Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant.

Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension

WHO O2CoV2: oxygen requirements and respiratory support in patients with COVID-19 in low-and-middle income countries—protocol for a multicountry, prospective, observational cohort study

Introduction SARS-CoV-2 has been identified as the cause of the disease officially named COVID-19, primarily a respiratory illness. COVID-19 was characterised as a pandemic on 11 March 2020. It has been estimated that approximately 20% of people with COVID-19 require oxygen therapy. Oxygen has been listed on the WHO Model List of Essential Medicines List and Essential Medicines List for Children for almost two decades. The COVID-19 pandemic has highlighted, more than ever, the acute need for scale-up of oxygen therapy. Detailed data on the use of oxygen therapy in low-and-middle income countries at the patient and facility level are needed to target interventions better globally.Methods and analysis We aim to describe the requirements and use of oxygen at the facility and patient level of approximately 4500 patients with COVID-19 in 30 countries. Our objectives are specifically to characterise type and duration of different modalities of oxygen therapy delivered to patients; describe demographics and outcomes of hospitalised patients with COVID-19; and describe facility-level oxygen production and support. Primary analyses will be descriptive in nature. Respiratory support transitions will be described in Sankey plots, and Kaplan-Meier models will be used to estimate probability of each transition. A multistate model will be used to study the course of hospital stay of the study population, evaluating transitions of escalating respiratory support transitions to the absorbing states.Ethics and dissemination WHO Ad Hoc COVID-19 Research Ethics Review Committee (ERC) has approved this global protocol. When this protocol is adopted at specific country sites, national ERCs may make require adjustments in accordance with their respective national research ethics guidelines. Dissemination of this protocol and global findings will be open access through peer-reviewed scientific journals, study website, press and online media.Trial registration number NCT04918875