some subtypes of PTL is feasible and preferred in cases that are particularly high risk for general anesthesia. This minimally invasive technique is safe and cost effective as it avoids expenses associated with operative intervention.

Localized modulation of DNA supercoiling, triggered by the Shigella anti-silencer VirB, is sufficient to relieve H-NS-mediated silencing.

In Bacteria, nucleoid structuring proteins govern nucleoid dynamics and regulate transcription. In Shigella spp., at ≤30°C, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences many genes on the large virulence plasmid. Upon a switch to 37°C, VirB, a DNA binding protein and key transcriptional regulator of Shigella virulence, is produced. VirB functions to counter H-NS-mediated silencing in a process called transcriptional anti-silencing. Here, we show that VirB mediates a loss of negative DNA supercoils from our plasmid-borne, VirB-regulated PicsP-lacZ reporter in vivo. The changes are not caused by a VirB-dependent increase in transcription, nor do they require the presence of H-NS. Instead, the VirB-dependent change in DNA supercoiling requires the interaction of VirB with its DNA binding site, a critical first step in VirB-dependent gene regulation. Using two complementary approaches, we show that VirB:DNA interactions in vitro introduce positive supercoils in plasmid DNA. Subsequently, by exploiting transcription-coupled DNA supercoiling, we reveal that a localized loss of negative supercoils is sufficient to alleviate H-NS-mediated transcriptional silencing independently of VirB. Together, our findings provide novel insight into VirB, a central regulator of Shigella virulence and, more broadly, a molecular mechanism that offsets H-NS-dependent silencing of transcription in bacteria.

Localized modulation of DNA supercoiling, triggered by the Shigella anti-silencer VirB, is sufficient to relieve H-NS-mediated silencing.

In Bacteria, nucleoid structuring proteins govern nucleoid dynamics and regulate transcription. In Shigella spp ., at ≤ 30 °C, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences many genes on the large virulence plasmid. Upon a switch to 37 °C, VirB, a DNA binding protein and key transcriptional regulator of Shigella virulence, is produced. VirB functions to counter H-NS-mediated silencing in a process called transcriptional anti-silencing. Here, we show that VirB mediates a loss of negative DNA supercoils from our plasmid-borne, VirB-regulated PicsP-lacZ reporter, in vivo . The changes are not caused by a VirB-dependent increase in transcription, nor do they require the presence of H-NS. Instead, the VirB-dependent change in DNA supercoiling requires the interaction of VirB with its DNA binding site, a critical first step in VirB-dependent gene regulation. Using two complementary approaches, we show that VirB:DNA interactions in vitro introduce positive supercoils in plasmid DNA. Subsequently, by exploiting transcription-coupled DNA supercoiling, we reveal that a localized loss of negative supercoils is sufficient to alleviate H-NS-mediated transcriptional silencing, independently of VirB. Together, our findings provide novel insight into VirB, a central regulator of Shigella virulence and more broadly, a molecular mechanism that offsets H-NS-dependent silencing of transcription in bacteria.

Curated multiple sequence alignment for the Adenomatous Polyposis Coli (APC) gene and accuracy of in silico pathogenicity predictions.

Computational algorithms are often used to assess pathogenicity of Variants of Uncertain Significance (VUS) that are found in disease-associated genes. Most computational methods include analysis of protein multiple sequence alignments (PMSA), assessing interspecies variation. Careful validation of PMSA-based methods has been done for relatively few genes, partially because creation of curated PMSAs is labor-intensive. We assessed how PMSA-based computational tools predict the effects of the missense changes in the APC gene, in which pathogenic variants cause Familial Adenomatous Polyposis. Most Pathogenic or Likely Pathogenic APC variants are protein-truncating changes. However, public databases now contain thousands of variants reported as missense. We created a curated APC PMSA that contained >3 substitutions/site, which is large enough for statistically robust in silico analysis. The creation of the PMSA was not easily automated, requiring significant querying and computational analysis of protein and genome sequences. Of 1924 missense APC variants in the NCBI ClinVar database, 1800 (93.5%) are reported as VUS. All but two missense variants listed as P/LP occur at canonical splice or Exonic Splice Enhancer sites. Pathogenicity predictions by five computational tools (Align-GVGD, SIFT, PolyPhen2, MAPP, REVEL) differed widely in their predictions of Pathogenic/Likely Pathogenic (range 17.5-75.0%) and Benign/Likely Benign (range 25.0-82.5%) for APC missense variants in ClinVar. When applied to 21 missense variants reported in ClinVar and securely classified as Benign, the five methods ranged in accuracy from 76.2-100%. Computational PMSA-based methods can be an excellent classifier for variants of some hereditary cancer genes. However, there may be characteristics of the APC gene and protein that confound the results of in silico algorithms. A systematic study of these features could greatly improve the automation of alignment-based techniques and the use of predictive algorithms in hereditary cancer genes.

Opioid-Related Sleep-Disordered Breathing: An Update for Clinicians.

Opioids are an effective treatment for patients with intractable pain. Long-term administration of opioids for pain relief is being delivered by an increasing number of medical providers in the United States including primary care physicians and nonspecialists. One common complication of chronic opioid use is sleep-disordered breathing which can result in various morbidities as well as an increase in all-cause mortality. It is important for providers to understand the relationship between opioids and sleep-disordered breathing as well as methods to improve diagnosis and strategies for treatment. This review aims to update clinicians on the mechanism, diagnosis, and treatment of opioid-related sleep-disordered breathing in order to improve the quality of care for patients with chronic pain.

Pulmonary Haemosiderosis Secondary to Hereditary Haemochromatosis; a Case Report.

Introduction: Hereditary haemochromatosis (HH) is an autosomal recessive disease of increased intestinal absorption of iron, leading to accumulation in tissues which may progress to organ damage, most commonly in the liver. Iron deposition in the liver can lead to cirrhosis and hepatocellular carcinoma. Other common manifestations of haemochromatosis include diabetes, bronzing of the skin, arthropathy and cardiomyopathy. Here, we describe a case of pulmonary haemosiderosis secondary to HH. Case Description: A 49-year-old male with no medical history or family history of iron overload presented with fatigue, shortness of breath and chest pain after a recent finding of elevated ferritin. The patient was found to have biallelic C282Y mutations of the human homeostatic iron regulator protein (HFE) protein and after further workup with laboratory tests and imaging was diagnosed with HH with secondary pulmonary haemosiderosis. The patient is receiving twice weekly phlebotomies and has had an overall improvement in his symptoms. Practical Implications: The presentation of haemochromatosis can vary widely depending on the severity of iron overload and the presence of conditions that predispose organ dysfunction. Pulmonary haemosiderosis is a very rare manifestation of HH. This report illustrates the various manifestations of this disease and provides insight into this rare presentation to improve the diagnosis of this disease.

Investigating the DNA-Binding Site for VirB, a Key Transcriptional Regulator of Shigella Virulence Genes, Using an In Vivo Binding Tool.

The transcriptional anti-silencing and DNA-binding protein, VirB, is essential for the virulence of Shigella species and, yet, sequences required for VirB-DNA binding are poorly understood. While a 7-8 bp VirB-binding site has been proposed, it was derived from studies at a single VirB-dependent promoter, icsB. Our previous in vivo studies at a different VirB-dependent promoter, icsP, found that the proposed VirB-binding site was insufficient for regulation. Instead, the required site was found to be organized as a near-perfect inverted repeat separated by a single nucleotide spacer. Thus, the proposed 7-8 bp VirB-binding site needed to be re-evaluated. Here, we engineer and validate a molecular tool to capture protein-DNA binding interactions in vivo. Our data show that a sequence organized as a near-perfect inverted repeat is required for VirB-DNA binding interactions in vivo at both the icsB and icsP promoters. Furthermore, the previously proposed VirB-binding site and multiple sites found as a result of its description (i.e., sites located at the virB, virF, spa15, and virA promoters) are not sufficient for VirB to bind in vivo using this tool. The implications of these findings are discussed.

Insights into transcriptional silencing and anti-silencing in Shigella flexneri: a detailed molecular analysis of the icsP virulence locus.

Transcriptional silencing and anti-silencing mechanisms modulate bacterial physiology and virulence in many human pathogens. In Shigella species, many virulence plasmid genes are silenced by the histone-like nucleoid structuring protein H-NS and anti-silenced by the virulence gene regulator VirB. Despite the key role that these regulatory proteins play in Shigella virulence, their mechanisms of transcriptional control remain poorly understood. Here, we characterize the regulatory elements and their relative spacing requirements needed for the transcriptional silencing and anti-silencing of icsP, a locus that requires remotely located regulatory elements for both types of transcriptional control. Our findings highlight the flexibility of the regulatory elements' positions with respect to each other, and yet, a molecular roadblock docked between the VirB binding site and the upstream H-NS binding region abolishes transcriptional anti-silencing by VirB, providing insight into transcriptional anti-silencing. Our study also raises the need to re-evaluate the currently proposed VirB binding site. Models of transcriptional silencing and anti-silencing at this genetic locus are presented, and the implications for understanding these regulatory mechanisms in bacteria are discussed.