ASHG 2020 Curt Stern Award introduction: Fowzan Sami Alkuraya

This article is based on the address given by the author at the 2020 virtual meeting of the American Society of Human Genetics (ASHG) on October 26, 2020. The video of the original address can be found at the ASHG website.     

Split-wrmScarlet and split-sfGFP: tools for faster,easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663     

Correction to: Sex and race differences of cerebrospinal fluid metabolites in healthy individuals

Metabolomics - Metabolomics applications to the aquaculture research are increasing steadily. The use of standardized proton nuclear magnetic resonance (1H NMR) spectroscopy can provide the...     

Pyridostigmine improves cardiac function and rhythmicity through RyR2 stabilization and inhibition of STIM1-mediated calcium entry in heart failure

Heart failure (HF) is characterized by asymmetrical autonomic balance. Treatments to restore parasympathetic activity in human heart failure trials have shown beneficial effects. However, mechanisms of parasympathetic-mediated improvement in cardiac function remain unclear. The present study examined the effects and underpinning mechanisms of chronic treatment with the cholinesterase inhibitor, pyridostigmine (PYR), in pressure overload HF induced by transverse aortic constriction (TAC) in mice. TAC mice exhibited characteristic adverse structural (left ventricular hypertrophy) and functional remodelling (reduced ejection fraction, altered myocyte calcium (Ca) handling, increased arrhythmogenesis) with enhanced predisposition to arrhythmogenic aberrant sarcoplasmic reticulum (SR) Ca release, cardiac ryanodine receptor (RyR2) hyper-phosphorylation and up-regulated store-operated Ca entry (SOCE). PYR treatment resulted in improved cardiac contractile performance and rhythmic activity relative to untreated TAC mice. Chronic PYR treatment inhibited altered intracellular Ca handling by alleviating aberrant Ca release and diminishing pathologically enhanced SOCE in TAC myocytes. At the molecular level, these PYR-induced changes in Ca handling were associated with reductions of pathologically enhanced phosphorylation of RyR2 serine-2814 and STIM1 expression in HF myocytes. These results suggest that chronic cholinergic augmentation alleviates HF via normalization of both canonical RyR2-mediated SR Ca release and non-canonical hypertrophic Ca signaling via STIM1-dependent SOCE.     

Rapid high‐yield N‐acylation of aminothiols: N‐acetylglutathione and N‐acetylhomocysteine and their thiol pKa values

Methodology for the rapid N‐acylation of aminothiols in aqueous solution using procedures commonly employed in biochemical studies is described here. Glutathione disulfide (GSSG) and homocystine were diN‐acetylated in ~100% yield in 0.1 M aqueous NaHCO₃ (pH 8.5) at room temperature by 2.5 equiv of the activated ester, N‐hydroxysulfosuccinimidyl acetate, an efficient water‐soluble acetylating reagent. Following acetone precipitation, diN‐acetylGSSG was further purified and desalted on a strong anion‐exchange (SAX) cartridge. DiN‐acetylhomocystine was simultaneously purified and desalted on a C₁₈ cartridge. The N‐acetylated aminothiols were generated using gel‐immobilized tris(2‐carboxyethyl)phosphine as a reductant, which obviated the need for further purification. Alternatively, disulfide exchange with dissolved dithiothreitol yielded N‐acetylglutathione, which was purified on the SAX cartridge. pH titrations of N‐acetylglutathione (8.99) and N‐acetylhomocysteine (9.66) as well as those of commercially available N‐acetylcysteine (9.53) and N‐acetylpenicillamine (10.21) yielded pK_a(SH) values of importance for biological studies. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Adenocarcinoma of the Ileocolic Junction and Multifocal Hepatic Sarcomas in an Aged Rhesus Macaque (Macaca mulatta)

An aged male rhesus macaque in our colony had decreased appetite and a loss of interest in behavioral testing. CBC analysis revealed a regenerative, microcytic, hypochromic anemia with thrombocytosis, consistent with iron deficiency. A fecal occult blood test was positive. Ultrasound imaging revealed numerous, vascularized focal liver lesions that suggested metastases. The macaque''s appetite continued to decrease, and he became more lethargic. At this point, the investigator elected to euthanize the macaque. At necropsy, the ileocolic junction was white and abnormally thickened, and the liver was pale tan with approximately 18 discrete white masses randomly scattered throughout the hepatic parenchyma. Histologically, the mass at the ileocolic junction was identified as an intestinal adenocarcinoma, whereas the liver masses were confirmed to be undifferentiated hepatic sarcomas. This case report describes a rhesus macaque that had 2 unrelated primary neoplasms. A review of the literature indicates that this rhesus macaque is the first reported to have an adenocarcinoma of the ileocolic junction and multiple hepatic sarcomas simultaneously.Rhesus macaques (Macaca mulatta) are genetically similar to humans, have a similar aging phenotype at approximately 3 times the rate of those in humans, and develop spontaneous cancers similar to those in humans.³⁶ In humans, gastrointestinal carcinomas are relatively common, but most of these lesions arise in the colon and rectum with only a small percentage in the small intestine and ileum.^4,12,15,18 Although the ileocolic junction is considered a common site for intestinal adenocarcinomas in aged rhesus macaques, this tumor has also been found in the duodenum, jejunum, distal ileum, cecum, and colon.^{6,13,21-23,25,39} Intestinal adenocarcinomas also occur in aged cynomologus macaques (Macaca fasicularis),³⁹ cotton-top marmosets (Saguinus oedipus),^6,10 common marmosets (Callithrix jacchus),^6,27 and a squirrel monkey (Saimiri sciureus).²⁴ Cotton-top marmosets often develop adenocarcinomas of the colon, including the cecum–colon, and rectum.^6,10 Common marmosets have been reported to develop adenocarcinomas of the small intestine.^6,27 Adenocarcinoma of the cecum in a squirrel monkey has been reported.²⁴Spontaneous hepatic tumors unrelated to carcinogenic factors, such as aflatoxin B1,³³ occur only rarely in nonhuman primates. In the United States, primary malignant hepatic tumors in humans are rare, and fewer than 1% are reported to be hepatic sarcomas.^1,16,40 Review of the nonhuman primate literature revealed reports of hepatic cholangiocarcinoma in a 25-y-old male capuchin monkey (Cebus albifrons),⁷ hepatocellular carcinoma in a 24-y-old male squirrel monkey (Saimiri boliviensis)⁵ and in a female squirrel monkey (Saimiri sciureus) older than 13 y,²⁸ and hepatocellular carcinoma and cholangiocarcinoma in an African green monkey (Cercopithecus aethiops).³⁴ Spontaneous hepatocellular carcinomas were reported to occur in 2 adolescent male cynomologus macaques younger than 5 y.³¹ Hepatic hemangiosarcoma was diagnosed in 3-y-old female rhesus macaque,²⁶ and hepatic cholangiocarcinoma was found in a rhesus macaque that also had an intestinal adenocarcinoma.³⁹The aged male rhesus macaque (Macaca mulatta) in the current case study was found to have adenocarcinoma of the ileocolic junction and multiple, random, discrete neoplasms in the liver, which were identified as undifferentiated sarcomas. No metastases from the intestinal adenocarcinoma were detected, but neoplastic cells similar to those of the undifferentiated hepatic cells were identified in an intestinal artery. The frequency of multiple tumor types in aged nonhuman primates is relevant to the use of older animals in research.     

Effects of grazing on grassland soil carbon: a global review

Soils of grasslands represent a large potential reservoir for storing CO₂, but this potential likely depends on how grasslands are managed for large mammal grazing. Previous studies found both strong positive and negative grazing effects on soil organic carbon (SOC) but explanations for this variation are poorly developed. Expanding on previous reviews, we performed a multifactorial meta‐analysis of grazer effects on SOC density on 47 independent experimental contrasts from 17 studies. We explicitly tested hypotheses that grazer effects would shift from negative to positive with decreasing precipitation, increasing fineness of soil texture, transition from dominant grass species with C₃ to C₄ photosynthesis, and decreasing grazing intensity, after controlling for study duration and sampling depth. The six variables of soil texture, precipitation, grass type, grazing intensity, study duration, and sampling depth explained 85% of a large variation (±150 g m^?2 yr^?1) in grazing effects, and the best model included significant interactions between precipitation and soil texture (P = 0.002), grass type, and grazing intensity (P = 0.012), and study duration and soil sampling depth (P = 0.020). Specifically, an increase in mean annual precipitation of 600 mm resulted in a 24% decrease in grazer effect size on finer textured soils, while on sandy soils the same increase in precipitation produced a 22% increase in grazer effect on SOC. Increasing grazing intensity increased SOC by 6–7% on C₄‐dominated and C₄–C₃ mixed grasslands, but decreased SOC by an average 18% in C₃‐dominated grasslands. We discovered these patterns despite a lack of studies in natural, wildlife‐dominated ecosystems, and tropical grasslands. Our results, which suggest a future focus on why C₃ vs. C₄‐dominated grasslands differ so strongly in their response of SOC to grazing, show that grazer effects on SOC are highly context‐specific and imply that grazers in different regions might be managed differently to help mitigate greenhouse gas emissions.     

A Metagenomic Framework for the Study of Airborne Microbial Communities

Understanding the microbial content of the air has important scientific, health, and economic implications. While studies have primarily characterized the taxonomic content of air samples by sequencing the 16S or 18S ribosomal RNA gene, direct analysis of the genomic content of airborne microorganisms has not been possible due to the extremely low density of biological material in airborne environments. We developed sampling and amplification methods to enable adequate DNA recovery to allow metagenomic profiling of air samples collected from indoor and outdoor environments. Air samples were collected from a large urban building, a medical center, a house, and a pier. Analyses of metagenomic data generated from these samples reveal airborne communities with a high degree of diversity and different genera abundance profiles. The identities of many of the taxonomic groups and protein families also allows for the identification of the likely sources of the sampled airborne bacteria.