A critical component controlling bacterial virulence is the delivery of pathogen effectors into plant cells during infection. Effectors alter host metabolism and immunity for the benefit of pathogens. Multiple effectors are phosphorylated by host kinases, and this posttranslational modification is important for their activity. We sought to identify host kinases involved in effector phosphorylation. Multiple phosphorylated effector residues matched the proposed consensus motif for the plant calcium-dependent protein kinase (CDPK) and Snf1-related kinase (SnRK) superfamily. The conserved Pseudomonas effector AvrPtoB acts as an E3 ubiquitin ligase and promotes bacterial virulence. In this study, we identified a member of the Arabidopsis SnRK family, SnRK2.8, which interacts with AvrPtoB in yeast and in planta. We showed that SnRK2.8 was required for AvrPtoB virulence functions, including facilitating bacterial colonization, suppression of callose deposition, and targeting the plant defense regulator NPR1 and analyses receptor FLS2. Mass spectrometry analysis revealed that AvrPtoB phosphorylation occurs at multiple serine residues in planta, with S258 phosphorylation significantly reduced in the snrk2.8 knockout. AvrPtoB phospho-null mutants exhibited compromised virulence functions and were unable to suppress NPR1 accumulation, FLS2 accumulation, or inhibit FLS2-BAK1 complex formation upon flagellin perception. Taken together, these data identify a conserved plant kinase utilized by a pathogen effector to promote disease.

Germ cells develop as a cyst of interconnected sibling cells in a broad range of organisms in both sexes. A well-established function of intercellular connectivity is to transport cytoplasmic materials from nurse cells to oocytes, a critical process for developing functional oocytes in ovaries of many species. However, there are situations where connectivity exists without a nursing mechanism, and the biological meaning of such connectivity remains obscure. In this review, we summarize current knowledge on the formation of intercellular connectivity, and discuss its meaning by visiting multiple examples of germ cell connectivity observed in evolutionarily distant species.

Background

Based on CDC estimates in the United States, the prevalence of obesity was 42.4% in 2017-2018, and the annual cost of obesity was $147 billion in 2008. Yet studies estimate that only 20-40% of adults with obesity received counseling from their primary care providers. Recent studies using shared medical appointments (SMA), where patients are seen by a multidisciplinary team, have shown promising results in obesity management. We developed an insurance-based weight loss program incorporating SMA, called the Program for Reducing Obesity (PRO), and report our findings here.

Methods

Enrollment began in January 2019 at the UCLA Health Thousand Oaks clinic. Patients age ≥18 years with BMI ≥30 kg/m² were eligible by referral to PRO, a program consisting of individual visits and SMAs with an obesity medicine board certified endocrinologist and registered dietitian. Primary outcomes were change in weight after 3, 6, and 12 months. Secondary outcomes included proportion that achieved ≥5% weight loss, change in percent body fat, HbA1c, HDL, triglycerides, and blood pressure.

Results

102 patients (mean age 59.7 years, 72% women, mean weight 103.6 kg, mean BMI 36.6 kg/m²) have been analyzed, with 91 patients completing at least 12 months of the program. Patients achieved significant weight loss: 3.0%, 5.0%, and 7.8% of their baseline weight after 3, 6, and 12 months respectively. 52% of patients lost ≥5% of their baseline weight after 12 months. Patients had significant reductions in body fat: 2.1%, 7.4%, and 6.7% of their baseline (all p ≤ 0.01) after 3, 6, and 12 months respectively. Improvements were also seen in HbA1c (p ≤ 0.01), triglycerides (p ≤ 0.04), and systolic blood pressure (p ≤ 0.07) after 12 months although not all results achieved statistical significance.

Conclusion

Our institutional review of PRO, an insurance-based obesity program utilizing SMA, demonstrates a successful approach to promoting weight loss in a community-based setting.

Plant small RNAs play important roles in gene regulation during pathogen infection. Here we show that miR863-3p is induced by the bacterial pathogen Pseudomonas syringae carrying various effectors. Early during infection, miR863-3p silences two negative regulators of plant defence, atypical receptor-like pseudokinase1 (ARLPK1) and ARLPK2, both lacking extracellular domains and kinase activity, through mRNA degradation to promote immunity. ARLPK1 associates with, and may function through another negative immune regulator ARLPK1-interacting receptor-like kinase 1 (AKIK1), an active kinase with an extracellular domain. Later during infection, miR863-3p silences SERRATE, which is essential for miRNA accumulation and positively regulates defence, through translational inhibition. This results in decreased miR863-3p levels, thus forming a negative feedback loop to attenuate immune responses after successful defence. This is an example of a miRNA that sequentially targets both negative and positive regulators of immunity through two modes of action to fine-tune the timing and amplitude of defence responses.

Protein-based biosensors or activators have been engineered to visualize molecular signals or manipulate cellular functions. Here we integrate these two functionalities into one protein molecule, an integrated sensing and activating protein (iSNAP). A prototype that can detect tyrosine phosphorylation and immediately activate auto-inhibited Shp2 phosphatase, Shp2-iSNAP, is designed through modular assembly. When Shp2-iSNAP is fused to the SIRPα receptor which typically transduces anti-phagocytic signals from the 'don't eat me' CD47 ligand through negative Shp1 signaling, the engineered macrophages not only allow visualization of SIRPα phosphorylation upon CD47 engagement but also rewire the CD47-SIRPα axis into the positive Shp2 signaling, which enhances phagocytosis of opsonized tumor cells. A second SIRPα Syk-iSNAP with redesigned sensor and activator modules can likewise rewire the CD47-SIRPα axis to the pro-phagocytic Syk kinase activation. Thus, our approach can be extended to execute a broad range of sensing and automated reprogramming actions for directed therapeutics.Protein-based biosensors have been engineered to interrogate cellular signaling and manipulate function. Here the authors demonstrate iSNAP, a tool to detect tyrosine phosphorylation and activate desired protein enzymes allowing the control of phagocytosis in macrophages.

Peroxisomes account for ~35% of total H2O2 generation in mammalian tissues. Peroxisomal ACOX1 (acyl-CoA oxidase 1) is the first and rate-limiting enzyme in fatty acid β-oxidation and a major producer of H2O2 ACOX1 dysfunction is linked to peroxisomal disorders and hepatocarcinogenesis. Here, we show that the deacetylase sirtuin 5 (SIRT5) is present in peroxisomes and that ACOX1 is a physiological substrate of SIRT5. Mechanistically, SIRT5-mediated desuccinylation inhibits ACOX1 activity by suppressing its active dimer formation in both cultured cells and mouse livers. Deletion of SIRT5 increases H2O2 production and oxidative DNA damage, which can be alleviated by ACOX1 knockdown. We show that SIRT5 downregulation is associated with increased succinylation and activity of ACOX1 and oxidative DNA damage response in hepatocellular carcinoma (HCC). Our study reveals a novel role of SIRT5 in inhibiting peroxisome-induced oxidative stress, in liver protection, and in suppressing HCC development.

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway (PPP) and plays an essential role in the oxidative stress response by producing NADPH, the main intracellular reductant. G6PD deficiency is the most common human enzyme defect, affecting more than 400 million people worldwide. Here, we show that G6PD is negatively regulated by acetylation on lysine 403 (K403), an evolutionarily conserved residue. The K403 acetylated G6PD is incapable of forming active dimers and displays a complete loss of activity. Knockdown of G6PD sensitizes cells to oxidative stress, and re-expression of wild-type G6PD, but not the K403 acetylation mimetic mutant, rescues cells from oxidative injury. Moreover, we show that cells sense extracellular oxidative stimuli to decrease G6PD acetylation in a SIRT2-dependent manner. The SIRT2-mediated deacetylation and activation of G6PD stimulates PPP to supply cytosolic NADPH to counteract oxidative damage and protect mouse erythrocytes. We also identified KAT9/ELP3 as a potential acetyltransferase of G6PD. Our study uncovers a previously unknown mechanism by which acetylation negatively regulates G6PD activity to maintain cellular NADPH homeostasis during oxidative stress.

As one of the most induced genes in activated macrophages, immune-responsive gene 1 (IRG1) encodes a mitochondrial metabolic enzyme catalysing the production of itaconic acid (ITA). Although ITA has an anti-inflammatory property, the underlying mechanisms are not fully understood. Here we show that ITA is a potent inhibitor of the TET-family DNA dioxygenases. ITA binds to the same site on TET2 as the co-substrate α-ketoglutarate, inhibiting TET2 catalytic activity. Lipopolysaccharide treatment, which induces Irg1 expression and ITA accumulation, inhibits Tet activity in macrophages. Transcriptome analysis reveals that TET2 is a major target of ITA in suppressing lipopolysaccharide-induced genes, including those regulated by the NF-κB and STAT signalling pathways. In vivo, ITA decreases the levels of 5-hydroxymethylcytosine, reduces lipopolysaccharide-induced acute pulmonary oedema as well as lung and liver injury, and protects mice against lethal endotoxaemia, depending on the catalytic activity of Tet2. Our study thus identifies ITA as an immune modulatory metabolite that selectively inhibits TET enzymes to dampen the inflammatory responses.

The TET2 DNA dioxygenase regulates gene expression by catalyzing demethylation of 5-methylcytosine, thus epigenetically modulating the genome. TET2 does not contain a sequence-specific DNA-binding domain, and how it is recruited to specific genomic sites is not fully understood. Here we carried out a mammalian two-hybrid screen and identified multiple transcriptional regulators potentially interacting with TET2. The SMAD nuclear interacting protein 1 (SNIP1) physically interacts with TET2 and bridges TET2 to bind several transcription factors, including c-MYC. SNIP1 recruits TET2 to the promoters of c-MYC target genes, including those involved in DNA damage response and cell viability. TET2 protects cells from DNA damage-induced apoptosis dependending on SNIP1. Our observations uncover a mechanism for targeting TET2 to specific promoters through a ternary interaction with a co-activator and many sequence-specific DNA-binding factors. This study also reveals a TET2-SNIP1-c-MYC pathway in mediating DNA damage response, thereby connecting epigenetic control to maintenance of genome stability.