Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE) in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE–binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms) is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE–containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms.

Highlights d A system for membrane repair, removal, and replacement is coordinated by galectins d Galectin-3 recruits ESCRT components to damaged lysosomes to repair them d Galectins induce autophagy to remove damaged lysosomes and activate their biogenesis d The galectin-directed systems protect against M. tuberculosis and neurotoxic tau

The Ser/Thr protein kinase mTOR controls metabolic pathways, including the catabolic process of autophagy. Autophagy plays additional, catabolism-independent roles in homeostasis of cytoplasmic endomembranes and whole organelles. How signals from endomembrane damage are transmitted to mTOR to orchestrate autophagic responses is not known. Here we show that mTOR is inhibited by lysosomal damage. Lysosomal damage, recognized by galectins, leads to association of galectin-8 (Gal8) with the mTOR apparatus on the lysosome. Gal8 inhibits mTOR activity through its Ragulator-Rag signaling machinery, whereas galectin-9 activates AMPK in response to lysosomal injury. Both systems converge upon downstream effectors including autophagy and defense against Mycobacterium tuberculosis. Thus, a novel galectin-based signal-transduction system, termed here GALTOR, intersects with the known regulators of mTOR on the lysosome and controls them in response to lysosomal damage. VIDEO ABSTRACT.

COVID-19 patients frequently develop neurological symptoms, but the biological underpinnings of these phenomena are unknown. Through single cell RNA-seq and cytokine analyses of CSF and blood from COVID-19 patients with neurological symptoms, we find compartmentalized, CNS specific T cell activation and B cell responses. All COVID-19 cases had CSF anti-SARS-CoV-2 antibodies whose target epitopes diverged from serum antibodies. In an animal model, we find that intrathecal SARS-CoV-2 antibodies are found only during brain infection, and are not elicited by pulmonary infection. We produced CSF-derived monoclonal antibodies from a COVID-19 patient, and find that these mAbs target both anti-viral and anti-neural antigens—including one mAb that reacted to both spike protein and neural tissue. Overall, CSF IgG from 5/7 patients contains anti-neural reactivity. This immune survey reveals evidence of a compartmentalized immune response in the CNS of COVID-19 patients and suggests a role for autoimmunity in neurologic sequelae of COVID-19.

Amelogenin genes are located on both X and Y sex chromosomes in humans and are a major focus of DNA-based sex estimation methods. Amelogenin proteins, AMELX_HUMAN and AMELY_HUMAN, are expressed in the tooth organ and play a major role in mineralization of enamel, the most taphonomically resistant, archaeologically persistent human tissue. We describe shotgun liquid chromatography mass spectrometry analysis of 40 enamel samples representing 25 individuals, including modern third molars and archaeological teeth from open-air contexts including permanent adult (400-7300 BP) and deciduous teeth (100-1000 BP). Peptides specific to the X-chromosome isoform of amelogenin were detected in all samples. Peptides specific to the sexually dimorphic Y-chromosome isoform were also detected in 26 samples from 13 individuals, across all time periods, including previously unsexed deciduous teeth from archaeological contexts. While the signal of each gene product can vary by more than an order of magnitude, we show close agreement between osteological and amelogenin-based sex estimation and thus demonstrate that the protein-based signal can reliably be obtained from open-air archaeological contexts dating to at least 7300 years ago. While samples with AMELY_HUMAN peptides are unambiguously male, samples with no AMELY_HUMAN signal may either be low signal male false negative samples or female samples. In order to estimate sex in these samples we developed a probability curve of female sex as a function of the logarithm of AMELX_HUMAN signal (p < 0.0001) using logistic regression. This is also the first demonstration using proteomics to estimate sex in deciduous teeth and pushes back the application of the method to teeth that are at least 7300 years old.

The 2019 novel coronavirus infectious disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created an unsustainable need for molecular diagnostic testing. Molecular approaches such as reverse transcription (RT) polymerase chain reaction (PCR) offers highly sensitive and specific means to detect SARS-CoV-2 RNA, however, despite it being the accepted “gold standard”, molecular platforms often require a tradeoff between speed versus throughput. Matrix assisted laser desorption ionization (MALDI)—time of flight (TOF)—mass spectrometry (MS) has been proposed as a potential solution for COVID-19 testing and finding a balance between analytical performance, speed, and throughput, without relying on impacted supply chains. Combined with machine learning (ML), this MALDI-TOF-MS approach could overcome logistical barriers encountered by current testing paradigms. We evaluated the analytical performance of an ML-enhanced MALDI-TOF-MS method for screening COVID-19. Residual nasal swab samples from adult volunteers were used for testing and compared against RT-PCR. Two optimized ML models were identified, exhibiting accuracy of 98.3%, positive percent agreement (PPA) of 100%, negative percent agreement (NPA) of 96%, and accuracy of 96.6%, PPA of 98.5%, and NPA of 94% respectively. Machine learning enhanced MALDI-TOF-MS for COVID-19 testing exhibited performance comparable to existing commercial SARS-CoV-2 tests.

The Ser/Thr protein kinase MTOR (mechanistic target of rapamycin kinase) regulates cellular metabolism and controls macroautophagy/autophagy. Autophagy has both metabolic and quality control functions, including recycling nutrients at times of starvation and removing dysfunctional intracellular organelles. Lysosomal damage is one of the strongest inducers of autophagy, and yet mechanisms of its activation in response to lysosomal membrane damage are not fully understood. Our recent study has uncovered a new signal transduction system based on cytosolic galectins that elicits autophagy by controlling master regulators of metabolism and autophagy, MTOR and AMPK, in response to lysosomal damage. Thus, intracellular galectins are not, as previously thought, passive tags recognizing damage to guide selective autophagy receptors, but control the activation state of AMPK and MTOR in response to endomembrane damage. Abbreviations: MTOR: mechanistic target of rapamycin kinase; AMPK: AMP-activated protein kinase / Protein Kinase AMP-Activated; SLC38A9: Solute Carrier Family 38 Member 9; APEX2: engineered ascorbate peroxidase 2; RRAGA/B: Ras Related GTP Binding A or B; LAMTOR1: Late Endosomal/Lysosomal Adaptor, MAPK and MTOR Activator 1; LGALS8: Lectin, Galactoside-Binding, Soluble, 8 / Galectin 8; LGALS9: Lectin, Galactoside-Binding, Soluble, 9 / Galectin 9; TAK1: TGF-Beta Activated Kinase 1 / Mitogen-Activated Protein Kinase Kinase Kinase 7 (MAP3K7); STK11/LKB1: Serine/Threonine Kinase 11 / Liver Kinase B1; ULK1: Unc-51 Like Autophagy Activating Kinase 1.

(Fig. 1D, upper panel).Similar to pnc1∆ mutant (Fig. 1B), both nat3∆and mdm20∆ released mostly NAM not NA (Fig.1D, lower panel). Fig. 1E (Fig. 2E, top panel) had a lesser effect (Fig. 2E, bottom (Fig. 3B). Next, we tested whether deleting ATG14 was sufficient to restore NAD + levels in the NatB mutants. As shown in Fig. 3C (left panel), deleting ATG14did not rescue the low NAD + levels in nat3∆ cells. Similarly, deleting other factors contributing to NR and NAM production ( Fig. 1A and Fig. 2E) in nat3∆ cells also failed to restore the NAD + level back to wild type ( shown to restore actin cable formation in NatB mutants (23,24,28,29). Interestingly, both TPM1-oe and TPM1-5 largely blocked NA/NAM release in the nat3∆ mutant (Fig. 3D) 1A). N-terminal acetyltransferases have specific targets that are largely determined by the sequence of the first two amino acids. NatB acetylates proteins with a MET-retained residue, followed by ASP, GLU, GLN, or ASN as the second residue (25,26,39,40). Nma1 and Nma2 have a MET-ASP N-terminus, but neither protein has been identified as a NatB target.Similar to nat3∆ mutant, nma1∆ cells showed decreased NAD + levels, which could not be rescued by supplementing NR (Fig. 4B). As for the controls, NR efficiently restored the NAD + level in the npt1∆ mutant, which has functional NR salvage (Fig. 4B). The similarities between nma1∆ and nat3∆ mutants suggested that Nma1and Nma2 activities are likely reduced in nat3∆ cells. Since N-terminal acetylation may affect the turnover of target proteins, we first determined whether reduced Nma1/Nma2 activities were due to decreased Nma1/Nma2 protein levels. As shown in Fig. 4C, Nma1 and Nma2 protein levels were indeed decreased in nat3∆ cells (Fig. 4C). In addition to Nma1 and Nma2, NAD + homeostasis factors Bna2, Bna5and Hst1 are also potential NatB targets. Bna2and Bna5 are biosynthesis enzymes in the de novo pathway (Fig. 1A) DISCUSSIONIn this study we characterized NatB complex as a novel NAD + homeostasis factor.Mutants lacking components of the NatB complex, NAT3 and MDM20, produce and release excess amount of NA and NAM. Our studies showed two pathways downstream of NatB contribute to NAD + homeostasis (Fig. 5).In one, NatB is important for proper NAD + biosynthesis by regulating Nma1/Nma2. NatB mutants have low NAD + levels (Fig. 2C), and all NAD + precursors examined failed to restore the NAD + levels ( Fig. 4A and 4B). This suggests that a NAD + biosynthesis factor(s) required for utilization of all NAD + precursors is defected in NatB mutants. Nma1 and Nma2 are such targets because they are the only factors required for all three NAD + biosynthesis pathways (de novo, NA/NAM salvage, and NR salvage) (Fig. 1A), and the N-terminal amino acid sequences are a match for NatB acetylation. In addition, nma1∆and nat3∆ mutants showed similar NAD + utilization defects (Fig. 4B). Although Nma2 is present in both mutants, Nma2 is known to play a minor role in NAD + metabolism. Supporting this model, decreased Nma1 protein level was observed in t...