Introducing NICE guidelines for intravenous fluid therapy into a district general hospital

Peer reviewedPublisher PD

An ∼140-kb Deletion Associated with Feline Spinal Muscular Atrophy Implies an Essential LIX1 Function for Motor Neuron Survival

The leading genetic cause of infant mortality is spinal muscular atrophy (SMA), a clinically and genetically heterogeneous group of disorders. Previously we described a domestic cat model of autosomal recessive, juvenile-onset SMA similar to human SMA type III. Here we report results of a whole-genome scan for linkage in the feline SMA pedigree using recently developed species-specific and comparative mapping resources. We identified a novel SMA gene candidate, LIX1, in an ~140-kb deletion on feline chromosome A1q in a region of conserved synteny to human chromosome 5q15. Though LIX1 function is unknown, the predicted secondary structure is compatible with a role in RNA metabolism. LIX1 expression is largely restricted to the central nervous system, primarily in spinal motor neurons, thus offering explanation of the tissue restriction of pathology in feline SMA. An exon sequence screen of 25 human SMA cases, not otherwise explicable by mutations at the SMN1 locus, failed to identify comparable LIX1 mutations. Nonetheless, a LIX1-associated etiology in feline SMA implicates a previously undetected mechanism of motor neuron maintenance and mandates consideration of LIX1 as a candidate gene in human SMA when SMN1 mutations are not found

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

A novel actin mRNA splice variant regulates ACTG1 expression.

Cytoplasmic actins are abundant, ubiquitous proteins in nucleated cells. However, actin expression is regulated in a tissue- and development-specific manner. We identified a novel cytoplasmic-γ-actin (Actg1) transcript that includes a previously unidentified exon (3a). Inclusion of this exon introduces an in-frame termination codon. We hypothesized this alternatively-spliced transcript down-regulates γ-actin production by targeting these transcripts for nonsense-mediated decay (NMD). To address this, we investigated conservation between mammals, tissue-specificity in mice, and developmental regulation using C2C12 cell culture. Exon 3a is 80% similar among mammals and varies in length from 41 nucleotides in humans to 45 in mice. Though the predicted amino acid sequences are not similar between all species, inclusion of exon 3a consistently results in the in the introduction of a premature termination codon within the alternative Actg1 transcript. Of twelve tissues examined, exon 3a is predominantly expressed in skeletal muscle, cardiac muscle, and diaphragm. Splicing to include exon 3a is concomitant with previously described down-regulation of Actg1 in differentiating C2C12 cells. Treatment of differentiated C2C12 cells with an inhibitor of NMD results in a 7-fold increase in exon 3a-containing transcripts. Therefore, splicing to generate exon 3a-containing transcripts may be one component of Actg1 regulation. We propose that this post-transcriptional regulation occurs via NMD, in a process previously described as "regulated unproductive splicing and translation" (RUST)

Cycloheximide (CHX) treatment of myotubes results in a 7-fold increase in alternative <i>Actg1</i>.

<p>(<b>A,B</b>) Competitive end-point PCR and (<b>C</b>) qPCR were used to evaluate relative abundance in untreated (<b>A,C</b>) and CHX treated cells (<b>B,C</b>). Expression of both the normal and alternative transcripts were normalized to <i>Ppia</i> and are presented as fold-difference compared to skeletal muscle. A two-tailed type 2 Student's T-test was used to compare expression differences between samples. * p<0.05, ** p<0.001.</p

Splicing to include exon 3a is a developmentally regulated event in skeletal muscle.

<p>(<b>A</b>) Microscopy of cell cultures before, during and after differentiation. C2C12 myoblasts were grown to 70% confluence and induced to differentiate in DMEM+10% horse serum. Partially differentiated cultures containing both myoblasts and myotubes were observed by 2 days post-differentiation. After 48 hours, medium was replaced with DMEM+2% horse serum and 10 µM Ara-C and cultured for an additional 4 days. (<b>B</b>) qPCR of RNA harvested in Trizol showed that concurrent with a decrease in normal <i>Actg1</i>, splicing to generate alternative <i>Actg1</i> increases during differentiation into myotubes. Expression of both the normal and alternative transcripts was normalized to <i>Ppia</i> and is presented as fold-difference compared to skeletal muscle. A two-tailed type 2 Student's T-test was used to compare expression differences between time points. For all time points compared, p<0.0001.</p

Splicing to include exon 3a into the <i>Actg1</i> transcript is tissue specific.

<p>(<b>A</b>) Two PCR-based assays were employed to screen for the presence of <i>Actg1</i> – competitive end-point PCR and splice-specific qPCR. <i>Actg1</i>-specific primers were designed to amplify either both the normal and alternative transcripts in a single reaction (competitive end-point PCR), or specifically the normal or alternative transcripts in separate reactions (splice-specific qPCR). (<b>B</b>) The competitive end-point PCR assay was used to amplify all <i>Actg1</i> transcripts in various tissues from adult mouse. The larger PCR products at ∼390 bp and ∼290 bp are intermediate spliceforms of <i>Actg1</i>. The 147 bp product represents alternatively spliced, exon 3a-containing <i>Actg1</i> transcripts, while the 102 bp product represents normally spliced <i>Actg1</i> transcripts. (<b>C</b>) Expression data from qPCR to amplify normal <i>Actg1</i> (exon 3 – exon 4) shows a high degree of variability of γ-actin expression between tissues. (<b>D</b>) Splicing to include exon 3a, as measured by qPCR is primarily limited to skeletal and cardiac muscle, with very low levels in the brain, eye, and intestine. All qPCR data is normalized to <i>Rplp0</i> and <i>Rrn18s</i> expression and presented as a fold-difference to skeletal muscle.</p

Exon 3a containing alternatively spliced <i>Actg1</i> is exported to the cytoplasm, but does not produce a stable protein.

<p>Nuclear (<b>A</b>) and cytoplasmic (<b>B</b>) RNA fractions were harvested from C2C12 myotubes and evaluated for the presence of alternative <i>Actg1</i> using competitive, end-point PCR. All <i>Actg1</i> spliceforms, including partially spliced transcripts, were present in the nuclear fraction, however, only the normal and exon 3a transcripts were observed in the cytoplasmic fraction. (<b>C</b>) Western blot using an anti-γ-actin specific antibody was used to probe mouse skeletal muscle lysate for the presence of a protein product corresponding to the inclusion of exon 3a. Usage of the in-frame stop codon in exon 3a would generate a 15 kDa protein. Alternatively, read-through of the stop codon would increase the size of the ACTG1 protein by 2 kDa, resulting in a 44 kDa protein. Neither of these protein products is present. A γ-actin protein at 52 kDa is observed and likely represents a post-translational modification. Molecular weight marker is prestained Low Range (BioRad).</p

Ct values for a skeletal muscle cDNA control demonstrate no plate-to-plate variability.

<p>For each qPCR plate, a skeletal muscle cDNA control sample was included to assess plate-to-plate variability for every primer pair. For the <i>Tissue Panel</i> experiment Ct values represent three skeletal muscle cDNA biological replicates in addition to the control skeletal muscle cDNA sample. Alternatively spliced transcripts constitute approximately 30% of steady-state <i>Actg1</i> mRNA in adult skeletal muscle. Two technical replicates were averaged for every biological replicate in qPCR experiments.</p