Three forms of phosphatase type 1 in Swiss 3T3 fibroblasts. Free catalytic subunit appears to mediate s6 dephosphorylation

The major 40 S ribosomal protein S6 phosphatase in Swiss mouse 3T3 fibroblasts is a type 1 enzyme (Olivier, A. R., Ballou, L. M., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4720-4724). Polyclonal antibodies were raised against a synthetic peptide containing the carboxyl-terminal 14 amino acids of the catalytic subunit of phosphatase 1 (PP-1C). Results from Western blot analysis and immunoprecipitation show that the peptide antiserum specifically recognizes PP-1C in cell extracts. Anion-exchange chromatography of cell extracts and Western blot analysis revealed three peaks of PP-1C termed A, B, and C. Peaks A and C are associated with the major type 1 S6 phosphatase activities, but peak B exhibits little activity. The phosphatase in peak A (Mr 39,000) appears to represent the free catalytic subunit, whereas the enzymes in peaks B and C display sizes of 68,000-140,000. Peak B contains two additional proteins of Mr 26,000 and 48,000 that co-immunoprecipitate with PP-1C, while peak C has a single additional protein of Mr 100,000. Fifteen min after serum withdrawal there is a 2-fold stimulation of S6 phosphatase activity in peak A that can be accounted for by an increase in the amount of PP-1C. The amount of PP-1C in the inactive peak B fraction also increases during this time and this increase is associated with changes in the phosphorylation state of the Mr 26,000 and 48,000 proteins. The results are discussed in relation to regulatory mechanisms which are thought to modulate the activity of type 1 phosphatase.     

The AMA1-RON complex drives Plasmodium sporozoite invasion in the mosquito and mammalian hosts

Plasmodium sporozoites that are transmitted by blood-feeding female Anopheles mosquitoes invade hepatocytes for an initial round of intracellular replication, leading to the release of merozoites that invade and multiply within red blood cells. Sporozoites and merozoites share a number of proteins that are expressed by both stages, including the Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck Proteins (RONs). Although AMA1 and RONs are essential for merozoite invasion of erythrocytes during asexual blood stage replication of the parasite, their function in sporozoites was still unclear. Here we show that AMA1 interacts with RONs in mature sporozoites. By using DiCre-mediated conditional gene deletion in P. berghei, we demonstrate that loss of AMA1, RON2 or RON4 in sporozoites impairs colonization of the mosquito salivary glands and invasion of mammalian hepatocytes, without affecting transcellular parasite migration. Three-dimensional electron microscopy data showed that sporozoites enter salivary gland cells through a ring-like structure and by forming a transient vacuole. The absence of a functional AMA1-RON complex led to an altered morphology of the entry junction, associated with epithelial cell damage. Our data establish that AMA1 and RONs facilitate host cell invasion across Plasmodium invasive stages, and suggest that sporozoites use the AMA1-RON complex to efficiently and safely enter the mosquito salivary glands to ensure successful parasite transmission. These results open up the possibility of targeting the AMA1-RON complex for transmission-blocking antimalarial strategies.     

Sensitivity of the S1 neuronal calcium network to insulin and Bay‐K 8644 in vivo: Relationship to gait,motivation, and aging processes

Neuronal hippocampal Ca²⁺ dysregulation is a critical component of cognitive decline in brain aging and Alzheimer''s disease and is suggested to impact communication and excitability through the activation of a larger after hyperpolarization. However, few studies have tested for the presence of Ca²⁺ dysregulation in vivo, how it manifests, and whether it impacts network function across hundreds of neurons. Here, we tested for neuronal Ca²⁺ network dysregulation in vivo in the primary somatosensory cortex (S1) of anesthetized young and aged male Fisher 344 rats using single‐cell resolution techniques. Because S1 is involved in sensory discrimination and proprioception, we tested for alterations in ambulatory performance in the aged animal and investigated two potential pathways underlying these central aging‐ and Ca²⁺‐dependent changes. Compared to young, aged animals displayed increased overall activity and connectivity of the network as well as decreased ambulatory speed. In aged animals, intranasal insulin (INI) increased network synchronicity and ambulatory speed. Importantly, in young animals, delivery of the L‐type voltage‐gated Ca²⁺ channel modifier Bay‐K 8644 altered network properties, replicating some of the changes seen in the older animal. These results suggest that hippocampal Ca²⁺ dysregulation may be generalizable to other areas, such as S1, and might engage modalities that are associated with locomotor stability and motivation to ambulate. Further, given the safety profile of INI in the clinic and the evidence presented here showing that this central dysregulation is sensitive to insulin, we suggest that these processes can be targeted to potentially increase motivation and coordination while also reducing fall frequency with age.     

Plasmodium berghei leucine-rich repeat protein 1 downregulates protein phosphatase 1 activity and is required for efficient oocyst development

Protein phosphatase 1 (PP1) is a key enzyme for Plasmodium development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the Plasmodium berghei leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of Plasmodium PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1–LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of Plasmodium oocyst development through its interaction with PbLRR1.     

The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. These cells possess a photosensitive outer segment linked to the cell body through the connecting cilium (CC). While structural defects of the CC have been associated with retinal degeneration, its nanoscale molecular composition, assembly, and function are barely known. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC inner scaffold containing POC5, CENTRIN, and FAM161A. Dissecting CC inner scaffold assembly during photoreceptor development in mouse revealed that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Furthermore, we show that Fam161a disruption in mouse leads to specific CC inner scaffold loss and triggers microtubule doublet spreading, prior to outer segment collapse and photoreceptor degeneration, suggesting a molecular mechanism for a subtype of retinitis pigmentosa.

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. Ultrastructure expansion microscopy on mouse retina reveals the presence of a novel structure inside the photoreceptor connecting cilium, the inner scaffold, that protects the outer segment against degeneration.     

Measuring the unknown: An estimator and simulation study for assessing case reporting during epidemics

The fraction of cases reported, known as ‘reporting’, is a key performance indicator in an outbreak response, and an essential factor to consider when modelling epidemics and assessing their impact on populations. Unfortunately, its estimation is inherently difficult, as it relates to the part of an epidemic which is, by definition, not observed. We introduce a simple statistical method for estimating reporting, initially developed for the response to Ebola in Eastern Democratic Republic of the Congo (DRC), 2018–2020. This approach uses transmission chain data typically gathered through case investigation and contact tracing, and uses the proportion of investigated cases with a known, reported infector as a proxy for reporting. Using simulated epidemics, we study how this method performs for different outbreak sizes and reporting levels. Results suggest that our method has low bias, reasonable precision, and despite sub-optimal coverage, usually provides estimates within close range (5–10%) of the true value. Being fast and simple, this method could be useful for estimating reporting in real-time in settings where person-to-person transmission is the main driver of the epidemic, and where case investigation is routinely performed as part of surveillance and contact tracing activities.     

Application of silica aerogel encapsulated lipases in the synthesis of biodiesel by transesterification reactions

Two types of commercial lipases preparations, one from Burkholderia cepacia, the other one from Candida antartica, were encapsulated in silica aerogels reinforced with silica quartz fibre felt and dried by the CO₂ supercritical technique. These immobilized biocatalysts were applied in biodiesel synthesis by transesterification of sunflower seed oil with methyl acetate. They were found to be efficient even with mixtures of both substrates without any solvent addition. The aerogel encapsulation technique made it possible to maintain the enzymes in a dispersion state similar to the dispersion prevailing in an aqueous solution, even for further use in organic hydrophobic media. In transesterification in excess iso-octane, the two lipases encapsulated in aerogels made from 40% MTMS, were found to have activities relatively close to each other and comparable with commercial Novozyme 435. On the other in transesterification with mixture of oil and methyl acetate without any solvent, the kinetics were severely limited by substrate diffusion inside the aerogels. This was particularly true with the C. antartica, so that the corresponding aerogel encapsulated enzyme was much less active than commercial Novozyme 435, although it improved after a few tests.     

Stop codon recognition in ciliates: Euplotes release factor does not respond to reassigned UGA codon

In eukaryotes, the polypeptide release factor 1 (eRF1) is involved in translation termination at all three stop codons. However, the mechanism for decoding stop codons remains unknown. A direct interaction of eRF1 with the stop codons has been postulated. Recent studies focus on eRF1 from ciliates in which some stop codons are reassigned to sense codons. Using an in vitro assay based on mammalian ribosomes, we show that eRF1 from the ciliate Euplotes aediculatus responds to UAA and UAG as stop codons and lacks the capacity to decipher the UGA codon, which encodes cysteine in this organism. This result strongly suggests that in ciliates with variant genetic codes eRF1 does not recognize the reassigned codons. Recent hypotheses describing stop codon discrimination by eRF1 are not fully consistent with the set of eRF1 sequences available so far and require direct experimental testing.