Alternative poly-adenylation modulates α1-antitrypsin expression in chronic obstructive pulmonary disease

α1-anti-trypsin (A1AT), encoded by SERPINA1, is a neutrophil elastase inhibitor that controls the inflammatory response in the lung. Severe A1AT deficiency increases risk for Chronic Obstructive Pulmonary Disease (COPD), however, the role of A1AT in COPD in non-deficient individuals is not well known. We identify a 2.1-fold increase (p = 2.5x10-6) in the use of a distal poly-adenylation site in primary lung tissue RNA-seq in 82 COPD cases when compared to 64 controls and replicate this in an independent study of 376 COPD and 267 controls. This alternative polyadenylation event involves two sites, a proximal and distal site, 61 and 1683 nucleotides downstream of the A1AT stop codon. To characterize this event, we measured the distal ratio in human primary tissue short read RNA-seq data and corroborated our results with long read RNA-seq data. Integrating these results with 3’ end RNA-seq and nanoluciferase reporter assay experiments we show that use of the distal site yields mRNA transcripts with over 50-fold decreased translation efficiency and A1AT expression. We identified seven RNA binding proteins using enhanced CrossLinking and ImmunoPrecipitation precipitation (eCLIP) with one or more binding sites in the SERPINA1 3’ UTR. We combined these data with measurements of the distal ratio in shRNA knockdown experiments, nuclear and cytoplasmic fractionation, and chemical RNA structure probing. We identify Quaking Homolog (QKI) as a modulator of SERPINA1 mRNA translation and confirm the role of QKI in SERPINA1 translation with luciferase reporter assays. Analysis of single-cell RNA-seq showed differences in the distribution of the SERPINA1 distal ratio among hepatocytes, macrophages, αβ-Tcells and plasma cells in the liver. Alveolar Type 1,2, dendritic cells and macrophages also vary in their distal ratio in the lung. Our work reveals a complex post-transcriptional mechanism that regulates alternative polyadenylation and A1AT expression in COPD.

A SARS CoV-2 nucleocapsid vaccine protects against distal viral dissemination

The SARS CoV-2 pandemic has killed millions of people. This viral infection can also result in substantial morbidity, including respiratory insufficiency and neurological manifestations, such as loss of smell and psychiatric diseases. Most SARS CoV-2 vaccines are based on the spike antigen, and although they have shown extraordinary efficacy at preventing severe lung disease and death, they do not always confer sterilizing immune protection. We performed studies in K18-hACE2 mice to evaluate whether the efficacy of SARS CoV-2 vaccines could be augmented by incorporating nucleocapsid as a vaccine antigen. We vaccinated mice with adenovirus-based vaccines encoding spike antigen alone, nucleocapsid antigen alone, or combined spike and nucleocapsid antigens. Mice were then challenged intranasally with SARS CoV-2, and acute viral loads were quantified at a proximal site of infection (lung) and a distal site of infection (brain). Interestingly, the spike-based vaccine conferred acute protection in the lung, but not in the brain. The spike-based vaccine conferred acute protection in the brain only if combined with the nucleocapsid-based vaccine. These findings suggest that nucleocapsid-specific immunity is important for the distal control of SARS CoV-2, warranting the inclusion of nucleocapsid in next-generation COVID-19 vaccines.

Difference in Moxibustion-Induced Microcirculatory Responses between the Heart and Lung Meridians Assessed by Laser Doppler Flowmetry

Objective. By comparing the differences in microcirculatory responses of the heart and lung meridians induced by moxibustion on these two meridians, respectively, this study aimed to investigate the specificity for site-to-site association on body surface between different meridians. Methods. Eighty healthy adults were enrolled and divided into the lung meridian intervention group and heart meridian intervention group in a ratio of 1 : 1. Three-channel laser Doppler flowmetry was used to monitor microcirculatory responses for the heart and lung meridians. Primary outcome was change of blood perfusion units (PU) of three measurement sites along the two meridians. Results. In the lung meridian intervention group, following moxibustion performed at LU5 of the lung meridian, PU in the distal site of the lung meridian increased significantly. By contrast, the PU of HT3 in the heart meridian, which was nearest to the moxibustion site, did not change significantly. PU in the distal site of the heart meridian declined. Meanwhile, significant difference in PU change was detected between the distal site of the lung meridian and the other two control sites of the heart meridians during moxibustion and postmoxibustion. Alternatively, similar to the results of the lung meridian intervention group, the specificity of microcirculatory response between the heart and lung meridians was observed in the heart meridian intervention group. Conclusions. For the heart and lung meridians, the effect of moxibustion-induced microcirculatory response may be more related to meridian routes than the specific distance between two sites located at different meridians, thereby supporting possible specificity for site-to-site association on the body surface between these two meridians. Nevertheless, given that only two meridians and limited measurement sites were compared, all current findings are not sufficiently robust. Further research should be conducted to investigate more meridians and measurement sites.

Utilization of the autologous vein on the ulnar side of the dorsal hand for arteriovenous fistula creation

We previously described the success and usefulness of two operative techniques for creating a radial artery-first or second dorsal metacarpal vein arteriovenous fistula (AVF) in the first interdigital space of the dorsal hand using the most distal site and autologous veins in the upper limb. These techniques utilize the dorsal metacarpal veins on the radial side of the dorsal hand. Developing these ideas, we devised a novel operative technique for creating a transposed radial artery-third metacarpal vein AVF in the first interdigital space of the dorsal hand using the most distal vein on the ulnar side of the upper limb and most distal site in the upper limb. The distinctive advantage of this technique is that it can be applied to patients whose cephalic vein in the forearm and the dorsal metacarpal veins on the radial side of the dorsal hand are of a poor quality. We herein report the steps of this technique and describe its successful performance in a patient who has been on hemodialysis for 14 months without any additional vascular access interventions or postoperative complications. We consider this technique to be a valuable option in select patients who meet the applicable conditions. The creation of the first AVF as distally as possible is ideal, and it offers a further viable option of distal native vascular access that may be overlooked.

Effects of PACAP on Schwann Cells: Focus on Nerve Injury

Schwann cells, the most abundant glial cells of the peripheral nervous system, represent the key players able to supply extracellular microenvironment for axonal regrowth and restoration of myelin sheaths on regenerating axons. Following nerve injury, Schwann cells respond adaptively to damage by acquiring a new phenotype. In particular, some of them localize in the distal stump to form the Bungner band, a regeneration track in the distal site of the injured nerve, whereas others produce cytokines involved in recruitment of macrophages infiltrating into the nerve damaged area for axonal and myelin debris clearance. Several neurotrophic factors, including pituitary adenylyl cyclase-activating peptide (PACAP), promote survival and axonal elongation of injured neurons. The present review summarizes the evidence existing in the literature demonstrating the autocrine and/or paracrine action exerted by PACAP to promote remyelination and ameliorate the peripheral nerve inflammatory response following nerve injury.

From Outbreak to Compliance and Beyond: UAB Medicine’s Successful Implementation of a Water Safety Program

Background: Pre-emptive management of the water supply can reduce hospital-onset legionellosis associated with building water systems. In 2014, an outbreak of Legionella pneumonia occurred in a 1,150-bed academic medical center (with ∼0.557 km2 or ∼6 million ft2 of space) among hematology-oncology patients. A comprehensive water safety and management program was implemented after the outbreak was controlled. We describe our experience implementing this prevention program. Methods: After the Legionella outbreak was controlled, an air and water safety committee (AWSC) was established and cochaired by the healthcare epidemiologist and the chief facilities officer. The AWSC established protocols for proactive environmental testing and the development of the an infection control risk assessment (ICRA) dedicated to water safety known as the water system construction and renovation risk assessment (WSCRRA).The water system management plan (WSMP) was developed (prior to the publication of ASHRAE 188) to direct the risk assessments and mitigation of any risks throughout the campus. Results: The WSMP identifies critical control measures, points, and limits that need to be maintained to control and monitor Legionella growth in the water systems. A control point is any step in a process at which biological, chemical, or physical factors can be controlled. The UAB Medicine WSMP includes 7 control points that are monitored on a daily basis. Examples of these control steps include monitoring of the hot water temperatures at the water heaters and distal outlets, managing the levels of mono-chloramines and chlorines in the water system, and managing water system components. To validate the efficacy of the WSMP, >610 water samples are collected from 19 hospital buildings over a year to be tested for Legionella. The results of water testing have shown significant decrease in distal site positivity due to managing and controlling these control points. This WSMP also evaluated the efficacy of 2 different methods for disinfecting water systems on campus as a corrective measure to Legionella growth. These methods are hyperchlorination and temporary copper silver ionization; based on the culture results of the water samples collected post disinfection, WSMP data show that the copper silver ionization method was more effective than hyperchlorination in controlling Legionella growth and decreasing the distal site positivity. Conclusions: The WSMP has provided ongoing management of building water systems and proactive actions around construction and renovation projects that involve water systems to prevent healthcare-acquired legionellosis. We strongly recommend other healthcare facilities to implement a similar program to avoid outbreaks.Funding: NoneDisclosures: Rachael Anne Lee reports speaker honoraria from Prime Education, LLC.

Distal Unfolding of Ferricytochrome c Induced by the F82K Mutation

It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side chains under non-native conditions induced by alkaline pH, high temperature, or denaturing agents. Here, we showed a redox-dependent destabilization induced in human cytochrome c by substituting Phe82—conserved amino acid and a key actor in cytochrome c intermolecular interactions—with a Lys residue. Introducing a positive charge at position 82 did not significantly affect the structure of ferrous cytochrome c but caused localized unfolding of the distal site in the ferric state. As revealed by 1H NMR fingerprint, the ferric form of the F82K variant had axial coordination resembling the renowned alkaline species, where the detachment of the native Met80 ligand favored the formation of multiple conformations involving distal Lys residues binding to iron, but with more limited overall structural destabilization.

Bone metastasis of limb segments: Is mesometastasis another poor prognostic factor of cancer patients?

Objective In contrast to acrometastasis, defined as bone metastasis to the hand or foot, the frequency and prognosis of bone metastasis of other limb segments remain unclear. To compare prognosis according to sites of bone metastasis, we defined two new terms in this study: ‘mesometastasis’ and ‘rhizometastasis’ as bone metastasis of ‘forearm or lower leg’ and ‘arm or thigh’, respectively. Methods A total of 539 patients who were registered to the bone metastasis database of The University of Tokyo Hospital from April 2012 to May 2016 were retrospectively surveyed. All patients who were diagnosed to have bone metastases in our hospital are registered to the database. Patients were categorized into four groups according to the most distal site of bone metastases: ‘acrometastasis’, ‘mesometastasis’, ‘rhizometastasis’ and ‘body trunk metastasis’. Results The frequency of rhizometastasis (22.5%) or body trunk metastasis (73.1%) was significantly higher than that of acrometastasis (2.0%) or mesometastasis (2.4%). The median survival time after diagnosis of bone metastases for each group was as follows: 6.5 months in acrometastasis, 4.0 months in mesometastasis, 16 months in rhizometastasis, 17 months in body trunk metastasis and 16 months overall. In survival curve, there was a statistically significant difference between mesometastasis and body trunk metastasis. Conclusions Our findings suggest that ‘mesometastasis’ could be another poor prognostic factor in cancer patients and that patients with mesometastasis should receive appropriate treatments according to their expected prognosis.