Building momentum through networks: Bioimaging across the Americas.

In September 2023, the two largest bioimaging networks in the Americas, Latin America Bioimaging (LABI) and BioImaging North America (BINA), came together during a 1-week meeting in Mexico. This meeting provided opportunities for participants to interact closely with decision-makers from imaging core facilities across the Americas. The meeting was held in a hybrid format and attended in-person by imaging scientists from across the Americas, including Canada, the United States, Mexico, Colombia, Peru, Argentina, Chile, Brazil and Uruguay. The aims of the meeting were to discuss progress achieved over the past year, to foster networking and collaborative efforts among members of both communities, to bring together key members of the international imaging community to promote the exchange of experience and expertise, to engage with industry partners, and to establish future directions within each individual network, as well as common goals. This meeting report summarises the discussions exchanged, the achievements shared, and the goals set during the LABIxBINA2023: Bioimaging across the Americas meeting.

The Caenorhabditis elegans TDRD5/7-like protein, LOTR-1, interacts with the helicase ZNFX-1 to balance epigenetic signals in the germline.

LOTUS and Tudor domain containing proteins have critical roles in the germline. Proteins that contain these domains, such as Tejas/Tapas in Drosophila, help localize the Vasa helicase to the germ granules and facilitate piRNA-mediated transposon silencing. The homologous proteins in mammals, TDRD5 and TDRD7, are required during spermiogenesis. Until now, proteins containing both LOTUS and Tudor domains in Caenorhabditis elegans have remained elusive. Here we describe LOTR-1 (D1081.7), which derives its name from its LOTUS and Tudor domains. Interestingly, LOTR-1 docks next to P granules to colocalize with the broadly conserved Z-granule helicase, ZNFX-1. The Tudor domain of LOTR-1 is required for its Z-granule retention. Like znfx-1 mutants, lotr-1 mutants lose small RNAs from the 3' ends of WAGO and mutator targets, reminiscent of the loss of piRNAs from the 3' ends of piRNA precursor transcripts in mouse Tdrd5 mutants. Our work shows that LOTR-1 acts with ZNFX-1 to bring small RNA amplifying mechanisms towards the 3' ends of its RNA templates.

Quantification of tissue-specific protein translation in whole C. elegans using O-propargyl-puromycin labeling and fluorescence microscopy.

The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to quantify protein translation in C. elegans, they have been limited to using lysate from whole worms. To achieve tissue-specific quantification of ribosome activity in intact C. elegans, we report the application of O-propargyl-puromycin in a cuticle defective mutant followed by conjugation of an azide fluorophore for detection using fluorescent confocal microscopy. We apply this technique to quantify translation in response to heat shock, cycloheximide, or knockdown of translation factors. Furthermore, we demonstrate that O-propargyl-puromycin can be used to quantify translation between tissues or within a tissue like the germline. This technique is expected to have a broad range of applications in determining how protein translation is altered in different tissues in response to stress or gene knockdowns or with age.

Single-cell imaging of the cell cycle reveals CDC25B-induced heterogeneity of G1 phase length in neural progenitor cells.

Although lengthening of the cell cycle and G1 phase is a generic feature of tissue maturation during development, the underlying mechanism remains poorly understood. Here, we develop a time-lapse imaging strategy to measure the four cell cycle phases in single chick neural progenitor cells in their endogenous environment. We show that neural progenitors are widely heterogeneous with respect to cell cycle length. This variability in duration is distributed over all phases of the cell cycle, with the G1 phase contributing the most. Within one cell cycle, each phase duration appears stochastic and independent except for a correlation between S and M phase duration. Lineage analysis indicates that the majority of daughter cells may have a longer G1 phase than mother cells, suggesting that, at each cell cycle, a mechanism lengthens the G1 phase. We identify that the CDC25B phosphatase known to regulate the G2/M transition indirectly increases the duration of the G1 phase, partly through delaying passage through the restriction point. We propose that CDC25B increases the heterogeneity of G1 phase length, revealing a previously undescribed mechanism of G1 lengthening that is associated with tissue development.

Neurogenic decisions require a cell cycle independent function of the CDC25B phosphatase.

A fundamental issue in developmental biology and in organ homeostasis is understanding the molecular mechanisms governing the balance between stem cell maintenance and differentiation into a specific lineage. Accumulating data suggest that cell cycle dynamics play a major role in the regulation of this balance. Here we show that the G2/M cell cycle regulator CDC25B phosphatase is required in mammals to finely tune neuronal production in the neural tube. We show that in chick neural progenitors, CDC25B activity favors fast nuclei departure from the apical surface in early G1, stimulates neurogenic divisions and promotes neuronal differentiation. We design a mathematical model showing that within a limited period of time, cell cycle length modifications cannot account for changes in the ratio of the mode of division. Using a CDC25B point mutation that cannot interact with CDK, we show that part of CDC25B activity is independent of its action on the cell cycle.

A Stochastic Hill Climbing Approach for Simultaneous 2D Alignment and Clustering of Cryogenic Electron Microscopy Images.

A critical step in the analysis of novel cryogenic electron microscopy (cryo-EM) single-particle datasets is the identification of homogeneous subsets of images. Methods for solving this problem are important for data quality assessment, ab initio 3D reconstruction, and analysis of population diversity due to the heterogeneous nature of macromolecules. Here we formulate a stochastic algorithm for identification of homogeneous subsets of images. The purpose of the method is to generate improved 2D class averages that can be used to produce a reliable 3D starting model in a rapid and unbiased fashion. We show that our method overcomes inherent limitations of widely used clustering approaches and proceed to test the approach on six publicly available experimental cryo-EM datasets. We conclude that, in each instance, ab initio 3D reconstructions of quality suitable for initialization of high-resolution refinement are produced from the cluster centers.

Cell cycle and cell fate in the developing nervous system: the role of CDC25B phosphatase.

Deciphering the core machinery of the cell cycle and cell division has been primarily the focus of cell biologists, while developmental biologists have identified the signaling pathways and transcriptional programs controlling cell fate choices. As a result, until recently, the interplay between these two fundamental aspects of biology have remained largely unexplored. Increasing data show that the cell cycle and regulators of the core cell cycle machinery are important players in cell fate decisions during neurogenesis. Here, we summarize recent data describing how cell cycle dynamics affect the switch between proliferation and differentiation, with an emphasis on the roles played by the cell cycle regulators, the CDC25 phosphatases.

Formalizing under-the-table payments to control out-of-pocket hospital expenditures in Cambodia.

Growing evidence has demonstrated that informal fees for health services comprise a large proportion of total health spending in some countries. In 1999, individual out-of-pocket payments for health in Cambodia were estimated at 27 US dollars per person, with a proportion paid as under-the-table fees at public facilities. By formalizing such payments and implementing resource management systems within a comprehensive health financing scheme, Takeo Referral Hospital controlled out-of-pocket patient expenditures, ensured patients of fixed prices, protected patients from the unpredictability of hospital fees and promoted financial sustainability. Utilization levels increased by more than 50% for inpatient and surgical services, and cost recovery from user fees averaged 33%. Furthermore, the hospital phased out external donor support gradually over 4 years and achieved financial sustainability.