Serine-arginine (SR) proteins are essential splicing factors containing a canonical RNA recognition motif (RRM), sometimes followed by a pseudo-RRM, and a C-terminal arginine/serine-rich (RS) domain that undergoes multisite phosphorylation. Phosphorylation regulates the localization and activity of SR proteins, and thus may provide insight into their differential biological roles. The phosphorylation mechanism of the prototypic SRSF1 by serine-arginine protein kinase 1 (SRPK1) has been well studied but little is known about the phosphorylation of other SR protein members. In the present study, interaction and kinetic assays unveiled how SRSF1 and the single RRM-containing SRSF3 are phosphorylated by SRPK2, another member of the SRPK family. We showed a conserved SRPK-specific substrate-docking groove in SRPK2 impacts the binding and phosphorylation of both SR proteins, and the localization of SRSF3. We identified a non-conserved residue within the groove that affects the kinase processivity. We demonstrated that, in contrast to SRSF1, for which SRPK-mediated phosphorylation is confined to the N-terminal region of the RS domain, SRSF3 phosphorylation sites are spread throughout its entire RS domain in vitro. Despite this, SRSF3 appears to be hypophosphorylated in cells at steady state. Our results suggest that the absence of a pseudo-RRM renders the single RRM-containing SRSF3 more susceptible to dephosphorylation by phosphatase. These findings suggest the single RRM- and two RRMs-containing SR proteins represent two subclasses of phosphoproteins which phosphorylation statuses are maintained by unique mechanisms, and pose new directions to explore the distinct roles of SR proteins in vivo.

While dromedaries are the immediate animal source of Middle East Respiratory Syndrome (MERS) epidemic, viruses related to MERS coronavirus (MERS-CoV) have also been found in bats as well as hedgehogs. To elucidate the evolution of MERS-CoV-related viruses and their interspecies transmission pathway, samples were collected from different mammals in China. A novel coronavirus related to MERS-CoV, Erinaceus amurensis hedgehog coronavirus HKU31 (Ea-HedCoV HKU31), was identified from two Amur hedgehogs. Genome analysis supported that Ea-HedCoV HKU31 represents a novel species under Merbecovirus, being most closely related to Erinaceus CoV from European hedgehogs in Germany, with 79.6% genome sequence identity. Compared to other members of Merbecovirus, Ea-HedCoV HKU31 possessed unique non-structural proteins and putative cleavage sites at ORF1ab. Phylogenetic analysis showed that Ea-HedCoV HKU31 and BetaCoV Erinaceus/VMC/DEU/2012 were closely related to NeoCoV and BatCoV PREDICT from African bats in the spike region, suggesting that the latter bat viruses have arisen from recombination between CoVs from hedgehogs and bats. The predicted HKU31 receptor-binding domain (RBD) possessed only one out of 12 critical amino acid residues for binding to human dipeptidyl peptidase 4 (hDPP4), the MERS-CoV receptor. The structural modeling of the HKU31-RBD-hDPP4 binding interphase compared to that of MERS-CoV and Tylonycteris bat CoV HKU4 (Ty-BatCoV HKU4) suggested that HKU31-RBD is unlikely to bind to hDPP4. Our findings support that hedgehogs are an important reservoir of Merbecovirus, with evidence of recombination with viruses from bats. Further investigations in bats, hedgehogs and related animals are warranted to understand the evolution of MERS-CoV-related viruses.

In zebrafish, the role of matrix metalloproteinases (MMPs) in the inflammatory phase of heart regeneration following cryoinjury remains poorly understood. Here, we demonstrated an increase in MMP enzymatic activity and elevated expression of mmp9 and mmp13 in the injured area (IA) of hearts from as early as 1 day post-cryoinjury (dpc). Treatment with the broad-spectrum MMP inhibitor, GM6001, during the first week after cryoinjury resulted in impaired heart regeneration, as indicated by the larger scar and reduced numbers of proliferating cardiomyocytes. GM6001 also significantly reduced the number of leukocytes to the IA at 0.5 dpc to 4 dpc. Specific inhibition of both MMP-9 and MMP-13 also resulted in impaired regeneration and leukocyte recruitment. However, chemokine rescue with recombinant CXCL8 and CCL2 restored the recruitment of macrophages and the cardiac regenerative capability in GM6001-treated fish. MMP-9 and MMP-13 cleaved zebrafish CXCL8 at the same site, and the truncated form was more chemotactic than the intact form. In contrast, CCL2 did not have an MMP-9 or MMP-13 cleavage site. Together, these data suggest that MMPs might play a key role in the inflammatory phase of heart regeneration in zebrafish, by mediating leukocyte recruitment via the activation of chemokines.

Multidrug-resistant Acinetobacter baumannii, a major hospital-acquired pathogen, is a serious health threat and poses a great challenge to healthcare providers. Although there have been many genomic studies on the evolution and antibiotic resistance of this species, there have been very limited transcriptome studies on its responses to antibiotics. We conducted a comparative transcriptomic study on 12 strains with different growth rates and antibiotic resistance profiles, including 3 fast-growing pan-drug-resistant strains, under separate treatment with 3 antibiotics, namely amikacin, imipenem, and meropenem. We performed deep sequencing using a strand-specific RNA-sequencing protocol, and used de novo transcriptome assembly to analyze gene expression in the form of polycistronic transcripts. Our results indicated that genes associated with transposable elements generally showed higher levels of expression under antibiotic-treated conditions, and many of these transposon-associated genes have previously been linked to drug resistance. Using co-expressed transposon genes as markers, we further identified and experimentally validated two novel genes of which overexpression conferred significant increases in amikacin resistance. To the best of our knowledge, this study represents the first comparative transcriptomic analysis of multidrug-resistant A. baumannii under different antibiotic treatments, and revealed a new relationship between transposons and antibiotic resistance.

Given the extremely limited regeneration potential of the heart, one of the most effective strategies to reduce the prevalence and mortality of coronary artery disease is prevention. Short-chain fatty acids (SCFAs), which are by-products of beneficial probiotics, have been reported to possess cardioprotective effects. Despite their beneficial roles, delivering SCFAs and maintaining their effective concentration in plasma present major challenges. Therefore, in the present study, we aimed to devise a strategy to prevent coronary heart disease effectively by using engineered probiotics to continuously release SCFAs in vivo. We engineered a novel probiotic cocktail, EcN_TL, from the commercially available Escherichia coli Nissle 1917 strain to continuously secrete SCFAs by introducing the propionate and butyrate biosynthetic pathways. Oral administration of EcN_TL enhanced and maintained an effective concentration of SCFAs in the plasma. As a preventative strategy, we observed that daily intake of EcN_TL for 14 days prior to ischemia-reperfusion injury significantly reduced myocardial injury and improved cardiac performance compared to EcN administration. We uncovered that EcN_TL’s protective mechanisms included reducing neutrophil infiltration into the infarct site and promoting the polarization of wound-healing macrophages. We further revealed that SCFAs at plasma concentration protected cardiomyocytes from inflammation by suppressing the NF-κB activation pathway. These data provide strong evidence to support the use of SCFA-secreting probiotics to prevent coronary heart disease. Since SCFAs also play a key role in other metabolic diseases, EcN_TL can potentially be used to treat a variety of other diseases.

While dromedary camels are the immediate animal source of MERS coronavirus (MERS-CoV) infection, the evolutionary origin of MERS-CoV remains obscure. We analyzed 219 camel and human MERS-CoV genome sequences available in GenBank. Phylogenetic analysis showed that 5 and 214 strains belong to clade A and B, respectively, with clade A further divided into lineage A1 (3 human strains) and lineage A2 (2 camel strains), and clade B divided into B1 to B6 (each containing both human and camel strains). Recombination analysis showed potential recombination events in five strains from dromedaries in Saudi Arabia, with recombination between lineage B5 and B3 in four strains, and between lineage B3 and B4 in one strain. The spike protein showed the highest number of amino acid substitutions, especially between A2 and other lineages, and contained positively selected codons. Notably, codon 1020 was positively selected among B and B5 strains, and can distinguish between clade A (Q1020) and B (R1020/H1020) strains, suggesting that this residue may play a role in the evolution of S protein during divergence of different lineages. The time of the most recent common ancestor of all MERS-CoV was dated to approximately 2010. The implications on the role of camels in the evolution of MERS-CoV are discussed.