A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

Next-generation sequencing: A new avenue to understand viral RNA–protein interactions

The genomes of RNA viruses present an astonishing source of both sequence and structural diversity. From intracellular viral RNA-host interfaces to interactions between the RNA genome and structural proteins in virus particles themselves, almost the entire viral lifecycle is accompanied by a myriad of RNA–protein interactions that are required to fulfill their replicative potential. It is therefore important to characterize such rich and dynamic collections of viral RNA–protein interactions to understand virus evolution and their adaptation to their hosts and environment. Recent advances in next-generation sequencing technologies have allowed the characterization of viral RNA–protein interactions, including both transient and conserved interactions, where molecular and structural approaches have fallen short. In this review, we will provide a methodological overview of the high-throughput techniques used to study viral RNA–protein interactions, their biochemical mechanisms, and how they evolved from classical methods as well as one another. We will discuss how different techniques have fueled virus research to characterize how viral RNA and proteins interact, both locally and on a global scale. Finally, we will present examples on how these techniques influence the studies of clinically important pathogens such as HIV-1 and SARS-CoV-2.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Recurrent hypoglycemia reduces the glucose sensitivity of glucose-inhibited neurons in the ventromedial hypothalamus nucleus

Recurrent hypoglycemia blunts the brain's ability to sense and respond to subsequent hypoglycemic episodes. Glucose-sensing neurons in the ventromedial hypothalamus nucleus (VMN) are well situated to play a role in hypoglycemia detection. VMN glucose-inhibited (GI) neurons, which decrease their firing rate as extracellular glucose increases, are extremely sensitive to decreased extracellular glucose. We hypothesize that recurrent hypoglycemia decreases the glucose sensitivity of VMN GI neurons. To test our hypothesis, 14- to 21-day-old Sprague-Dawley rats were subcutaneously injected with regular human insulin (4 U/kg) or saline (control) for three consecutive days. Blood glucose levels 1 h after insulin injection on day 3 were significantly lower than on day 1, reflecting an impaired ability to counteract hypoglycemia. On day 4, the glucose sensitivity of VMN GI neurons was measured using conventional whole cell current-clamp recording. After recurrent insulin-induced hypoglycemia, VMN GI neurons only responded to a glucose decrease from 2.5 to 0.1, but not 0.5, mM. Additionally, lactate supplementation also decreased glucose sensitivity of VMN GI neurons. Thus our findings suggest that decreases in glucose sensitivity of VMN GI neurons may contribute to the impairments in central glucose-sensing mechanisms after recurrent hypoglycemia.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA-binding proteins during rat heart development

Alternative splicing (AS) contributes to the diversity of the proteome by producing multiple isoforms from a single gene. Although short-read RNA-sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full-length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin-binding protein required for cytoskeletal functions in non-muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non-muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full-length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long-read cDNA sequencing without gene-specific PCR amplification. In rat hearts, we identified full-length Tpm1 isoforms composed of distinct exons with specific exon linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected generating predominantly muscle-specific Tpm1 isoforms in adult hearts. We found that the RNA-binding protein RBFOX2 controls AS of rat Tpm1 exon 6a, which is important for cooperative actin binding. Furthermore, RBFOX2 regulates Tpm1 AS of exon 6a antagonistically to the RNA-binding protein PTBP1. In sum, we defined full-length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into the regulation of Tpm1 AS by RNA-binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full-length AS variants of muscle-enriched genes.     

Relationship between behavior, adrenal activity, and environment in zoo-housed western lowland gorillas (Gorilla gorilla gorilla)

Monitoring adrenal activity through noninvasive fecal hormone sampling is rapidly gaining popularity as a tool to assess zoo animal welfare. However, few studies have sought to investigate the interrelationships between behavior, adrenal activity, and environment, and ask whether both behavioral and adrenal monitoring strategies are required to assess welfare sufficiently. We present the findings of a 9-month study of a small group (one male, two females) of Western lowland gorillas, Gorilla gorilla gorilla. First, we examined the effect of environmental variables on gorilla behavior. Second, we examined the effect of environmental variables on the concentration of fecal glucocorticoid metabolites (FGC) and the relationship between behavior and FGC. Environmental variables had similar effects on all three gorillas. Negative vigilance of visitors (NVV; staring, posturing, and charging at visitors) significantly increased in all subjects as environmental noise levels increased, and food-related behavior significantly decreased in all subjects as crowd size increased. Exhibit modifications had a number of positive effects on behavior. Notably, when privacy screens were used, NVV significantly decreased in two subjects. We found no significant effects of environmental variables on FGC. However, we did find significant relationships between behavior and FGC in one female. Specifically, her NVV was significantly higher one day before, and on the same day as, raised FGC. Also, hair plucking significantly increased in the two days following raised FGC. Overall, this study demonstrates how concurrent noninvasive fecal and behavioral monitoring can be used for gorilla welfare assessment.