Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels

What Extent of Pancreatic Resection Do Patients with MEN-1 Require?

Context The surgical management of pancreatic endocrine tumors in patients with multiple endocrine neoplasia type 1 (MEN-1) is controversial and complicated by the fact that these tumors are frequently multifocal. The degree of tumor resection is determined by weighing the risk of malignancy or tumor recurrence against the risks of endocrine/exocrine insufficiency with complete gland removal. Methods A retrospective review was performed identifying 4 patients with MEN-1 and pancreatic endocrine tumors treated with pancreatic resection over a 2-year period at our institution. Results Mean age at operation was 35 years. Surgical approach was determined by size of tumor(s) and presence of multifocality. MRI and EUS were performed in all patients. While EUS identified a greater number of tumors when compared to MRI (median 5 versus 1), both studies grossly underestimated the total number of tumors found on final pathology. Three patients underwent laparoscopic total pancreatectomy for multifocal disease with diffuse pancreatic involvement, finding a median of 12 tumors. One patient underwent laparoscopic subtotal pancreatectomy for a presumed single pancreatic tail mass, but was found to have multifocal disease on final pathology consisting of 7 tumors. The average number of tumors found on final pathology was 13.5 with an average size of 2.6 cm. The median number of lymph nodes analyzed was 14. Diffuse, multifocal disease was present in all 4 patients. No major postoperative complications were observed. Conclusion In patients with MEN-1 and pancreatic endocrine tumors, preoperative workup underestimates extent of disease and total pancreatectomy should be considered for complete tumor removal.Image: Mayo Clinic Hospital, Jacksonville. FL, USA

The Role of Preoperative Bilateral Breast Magnetic Resonance Imaging in Patient Selection for Partial Breast Irradiation in Ductal Carcinoma In Situ

Purpose. Women with ductal carcinoma in situ (DCIS) are often candidates for breast-conserving therapy, and one option for radiation treatment is partial breast irradiation (PBI). This study evaluates the use of preoperative breast magnetic resonance imaging (MRI) for PBI selection in DCIS patients. Methods. Between 2002 and 2009, 136 women with newly diagnosed DCIS underwent a preoperative bilateral breast MRI at Mayo Clinic in Florida. One hundred seventeen women were deemed eligible for PBI by the NSABP B-39 (National Surgical Adjuvant Breast and Bowel Project, Protocol B-39) inclusion criteria using physical examination, mammogram, and/or ultrasound. MRIs were reviewed for their impact on patient eligibility, and findings were pathologically confirmed. Results. Of the 117 patients, 23 (20%) were found ineligible because of pathologically proven MRI findings. MRI detected additional ipsilateral breast cancer in 21 (18%) patients. Of these women, 15 (13%) had more extensive disease than originally noted before MRI, and 6 (5%) had multicentric disease in the ipsilateral breast. In addition, contralateral breast cancer was detected in 4 (4%). Conclusions. Preoperative breast MRI altered the PBI recommendations for 20% of women. Bilateral breast MRI should be an integral part of the preoperative evaluation of all patients with DCIS being considered for PBI

Host-microbe multiomic profiling reveals age-dependent immune dysregulation associated with COVID-19 immunopathology

Age is a major risk factor for severe coronavirus disease 2019 (COVID-19), yet the mechanisms behind this relationship have remained incompletely understood. To address this, we evaluated the impact of aging on host immune response in the blood and the upper airway, as well as the nasal microbiome in a prospective, multicenter cohort of 1031 vaccine-naïve patients hospitalized for COVID-19 between 18 and 96 years old. We performed mass cytometry, serum protein profiling, anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody assays, and blood and nasal transcriptomics. We found that older age correlated with increased SARS-CoV-2 viral abundance upon hospital admission, delayed viral clearance, and increased type I interferon gene expression in both the blood and upper airway. We also observed age-dependent up-regulation of innate immune signaling pathways and down-regulation of adaptive immune signaling pathways. Older adults had lower naïve T and B cell populations and higher monocyte populations. Over time, older adults demonstrated a sustained induction of pro-inflammatory genes and serum chemokines compared with younger individuals, suggesting an age-dependent impairment in inflammation resolution. Transcriptional and protein biomarkers of disease severity differed with age, with the oldest adults exhibiting greater expression of pro-inflammatory genes and proteins in severe disease. Together, our study finds that aging is associated with impaired viral clearance, dysregulated immune signaling, and persistent and potentially pathologic activation of pro-inflammatory genes and proteins

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS

Ongoing Policy, Regulatory, and Competitive Challenges Facing Canada's Small Internet Service Providers

Abstract Many rural and remote Canadians continue to experience a digital divide. Internet Service Providers (ISPs) operating in these communities play a crucial role in connecting Canadians, yet they are understudied. This study aims to address this gap in the literature by focusing on policy, regulatory, and competitive challenges and issues facing small ISPs. Small ISPs face competitive pressures, but more importantly deal with regulatory challenges induced by the lack of a coherent national approach to rural broadband. Ten Canadian ISPs were interviewed as part of this study. The paper recommends the Government of Canada develop a national broadband strategy informed in part through consultation with small ISPs.</jats:p

Inpatient Growth in Infants Requiring Pharmacologic Treatment for Neonatal Opioid Withdrawal Syndrome

Conclusion: Infants with NOWS requiring pharmacologic treatment have a decrease in Z-scores for weight, length, and head circumference at birth and at hospital discharge. Infants with prenatal polysubstance exposure were at particular risk for poorer inpatient growth relative to infants exposed to SAO and LAO, indicated by lower Z-scores for length and occipital frontal circumference (OFC)