SPTLC3 Is Essential for Complex I Activity and Contributes to Ischemic Cardiomyopathy.

BACKGROUND: Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3). METHODS: The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3KO mice subjected to permanent nonreperfused myocardial infarction. RESULTS: Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity. CONCLUSIONS: Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.

Acute endoplasmic reticulum stress-induced mitochondria respiratory chain damage: The role of activated calpains.

The induction of acute endoplasmic reticulum (ER) stress damages the electron transport chain (ETC) in cardiac mitochondria. Activation of mitochondria-localized calpain 1 (CPN1) and calpain 2 (CPN2) impairs the ETC in pathological conditions, including aging and ischemia-reperfusion in settings where ER stress is increased. We asked if the activation of calpains causes the damage to the ETC during ER stress. Control littermate and CPNS1 (calpain small regulatory subunit 1) deletion mice were used in the current study. CPNS1 is an essential subunit required to maintain CPN1 and CPN2 activities, and deletion of CPNS1 prevents their activation. Tunicamycin (TUNI, 0.4 mg/kg) was used to induce ER stress in C57BL/6 mice. Cardiac mitochondria were isolated after 72 h of TUNI treatment. ER stress was increased in both control littermate and CPNS1 deletion mice with TUNI treatment. The TUNI treatment activated both cytosolic and mitochondrial CPN1 and 2 (CPN1/2) in control but not in CPNS1 deletion mice. TUNI treatment led to decreased oxidative phosphorylation and complex I activity in control but not in CPNS1 deletion mice compared to vehicle. The contents of complex I subunits, including NDUFV2 and ND5, were decreased in control but not in CPNS1 deletion mice. TUNI treatment also led to decreased oxidation through cytochrome oxidase (COX) only in control mice. Proteomic study showed that subunit 2 of COX was decreased in control but not in CPNS1 deletion mice. Our results provide a direct link between activation of CPN1/2 and complex I and COX damage during acute ER stress.

Aging-induced mitochondrial dysfunction: two distinct populations of mitochondria versus a combined population.

Mitochondrial function in aged hearts is impaired, and studies of isolated mitochondria are commonly used to assess their function. The two populations of cardiac mitochondria, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), are affected by aging. However, the yield of these mitochondria, particularly SSM, is limited in the mouse heart because of the smaller heart size. To address this issue, the authors developed a method to isolate a mixed population (MIX) of SSM and IFM mitochondria from a single mouse heart. The aim of the study was to compare the mitochondrial function between SSM, IFM, and the MIX population from young and aged mouse hearts. The MIX population had a higher yield of total protein and citrate synthase activity from both young and aged hearts compared with the individual yields of SSM or IFM. Oxidative phosphorylation (OXPHOS) decreased in aged SSM and IFM compared with young SSM and IFM, as well as in the MIX population isolated from aged hearts compared with young hearts, when using complex I or IV substrates. Furthermore, aging barely affected the sensitivity to mitochondrial permeability transition pore (MPTP) opening in SSM, whereas the sensitivity was increased in IFM isolated from aged hearts and in the MIX population from aged hearts compared with the corresponding populations isolated from young hearts. These results suggest that mitochondrial dysfunction exists in aged hearts and the isolation of a MIX population of mitochondria from the mouse heart is a potential approach to studying mitochondrial function in the mouse heart.NEW & NOTEWORTHY We developed two methods to isolate mitochondria from a single mouse heart. We compared mitochondrial function in young and aged mice using mitochondria isolated with different methods. Both methods can be successfully used to isolate cardiac mitochondria from single mouse hearts. Our results provide the flexibility to isolate mitochondria from a single mouse heart based on the purpose of the study.

Microglial STAT1-sufficiency is required for resistance to toxoplasmic encephalitis.

Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that establishes a life-long chronic infection largely restricted to the central nervous system (CNS). Constant immune pressure, notably IFN-γ-STAT1 signaling, is required for preventing fatal pathology during T. gondii infection. Here, we report that abrogation of STAT1 signaling in microglia, the resident immune cells of the CNS, is sufficient to induce a loss of parasite control in the CNS and susceptibility to toxoplasmic encephalitis during the early stages of chronic infection. Using a microglia-specific genetic labeling and targeting system that discriminates microglia from blood-derived myeloid cells that infiltrate the brain during infection, we find that, contrary to previous in vitro reports, microglia do not express inducible nitric-oxide synthase (iNOS) during T. gondii infection in vivo. Instead, transcriptomic analyses of microglia reveal that STAT1 regulates both (i) a transcriptional shift from homeostatic to "disease-associated microglia" (DAM) phenotype conserved across several neuroinflammatory models, including T. gondii infection, and (ii) the expression of anti-parasitic cytosolic molecules that are required for eliminating T. gondii in a cell-intrinsic manner. Further, genetic deletion of Stat1 from microglia during T. gondii challenge leads to fatal pathology despite largely equivalent or enhanced immune effector functions displayed by brain-infiltrating immune populations. Finally, we show that microglial STAT1-deficiency results in the overrepresentation of the highly replicative, lytic tachyzoite form of T. gondii, relative to its quiescent, semi-dormant bradyzoite form typical of chronic CNS infection. Our data suggest an overall protective role of CNS-resident microglia against T. gondii infection, illuminating (i) general mechanisms of CNS-specific immunity to infection (ii) and a clear role for IFN-STAT1 signaling in regulating a microglial activation phenotype observed across diverse neuroinflammatory disease states.

A micro-fabricated device (microICSI) improves porcine blastocyst development and procedural efficiency for both porcine intracytoplasmic sperm injection and human microinjection.

PURPOSE: Intracytoplasmic sperm injection (ICSI) imparts physical stress on the oolemma of the oocyte and remains among the most technically demanding skills to master, with success rates related to experience and expertise. ICSI is also time-consuming and requires workflow management in the laboratory. This study presents a device designed to reduce the pressure on the oocyte during injection and investigates if this improves embryo development in a porcine model. The impact of this device on laboratory workflow was also assessed. METHODS: Porcine oocytes were matured in vitro and injected with porcine sperm by conventional ICSI (C-ICSI) or with microICSI, an ICSI dish that supports up to 20 oocytes housed individually in microwells created through microfabrication. Data collected included set-up time, time to align the polar body, time to perform the injection, the number of hand adjustments between controllers, and degree of invagination at injection. Developmental parameters measured included cleavage and day 6 blastocyst rates. Blastocysts were differentially stained to assess cell numbers of the inner cell mass and trophectoderm. A pilot study with human donated MII oocytes injected with beads was also performed. RESULTS: A significant increase in porcine blastocyst rate for microICSI compared to C-ICSI was observed, while cleavage rates and blastocyst cell numbers were comparable between treatments. Procedural efficiency of microinjection was significantly improved with microICSI compared to C-ICSI in both species. CONCLUSION: The microICSI device demonstrated significant developmental and procedural benefits for porcine ICSI. A pilot study suggests human ICSI should benefit equally.

Lavender Harbors More Viruses than Previously Thought: First Report of Raspberry Ringspot Virus and Phlox Virus M in Lavandula × intermedia.

Plants of the genus Lavandula are thought to be rarely infected by viruses. To date, only alfalfa mosaic virus, cucumber mosaic virus, tobacco mosaic virus, and tomato spotted wilt virus have been reported in this host. In this study, we identified for the first time raspberry ringspot virus (RpRSV) and phlox virus M (PhlVM) in lavender using herbaceous indexing, enzyme-linked immunosorbent assay, and high-throughput sequencing. Nearly complete genome sequences for both viruses were determined. Phylogenetic and serological characterizations suggest that the obtained RpRSV isolate is a raspberry strain. A preliminary survey of 166 samples indicated RpRSV was spread only in the lavender cultivar 'Grosso', while PhlVM was detected in multiple lavender cultivars. Although RpRSV raspberry strain may have spread throughout Auckland and nearby areas in New Zealand, it is very likely restricted to the genus Lavandula or even to the cultivar 'Grosso' due to the absence or limited occurrence of the nematode vector. Interestingly, all infected lavender plants, regardless of their infection status (by RpRSV, PhlVM, or both) were asymptomatic. RpRSV is an important virus that infects horticultural crops including grapevine, cherry, berry fruits, and rose. It remains on the list of regulated pests in New Zealand. RpRSV testing is mandatory for imported Fragaria, Prunus, Ribes, Rosa, Rubus, and Vitis nursery stock and seeds for sowing, while this is not required for Lavandula importation. Our study revealed that lavender could play a role not only as a reservoir but also as an uncontrolled import pathway of viruses that pose a threat to New Zealand's primary industries.

Detection, Characterization, and Distribution of the First Case of Pepino Mosaic Virus in Aotearoa New Zealand.

Tomato (Solanum lycopersicum L., family Solanaceae) represents one of the most economically valuable horticultural crops worldwide. Tomato production is affected by numerous emerging plant viruses. We identified, for the first time in New Zealand (NZ), Pepino mosaic virus (PepMV) in greenhouse grown tomato crops using a combination of methods from electron microscopy and herbaceous indexing to RT-qPCR and high-throughput sequencing. Phylogenetic and genomic analysis of a near-complete PepMV genome determined that the detected strain belonged to the mild form of the CH2 lineage of the virus. Subsequently, a delimiting survey of PepMV was conducted, and PepMV was detected at four additional locations. PCR-derived sequences obtained from samples collected from different greenhouses and from herbaceous indicator plants were identical to the original sequence. Since PepMV has never been reported in NZ before, seed pathways are speculated to be the most likely source of entry into the country.

Urban flask measurements of CO2ff and CO to identify emission sources at different site types in Auckland, New Zealand.

As part of the CarbonWatch-NZ research programme, air samples were collected at 28 sites around Auckland, New Zealand, to determine the atmospheric ratio (RCO) of excess (local enhancement over background) carbon monoxide to fossil CO2 (CO2ff). Sites were categorized into seven types (background, forest, industrial, suburban, urban, downwind and motorway) to observe RCO around Auckland. Motorway flasks observed RCO of 14 ± 1 ppb ppm-1 and were used to evaluate traffic RCO. The similarity between suburban (14 ± 1 ppb ppm-1) and traffic RCO suggests that traffic dominates suburban CO2ff emissions during daytime hours, the period of flask collection. The lower urban RCO (11 ± 1 ppb ppm-1) suggests that urban CO2ff emissions are comprised of more than just traffic, with contributions from residential, commercial and industrial sources, all with a lower RCO than traffic. Finally, the downwind sites were believed to best represent RCO for Auckland City overall (11 ± 1 ppb ppm-1). We demonstrate that the initial discrepancy between the downwind RCO and Auckland's estimated daytime inventory RCO (15 ppb ppm-1) can be attributed to an overestimation in inventory traffic CO emissions. After revision based on our observed motorway RCO, the revised inventory RCO (12 ppb ppm-1) is consistent with our observations. This article is part of the Theo Murphy meeting issue 'Radiocarbon in the Anthropocene'.

First report of Streptocarpus flower break virus in Streptocarpus hybrids in Aotearoa New Zealand.

Streptocarpus (Cape primrose, family Gesneriaceae) is a genus of plants native to Southern Africa commonly grown indoors for their foliage and trumpet-shaped flowers. In Aoteroa New Zealand (NZ) to date, no viruses have been reported to infect plants of the Gesneriaceae (Veerakone et al. 2015). In September 2022, a plant of Streptocarpus hybrid exhibiting necrotic rings was observed in a hobbyist's greenhouse in Auckland, NZ. High-Throughput Sequencing (HTS) using MinIONTM (Oxford Nanopore Technologies), was applied as a first screen (Liefting et al. 2021). Phylogenetic analysis was performed using Geneious Prime 2021 (Biomatters Ltd, NZ). A BLASTn search with 622,847 obtained reads resulted in 3,260 and 4,340 matches to the sequences of Streptocarpus flower break tobamovirus (SFBV) and Impatiens necrotic spot orthotospovirus (INSV), respectively. A near-complete (98.5%) genome sequence of SFBV was obtained (GenBank accession No. OQ970154), which shared 99.52% nucleotide identity to a SFBV type isolate from Germany (GenBank accession No. NC_008365). A phylogenetic tree was also generated (e-Xtra). To confirm the presence of both viruses, leaf tissue was rub inoculated onto herbaceous indicator plants as described by Tang et al. (2013). Chenopodium amaranticolor and C. quinoa plants developed local lesions while Nicotiana occidentalis plants showed local necrosis followed by systemic leaf puckering by 14 days post inoculation (dpi). Nicotiana benthamiana and N. clevelandii plants showed systemic chlorosis but N. tabacum plants did not exhibit any symptoms by 28 dpi. Samples from indicators and Streptocarpus were tested by RT-PCR (SFBV) or RT-qPCR (INSV), using in-house designed primers: SFBV-forward (5'-GTCATCAGCCGGAGAGGTTC-3'), SFBV-reverse (5'-AGGGCGAGTCTCTTCCTCTG-3'), INSV-forward (5'-CAATCAGAGGGTGACTTGGAA-3'), INSV-reverse (5'-GACTTTCCGAAGACTTGATGC-3') and INSV-probe (5'-CCATTGTCCTTTATCATTCCAACAAG-3'). RT-PCR products (across MP and CP regions) of the expected size (357 bp) were amplified from the Streptocarpus sample and symptomatic indicators. All amplicons were sequenced in both directions and found to be identical to the obtained HTS sequence. The presence of INSV was confirmed in all Streptocarpus and inoculated indicators except N. tabacum by INSV-specific RT-qPCR. A further 77 Streptocarpus plants were collected from a greenhouse in Auckland that holds a collection of multiple Streptocarpus cultivars from across NZ and overseas. Twenty-five plants, either displaying flower colour-break (only one plant) or asymptomatic (24 plants), tested positive for SFBV by RT-PCR. All amplicons were sequenced and found to be identical. SFBV was first described from naturally infected Streptocarpus plants in 1995 in the Netherlands (Verhoeven et al. 1995), and then in Germany (Heinze et al. 2006) and the United States (Pappu & Druffel 2007). While INSV has been found in NZ in several plant genera (Veerakone et al., 2015), to our knowledge, this is the first report of SFBV in NZ. SFBV was thought to be associated with colour breaking of Streptocarpus flowers (Verhoeven et al. 1995) but the virus was detected in asymptomatic Streptocarpus plants in this study and in California (Pappu & Druffel 2007). Given SFBV-infected plants were purchased from several sources, and leaf cuttings for propagation are shared among hobbyists, SFBV is likely to have spread throughout NZ. How this will affect production is unclear at this stage.

First report of Fig virus B in Ficus carica in New Zealand.

Fig (Ficus carica) has been cultivated since ancient times, and is now grown worldwide, both for its fruit and as an ornamental plant. Several viruses and viroids are associated with Fig mosaic disease (FMD), a disease complex occurring worldwide (Preising et al. 2021). Fig mosaic virus (FMV), fig leaf mottle-associated virus 1 (FLMaV-1), fig mild mottle-associated virus (FMMaV), and fig badnavirus 1 (FBV-1) are known to infect fig in New Zealand (Minafra et al. 2012; Veerakone et al. 2015). In December 2020, leaf samples from a fig tree growing on the roadside at St Heliers, Auckland, showing dieback with foliar chlorotic mosaic symptoms, was received for virus testing. Total nucleic acid was extracted from the symptomatic leaves using a KingFisher™ mL Purification System (Thermofisher Scientific, Waltham, MA) with an InviMag Plant DNA Mini Kit (Invitek Molecular GmbH, Germany) and subjected to high-throughput sequencing on an Oxford Nanopore Technologies MinION device using the method described in Liefting et al. 2021. All sequence analysis was performed using Geneious Prime 2021.1.1 (https://www.geneious.com). A total of 355,858 reads that passed quality check were subjected to BLASTn search against the NCBI nt database as described in Liefting et al. 2021. The following viruses produced hits: FMV, FBV-1, FMMaV and a fig closterovirus. The presence of FMV, FBV-1 and FMMaV were confirmed by species specific RT-PCRs. To identify the closterovirus, reads were mapped to closteroviruses reported in fig including the recently identified tentative species fig virus A (FiVA; GenBank accession no MN817232) and fig virus B (FiVB; GenBank accession no. MN817233). Five viral contigs ranging from 939 to 2,340 nucleotides (nt) were obtained from mapping to FiVB. Subsequently, a 6.4 kb sequence (GenBank accession no. OQ968551) from the 3' region of the NZ isolate was amplified by overlapping RT-PCR using primers designed from the contig sequences. The sequence shared 79.5% nucleotide (nt) identity with FiVB The original sample and a further 25 symptomatic and 10 asymptomatic fig samples, collected from the Auckland area between 2016 and 2021, were tested using FiVB specific RT-PCR and Sanger sequencing using primers FiVB-F1 (5'-GAGGGAGAGATGTAGATGC-3') and FiVB-R2 (5'-TGTCGTCGATATCGTTGTGT-3'), designed to amplify a 725 nt fragment in the 70 kDa heat shock protein (HSP70) ORF. Products of the expected size were amplified from the original sample and three symptomatic samples and their sequences found to be identical. BLAST searches showed that the sequence (GenBank accession no. ON553403) shared 82.7% nt and 87.3% amino acid (aa) identity to an isolate of FiVB (GenBank accession no MN817233). These additional positive samples were collected from a small home nursery where the plants were propagated from cuttings and have been distributed locally, suggesting the virus is very likely to have a limited spread throughout the Auckland area. All three FiVB infected samples were also positive for FMV. However, the association of FiVB with FMD symptoms is unknown. FiVB was first identified from a latex sample exuded from a fig tree collected from Japan (Park et al. 2021) and is the only report of FiVB in the world to date. Although an identical sequence from Argentina, named fig closterovirus 1, was submitted to GenBank, the origin of this isolate is not known. To our knowledge, this is the first report of FiVB in New Zealand.

Somatostatin-Positive Interneurons Contribute to Seizures in SCN8A Epileptic Encephalopathy.

SCN8A epileptic encephalopathy is a devastating epilepsy syndrome caused by mutant SCN8A, which encodes the voltage-gated sodium channel NaV1.6. To date, it is unclear if and how inhibitory interneurons, which express NaV1.6, influence disease pathology. Using both sexes of a transgenic mouse model of SCN8A epileptic encephalopathy, we found that selective expression of the R1872W SCN8A mutation in somatostatin (SST) interneurons was sufficient to convey susceptibility to audiogenic seizures. Patch-clamp electrophysiology experiments revealed that SST interneurons from mutant mice were hyperexcitable but hypersensitive to action potential failure via depolarization block under normal and seizure-like conditions. Remarkably, GqDREADD-mediated activation of WT SST interneurons resulted in prolonged electrographic seizures and was accompanied by SST hyperexcitability and depolarization block. Aberrantly large persistent sodium currents, a hallmark of SCN8A mutations, were observed and were found to contribute directly to aberrant SST physiology in computational modeling and pharmacological experiments. These novel findings demonstrate a critical and previously unidentified contribution of SST interneurons to seizure generation not only in SCN8A epileptic encephalopathy, but epilepsy in general.SIGNIFICANCE STATEMENTSCN8A epileptic encephalopathy is a devastating neurological disorder that results from de novo mutations in the sodium channel isoform Nav1.6. Inhibitory neurons express NaV1.6, yet their contribution to seizure generation in SCN8A epileptic encephalopathy has not been determined. We show that mice expressing a human-derived SCN8A variant (R1872W) selectively in somatostatin (SST) interneurons have audiogenic seizures. Physiological recordings from SST interneurons show that SCN8A mutations lead to an elevated persistent sodium current which drives initial hyperexcitability, followed by premature action potential failure because of depolarization block. Furthermore, chemogenetic activation of WT SST interneurons leads to audiogenic seizure activity. These findings provide new insight into the importance of SST inhibitory interneurons in seizure initiation, not only in SCN8A epileptic encephalopathy, but for epilepsy broadly.

Reversing mitochondrial defects in aged hearts: role of mitochondrial calpain activation.

Aging chronically increases endoplasmic reticulum (ER) stress that contributes to mitochondrial dysfunction. Activation of calpain 1 (CPN1) impairs mitochondrial function during acute ER stress. We proposed that aging-induced ER stress led to mitochondrial dysfunction by activating CPN1. We posit that attenuation of the ER stress or direct inhibition of CPN1 in aged hearts can decrease cardiac injury during ischemia-reperfusion by improving mitochondrial function. Male young (3 mo) and aged mice (24 mo) were used in the present study, and 4-phenylbutyrate (4-PBA) was used to decrease the ER stress in aged mice. Subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) were isolated. Chronic 4-PBA treatment for 2 wk decreased CPN1 activation as shown by the decreased cleavage of spectrin in cytosol and apoptosis inducing factor (AIF) and the α1 subunit of pyruvate dehydrogenase (PDH) in mitochondria. Treatment improved oxidative phosphorylation in 24-mo-old SSM and IFM at baseline compared with vehicle. When 4-PBA-treated 24-mo-old hearts were subjected to ischemia-reperfusion, infarct size was decreased. These results support that attenuation of the ER stress decreased cardiac injury in aged hearts by improving mitochondrial function before ischemia. To challenge the role of CPN1 as an effector of the ER stress, aged mice were treated with MDL-28170 (MDL, an inhibitor of calpain 1). MDL treatment improved mitochondrial function in aged SSM and IFM. MDL-treated 24-mo-old hearts sustained less cardiac injury following ischemia-reperfusion. These results support that age-induced ER stress augments cardiac injury during ischemia-reperfusion by impairing mitochondrial function through activation of CPN1.

Analysis of the genome of grapevine red blotch virus and related grabloviruses indicates diversification prior to the arrival of Vitis vinifera in North America.

In this study 163 complete whole-genome sequences of the emerging pathogen grapevine red blotch virus (GRBV; genus Grablovirus, family Geminiviridae) were used to reconstruct phylogenies using Bayesian analyses on time-tipped (heterochronous) data. Using different combinations of priors, Bayes factors identified heterochronous datasets (3×200 million chains) generated from strict clock and exponential tree priors as being the most robust. Substitution rates of 3.2×10-5 subsitutions per site per year (95% HPD 4.3-2.1×10-5) across the whole of the GRBV genome were estimated, suggesting ancestral GRBV diverged from ancestral wild Vitis latent virus 1 around 9 000 years ago, well before the first documented arrival of Vitis vinifera in North America. Whole-genome analysis of GRBV isolates in a single infected field-grown grapevine across 12 years identified 12 single nucleotide polymorphisms none of which were fixed substitutions: an observation not discordant with the in silico estimate. The substitution rate estimated here is lower than those estimated for other geminiviruses and is the first for a woody-host-infecting geminivirus.

ICTV Virus Taxonomy Profile: Secoviridae 2022.

Members of the family Secoviridae are non-enveloped plant viruses with mono- or bipartite linear positive-sense ssRNA genomes with a combined genome of 9 to 13.7 kb and icosahedral particles 25-30 nm in diameter. They are related to picornaviruses and are members of the order Picornavirales. Genera in the family are distinguished by the host range, vector, genomic features and phylogeny of the member viruses. Most members infect dicotyledonous plants, and many cause serious disease epidemics. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Secoviridae, which is available at ictv.global/report/secoviridae.

The mitochondrial electron transport chain contributes to calpain 1 activation during ischemia-reperfusion.

Activation of calpain1 (CPN1) contributes to mitochondrial dysfunction during cardiac ischemia (ISC) - reperfusion (REP). Blockade of electron transport using amobarbital (AMO) protects mitochondria during ISC-REP, indicating that the electron transport chain (ETC) is a key source of mitochondrial injury. We asked if AMO treatment can decrease CPN1 activation as a potential mechanism of mitochondrial protection during ISC-REP. Buffer-perfused adult rat hearts underwent 25 min global ISC and 30 min REP. AMO (2.5 mM) or vehicle was administered for 1 min before ISC to block electron flow in the ETC. Hearts in the time control group were untreated and buffer perfused without ISC. Hearts were collected at the end of perfusion and used for mitochondrial isolation. ISC-REP increased both the cleavage of spectrin (indicating cytosolic CPN1 activation) in cytosol and the truncation of AIF (apoptosis inducing factor, indicating mitochondrial CPN1 activation) in subsarcolemmal mitochondria compared to time control. Thus, ISC-REP activated both cytosolic and mitochondrial CPN1. AMO treatment prevented the cleavage of spectrin and AIF during ISC-REP, suggesting that the transient blockade of electron transport during ISC decreases CPN1 activation. AMO treatment decreased the activation of PARP [poly(ADP-ribose) polymerase] downstream of AIF that triggers caspase-independent apoptosis. AMO treatment also decreased the release of cytochrome c from mitochondria during ISC-REP that prevented caspase 3 activation. These results support that the damaged ETC activates CPN1 in cytosol and mitochondria during ISC-REP, likely via calcium overload and oxidative stress. Thus, AMO treatment to mitigate mitochondrial-driven cardiac injury can decrease both caspase-dependent and caspase-independent programmed cell death during ISC-REP.

Optical imaging detects metabolic signatures associated with oocyte quality†.

Oocyte developmental potential is intimately linked to metabolism. Existing approaches to measure metabolism in the cumulus oocyte complex (COC) do not provide information on the separate cumulus and oocyte compartments. Development of an assay that achieves this may lead to an accurate diagnostic for oocyte quality. Optical imaging of the autofluorescent cofactors reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) provides a spatially resolved indicator of metabolism via the optical redox ratio (FAD/[NAD(P)H + FAD]). This may provide an assessment of oocyte quality. Here, we determined whether the optical redox ratio is a robust methodology for measuring metabolism in the cumulus and oocyte compartments compared with oxygen consumption in the whole COC. We also determined whether optical imaging could detect metabolic differences associated with poor oocyte quality (etomoxir-treated). We used confocal microscopy to measure NAD(P)H and FAD, and extracellular flux to measure oxygen consumption. The optical redox ratio accurately reflected metabolism in the oocyte compartment when compared with oxygen consumption (whole COC). Etomoxir-treated COCs showed significantly lower levels of NAD(P)H and FAD compared to control. We further validated this approach using hyperspectral imaging, which is clinically compatible due to its low energy dose. This confirmed lower NAD(P)H and FAD in etomoxir-treated COCs. When comparing hyperspectral imaged vs non-imaged COCs, subsequent preimplantation development and post-transfer viability were comparable. Collectively, these results demonstrate that label-free optical imaging of metabolic cofactors is a safe and sensitive assay for measuring metabolism and has potential to assess oocyte developmental competence.

Astrocytes promote a protective immune response to brain Toxoplasma gondii infection via IL-33-ST2 signaling.

It is of great interest to understand how invading pathogens are sensed within the brain, a tissue with unique challenges to mounting an immune response. The eukaryotic parasite Toxoplasma gondii colonizes the brain of its hosts, and initiates robust immune cell recruitment, but little is known about pattern recognition of T. gondii within brain tissue. The host damage signal IL-33 is one protein that has been implicated in control of chronic T. gondii infection, but, like many other pattern recognition pathways, IL-33 can signal peripherally, and the specific impact of IL-33 signaling within the brain is unclear. Here, we show that IL-33 is expressed by oligodendrocytes and astrocytes during T. gondii infection, is released locally into the cerebrospinal fluid of T. gondii-infected animals, and is required for control of infection. IL-33 signaling promotes chemokine expression within brain tissue and is required for the recruitment and/or maintenance of blood-derived anti-parasitic immune cells, including proliferating, IFN-γ-expressing T cells and iNOS-expressing monocytes. Importantly, we find that the beneficial effects of IL-33 during chronic infection are not a result of signaling on infiltrating immune cells, but rather on radio-resistant responders, and specifically, astrocytes. Mice with IL-33 receptor-deficient astrocytes fail to mount an adequate adaptive immune response in the CNS to control parasite burden-demonstrating, genetically, that astrocytes can directly respond to IL-33 in vivo. Together, these results indicate a brain-specific mechanism by which IL-33 is released locally, and sensed locally, to engage the peripheral immune system in controlling a pathogen.

Complete nucleotide sequence of sweetbriar rose curly-top associated virus, a tentative member of the genus Waikavirus.

A novel virus, tentatively named "sweetbriar rose curly-top associated virus" (SRCTaV), was identified in sweetbriar rose (Rosa rubiginosa) using high-throughput sequencing. The complete genome sequence of SRCTaV was determined and characterized. Phylogenetic analysis revealed that SRCTaV is closely related to members of the genus Waikavirus.

Complete genome sequence of a novel potyvirus infecting Miscanthus sinensis (silver grass).

Here, we describe the full-length genome sequence of a novel potyvirus, tentatively named "Miscanthus sinensis mosaic virus" (MsiMV), isolated from Miscanthus sinensis (silver grass) held in a post-entry quarantine facility after being imported into Western Australia, Australia. The MsiMV genome is 9604 nucleotides (nt) in length, encoding a 3071-amino-acid (aa) polyprotein with conserved sequence motifs. The MsiMV genome is most closely related to that of sorghum mosaic virus (SrMV), with 74% nt and 78.5% aa sequence identity to the SrMV polyprotein region. Phylogenetic analysis based on the polyprotein grouped MsiMV with SrMV, sugarcane mosaic virus (SCMV), and maize dwarf mosaic virus (MDMV). This is the first report of a novel monopartite ssRNA virus in Miscanthus sinensis related to members of the genus Potyvirus in the family Potyviridae.

Searching for the Mechanism that Mediates Mefenoxam-Acquired Resistance in Phytophthora infestans and How It Is Regulated.

Phytophthora infestans, the causal agent of late blight disease of potatoes, is mainly controlled by the use of fungicides. Isolates that are resistant to commonly used fungicides have been reported. Also, several studies show that originally mefenoxam-sensitive isolates acquire resistance to this fungicide when exposed to sublethal concentrations. This phenomenon, termed "mefenoxam-acquired resistance," has been observed in different Phytophthora species and seems to be unique to mefenoxam. In this study, we aimed to elucidate the molecular mechanism mediating this type of resistance as well as a possible regulatory process behind it. A combination of computational analyses and experimental approaches was used to identify differentially expressed genes with a potential association to the phenomenon. These genes were classified into seven functional groups. Most of them seem to be associated with a pleiotropic drug resistance (PDR) phenotype, typically involved in the expulsion of diverse metabolites, drugs, or other substances out of the cell. Despite the importance of RNA Polymerase I for the constitutive resistance of P. infestans to mefenoxam, our results indicate no clear interaction between this protein and the acquisition of mefenoxam resistance. Several small non-coding RNAs were found to be differentially expressed and specifically related to genes mediating the PDR phenotype, thus suggesting a possible regulatory process. We propose a model of the molecular mechanisms acting within the cell when P. infestans acquires resistance to mefenoxam after exposed to sublethal concentrations of the fungicide. This study provides important insights into P. infestans' cellular and regulatory functionalities.