Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With Toxoplasma gondii

Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii. Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii. Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors (n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome—with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)—but very limited changes in the small RNA transcriptome—totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors (n = 3), extracted by a different isolation protocol but similarly infected with T. gondii, to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii. Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis

State of the climate in 2018

In 2018, the dominant greenhouse gases released into Earth’s atmosphere—carbon dioxide, methane, and nitrous oxide—continued their increase. The annual global average carbon dioxide concentration at Earth’s surface was 407.4 ± 0.1 ppm, the highest in the modern instrumental record and in ice core records dating back 800 000 years. Combined, greenhouse gases and several halogenated gases contribute just over 3 W m−2 to radiative forcing and represent a nearly 43% increase since 1990. Carbon dioxide is responsible for about 65% of this radiative forcing. With a weak La Niña in early 2018 transitioning to a weak El Niño by the year’s end, the global surface (land and ocean) temperature was the fourth highest on record, with only 2015 through 2017 being warmer. Several European countries reported record high annual temperatures. There were also more high, and fewer low, temperature extremes than in nearly all of the 68-year extremes record. Madagascar recorded a record daily temperature of 40.5°C in Morondava in March, while South Korea set its record high of 41.0°C in August in Hongcheon. Nawabshah, Pakistan, recorded its highest temperature of 50.2°C, which may be a new daily world record for April. Globally, the annual lower troposphere temperature was third to seventh highest, depending on the dataset analyzed. The lower stratospheric temperature was approximately fifth lowest. The 2018 Arctic land surface temperature was 1.2°C above the 1981–2010 average, tying for third highest in the 118-year record, following 2016 and 2017. June’s Arctic snow cover extent was almost half of what it was 35 years ago. Across Greenland, however, regional summer temperatures were generally below or near average. Additionally, a satellite survey of 47 glaciers in Greenland indicated a net increase in area for the first time since records began in 1999. Increasing permafrost temperatures were reported at most observation sites in the Arctic, with the overall increase of 0.1°–0.2°C between 2017 and 2018 being comparable to the highest rate of warming ever observed in the region. On 17 March, Arctic sea ice extent marked the second smallest annual maximum in the 38-year record, larger than only 2017. The minimum extent in 2018 was reached on 19 September and again on 23 September, tying 2008 and 2010 for the sixth lowest extent on record. The 23 September date tied 1997 as the latest sea ice minimum date on record. First-year ice now dominates the ice cover, comprising 77% of the March 2018 ice pack compared to 55% during the 1980s. Because thinner, younger ice is more vulnerable to melting out in summer, this shift in sea ice age has contributed to the decreasing trend in minimum ice extent. Regionally, Bering Sea ice extent was at record lows for almost the entire 2017/18 ice season. For the Antarctic continent as a whole, 2018 was warmer than average. On the highest points of the Antarctic Plateau, the automatic weather station Relay (74°S) broke or tied six monthly temperature records throughout the year, with August breaking its record by nearly 8°C. However, cool conditions in the western Bellingshausen Sea and Amundsen Sea sector contributed to a low melt season overall for 2017/18. High SSTs contributed to low summer sea ice extent in the Ross and Weddell Seas in 2018, underpinning the second lowest Antarctic summer minimum sea ice extent on record. Despite conducive conditions for its formation, the ozone hole at its maximum extent in September was near the 2000–18 mean, likely due to an ongoing slow decline in stratospheric chlorine monoxide concentration. Across the oceans, globally averaged SST decreased slightly since the record El Niño year of 2016 but was still far above the climatological mean. On average, SST is increasing at a rate of 0.10° ± 0.01°C decade−1 since 1950. The warming appeared largest in the tropical Indian Ocean and smallest in the North Pacific. The deeper ocean continues to warm year after year. For the seventh consecutive year, global annual mean sea level became the highest in the 26-year record, rising to 81 mm above the 1993 average. As anticipated in a warming climate, the hydrological cycle over the ocean is accelerating: dry regions are becoming drier and wet regions rainier. Closer to the equator, 95 named tropical storms were observed during 2018, well above the 1981–2010 average of 82. Eleven tropical cyclones reached Saffir–Simpson scale Category 5 intensity. North Atlantic Major Hurricane Michael’s landfall intensity of 140 kt was the fourth strongest for any continental U.S. hurricane landfall in the 168-year record. Michael caused more than 30 fatalities and $25 billion (U.S. dollars) in damages. In the western North Pacific, Super Typhoon Mangkhut led to 160 fatalities and$6 billion (U.S. dollars) in damages across the Philippines, Hong Kong, Macau, mainland China, Guam, and the Northern Mariana Islands. Tropical Storm Son-Tinh was responsible for 170 fatalities in Vietnam and Laos. Nearly all the islands of Micronesia experienced at least moderate impacts from various tropical cyclones. Across land, many areas around the globe received copious precipitation, notable at different time scales. Rodrigues and Réunion Island near southern Africa each reported their third wettest year on record. In Hawaii, 1262 mm precipitation at Waipā Gardens (Kauai) on 14–15 April set a new U.S. record for 24-h precipitation. In Brazil, the city of Belo Horizonte received nearly 75 mm of rain in just 20 minutes, nearly half its monthly average. Globally, fire activity during 2018 was the lowest since the start of the record in 1997, with a combined burned area of about 500 million hectares. This reinforced the long-term downward trend in fire emissions driven by changes in land use in frequently burning savannas. However, wildfires burned 3.5 million hectares across the United States, well above the 2000–10 average of 2.7 million hectares. Combined, U.S. wildfire damages for the 2017 and 2018 wildfire seasons exceeded $40 billion (U.S. dollars)

Experiment A

Experiment

Data from: Complex spike synchrony dependent modulation of rat deep cerebellar nuclear activity

The rules determining cerebellar output are not fully understood, but must involve Purkinje cell activity (PCs), as PCs are the major input to deep cerebellar nuclear (DCN) cells (which form the majority of cerebellar output). Here, the influence of PC complex spikes (CSs) was investigated by simultaneously recording DCN activity with CSs from PC arrays in anesthetized rats. Crosscorrelograms were used to identify PCs that were presynaptic to recorded DCN cells (presynaptic PCs). Such PCs were located within rostrocaudal cortical strips and displayed synchronous CS activity. CS-associated modulation of DCN activity included a short-latency post-CS inhibition and long-latency excitations before and after the CS. The amplitudes of the post-CS responses correlated with the level of synchronization among presynaptic PCs. A temporal precision of {less than or equal to} 10 ms was generally required for CSs to be maximally effective. The results suggest that CS synchrony is a key control parameter of cerebellar output.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)

Experiment D

Experiment

Experiment C

Experiment

Modulation of Purkinje cell complex spike waveform by synchrony levels in the olivocerebellar system

Purkinje cells (PCs) generate complex spikes (CSs) when activated by the olivocerebellar system. Unlike most spikes, the CS waveform is highly variable, with the number, amplitude, and timing of the spikelets that comprise it varying with each occurrence. This variability suggests that CS waveform could be an important control parameter of olivocerebellar activity. The origin of this variation is not well known. Thus, we obtained extracellular recordings of CSs to investigate the possibility that the electrical coupling state of the inferior olive (IO) affects the CS waveform. Using multielectrode recordings from arrays of PCs we showed that the variance in the recording signal during the period when the spikelets occur is correlated with CS synchrony levels in local groups of PCs. The correlation was demonstrated under both ketamine and urethane, indicating that it is robust. Moreover, climbing fiber reflex evoked CSs showed an analogous positive correlation between spikelet-related variance and the number of cells that responded to a stimulus. Intra-IO injections of GABA-A receptor antagonists or the gap junction blocker carbenoxolone produced correlated changes in the variance and synchrony levels, indicating the presence of a causal relationship. Control experiments showed that changes in variance with synchrony were primarily due to changes in the CS waveform, as opposed to changes in the strength of field potentials from surrounding cells. Direct counts of spikelets showed that their number increased with synchronization of CS activity. In sum, these results provide evidence of a causal link between two of the distinguishing characteristics of the olivocerebellar system, its ability to generate synchronous activity and the waveform of the CS