ABSTRACT

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin (TOR) signaling, oxidative stress resistance, and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild-type cells recovering from phosphate starvation. Nonphosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar, GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. We posit that low substrate concentrations of beta-d-glucan- and chitin synthases, together with pharmacologic inhibition of their activity, diminish enzymatic reaction rates as well as the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-d-glucans or chitin. Hence, inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.

IMPORTANCE Candida species cause hundreds of thousands of invasive infections with high mortality each year. Developing novel antifungal agents is challenging due to the many similarities between fungal and human cells. Maintaining phosphate balance is essential for all organisms but is achieved completely differently by fungi and humans. A protein that imports phosphate into fungal cells, Pho84, is not present in humans and is required for normal cell wall stress resistance and cell wall integrity signaling in C. albicans. Nucleotide sugars, which are phosphate-containing building block molecules for construction of the cell wall, are diminished in cells lacking Pho84. Cell wall-constructing enzymes may be slowed by lack of these building blocks, in addition to being inhibited by drugs. Combined targeting of Pho84 and cell wall-constructing enzymes may provide a strategy for antifungal therapy by which two sequential steps of cell wall maintenance are blocked for greater potency.

ABSTRACT

Bacillus anthracis is a spore-forming bacterium that causes devastating infections and has been used as a bioterror agent. This pathogen can survive hostile environments through the signaling activity of two-component systems, which couple environmental sensing with transcriptional activation to initiate a coordinated response to stress. In this work, we describe the identification of a two-component system, EdsRS, which mediates the B. anthracis response to the antimicrobial compound targocil. Targocil is a cell envelope-targeting compound that is toxic to B. anthracis at high concentrations. Exposure to targocil causes damage to the cellular barrier and activates EdsRS to induce expression of a previously uncharacterized cardiolipin synthase, which we have named ClsT. Both EdsRS and ClsT are required for protection against targocil-dependent damage. Induction of clsT by EdsRS during targocil treatment results in an increase in cardiolipin levels, which protects B. anthracis from envelope damage. Together, these results reveal that a two-component system signaling response to an envelope-targeting antimicrobial induces production of a phospholipid associated with stabilization of the membrane. Cardiolipin is then used to repair envelope damage and promote B. anthracis viability.

IMPORTANCE Compromising the integrity of the bacterial cell barrier is a common action of antimicrobials. Targocil is an antimicrobial that is active against the bacterial envelope. We hypothesized that Bacillus anthracis, a potential weapon of bioterror, senses and responds to targocil to alleviate targocil-dependent cell damage. Here, we show that targocil treatment increases the permeability of the cellular envelope and is particularly toxic to B. anthracis spores during outgrowth. In vegetative cells, two-component system signaling through EdsRS is activated by targocil. This results in an increase in the production of cardiolipin via a cardiolipin synthase, ClsT, which restores the loss of barrier function, thereby reducing the effectiveness of targocil. By elucidating the B. anthracis response to targocil, we have uncovered an intrinsic mechanism that this pathogen employs to resist toxicity and have revealed therapeutic targets that are important for bacterial defense against structural damage.

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans and remains endemic in the Middle East since first being identified in 2012. There are currently no approved vaccines or therapies available for MERS-CoV. In this study, we evaluated parainfluenza virus 5 (PIV5)-based vaccine expressing the MERS-CoV envelope spike protein (PIV5/MERS-S) in a human DPP4 knockin C57BL/6 congenic mouse model (hDPP4 KI). Following a single-dose intranasal immunization, PIV5-MERS-S induced neutralizing antibody and robust T cell responses in hDPP4 KI mice. A single intranasal administration of 10⁴ PFU PIV5-MERS-S provided complete protection against a lethal challenge with mouse-adapted MERS-CoV (MERS_MA6.1.2) and improved virus clearance in the lung. In comparison, single-dose intramuscular immunization with 10⁶ PFU UV-inactivated MERS_MA6.1.2 mixed with Imject alum provided protection to only 25% of immunized mice. Intriguingly, an influx of eosinophils was observed only in the lungs of mice immunized with inactivated MERS-CoV, suggestive of a hypersensitivity-type response. Overall, our study indicated that PIV5-MERS-S is a promising effective vaccine candidate against MERS-CoV infection.

IMPORTANCE MERS-CoV causes lethal infection in humans, and there is no vaccine. Our work demonstrates that PIV5 is a promising vector for developing a MERS vaccine. Furthermore, success of PIV5-based MERS vaccine can be employed to develop a vaccine for emerging CoVs such as SARS-CoV-2, which causes COVID-19.

ABSTRACT

The human gut microbiota (HGM) has far-reaching impacts on human health and nutrition, which are fueled primarily by the metabolism of otherwise indigestible complex carbohydrates commonly known as dietary fiber. However, the molecular basis of the ability of individual taxa of the HGM to address specific dietary glycan structures remains largely unclear. In particular, the utilization of β(1,3)-glucans, which are widespread in the human diet as yeast, seaweed, and plant cell walls, had not previously been resolved. Through a systems-based approach, here we show that the symbiont Bacteroides uniformis deploys a single, exemplar polysaccharide utilization locus (PUL) to access yeast β(1,3)-glucan, brown seaweed β(1,3)-glucan (laminarin), and cereal mixed-linkage β(1,3)/β(1,4)-glucan. Combined biochemical, enzymatic, and structural analysis of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) illuminates a concerted molecular system by which B. uniformis recognizes and saccharifies these distinct β-glucans. Strikingly, the functional characterization of homologous β(1,3)-glucan utilization loci (1,3GUL) in other Bacteroides further demonstrated that the ability of individual taxa to utilize β(1,3)-glucan variants and/or β(1,3)/β(1,4)-glucans arises combinatorially from the individual specificities of SGBPs and GHs at the cell surface, which feed corresponding signals to periplasmic hybrid two-component sensors (HTCSs) via TonB-dependent transporters (TBDTs). These data reveal the importance of cooperativity in the adaptive evolution of GH and SGBP cohorts to address individual polysaccharide structures. We anticipate that this fine-grained knowledge of PUL function will inform metabolic network analysis and proactive manipulation of the HGM. Indeed, a survey of 2,441 public human metagenomes revealed the international, yet individual-specific, distribution of each 1,3GUL.

IMPORTANCE Bacteroidetes are a dominant phylum of the human gut microbiota (HGM) that target otherwise indigestible dietary fiber with an arsenal of polysaccharide utilization loci (PULs), each of which is dedicated to the utilization of a specific complex carbohydrate. Here, we provide novel insight into this paradigm through functional characterization of homologous PULs from three autochthonous Bacteroides species, which target the family of dietary β(1,3)-glucans. Through detailed biochemical and protein structural analysis, we observed an unexpected diversity in the substrate specificity of PUL glycosidases and glycan-binding proteins with regard to β(1,3)-glucan linkage and branching patterns. In combination, these individual enzyme and protein specificities support taxon-specific growth on individual β(1,3)-glucans. This detailed metabolic insight, together with a comprehensive survey of individual 1,3GULs across human populations, further expands the fundamental roadmap of the HGM, with potential application to the future development of microbial intervention therapies.

ABSTRACT

RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent of mitochondrial impairment in human cells affected by neurodegeneration. To fulfill all the criteria to qualify as a prion-like protein, horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1. Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1 wild type [RepA-WH1(WT)] and assayed its response to exposure to in vitro-assembled RepA-WH1(A31V) amyloid fibers. In parallel, murine cells releasing RepA-WH1(A31V) aggregates were cocultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibers and donor-derived RepA-WH1(A31V) aggregates induced, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells pointed to alterations in mitochondria, protein quality triage, signaling, and intracellular traffic. Thus, a synthetic prion-like protein can be propagated to, and become cytotoxic to, cells of organisms placed at such distant branches of the tree of life as bacteria and mammalia, suggesting that mechanisms of protein aggregate spreading and toxicity follow default pathways.

IMPORTANCE Proteotoxic amyloid seeds can be transmitted between mammalian cells, arguing that the intercellular exchange of prion-like protein aggregates can be a common phenomenon. RepA-WH1 is derived from a bacterial intracellular functional amyloid protein, engineered to become cytotoxic in Escherichia coli. Here, we have studied if such bacterial aggregates can also be transmitted to, and become cytotoxic to, mammalian cells. We demonstrate that RepA-WH1 is capable of entering naive cells, thereby inducing the cytotoxic aggregation of a soluble RepA-WH1 variant expressed in the cytosol, following the same trend that had been described in bacteria. These findings highlight the universality of one of the central principles underlying prion biology: No matter the biological origin of a given prion-like protein, it can be transmitted to a phylogenetically unrelated recipient cell, provided that the latter expresses a soluble protein onto which the incoming protein can readily template its amyloid conformation.

Objective

To investigate the effect of somatic, postzygotic, gain-of-function mutation of Endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) encoding hypoxia-inducible factor-2α (HIF-2α) on posterior fossa development and spinal dysraphism in EPAS1 gain-of-function syndrome, which consists of multiple paragangliomas, somatostatinoma, and polycythemia.

Heterozygous germline mutations in mammalian target of rapamycin (MTOR) (OMIM 601231) are known to underlie Smith-Kingsmore syndrome (SKS; OMIM 616638), an infrequent entity with autosomal dominant inheritance, also known as macrocephaly-intellectual disability-neurodevelopmental disorder-small thorax syndrome (ORPHA 457485).¹ Among the clinical features of SKS, the most common features include intellectual disability, macrocephaly, epilepsy, and facial dysmorphism. The aim of this case is to raise awareness of a distinct phenotypical presentation of SKS manifesting with bilateral cataracts and no history of seizures.

The chloroplast glutamyl-tRNA (tRNA^Glu) is unique in that it has two entirely different functions. In addition to acting in translation, it serves as the substrate of glutamyl-tRNA reductase (GluTR), the enzyme catalyzing the committed step in the tetrapyrrole biosynthetic pathway. How the tRNA^Glu pool is distributed between the two pathways and whether tRNA^Glu allocation limits tetrapyrrole biosynthesis and/or protein biosynthesis remains poorly understood. We generated a series of transplastomic tobacco (Nicotiana tabacum) plants to alter tRNA^Glu expression levels and introduced a point mutation into the plastid trnE gene, which has been reported to uncouple protein biosynthesis from tetrapyrrole biosynthesis in chloroplasts of the protist Euglena gracilis. We show that, rather than comparable uncoupling of the two pathways, the trnE mutation is lethal in tobacco because it inhibits tRNA processing, thus preventing translation of Glu codons. Ectopic expression of the mutated trnE gene uncovered an unexpected inhibition of glutamyl-tRNA reductase by immature tRNA^Glu. We further demonstrate that whereas overexpression of tRNA^Glu does not affect tetrapyrrole biosynthesis, reduction of GluTR activity through inhibition by tRNA^Glu precursors causes tetrapyrrole synthesis to become limiting in early plant development when active photosystem biogenesis provokes a high demand for de novo chlorophyll biosynthesis. Taken together, our findings provide insight into the roles of tRNA^Glu at the intersection of protein biosynthesis and tetrapyrrole biosynthesis.

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula x alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1. Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis.

Key Points

Staphylococcus aureus is a noted human and animal pathogen. Despite decades of research on this important bacterium, there are still many unanswered questions regarding the pathogenic mechanisms it uses to infect the mammalian host. This can be attributed to it possessing a plethora of virulence factors and complex virulence factor and metabolic regulation. PurR, the purine biosynthesis regulator, was recently also shown to regulate virulence factors in S. aureus, and mutations in purR result in derepression of fibronectin binding proteins (FnBPs) and extracellular toxins, required for a so-called hypervirulent phenotype. Here, we show that hypervirulent strains containing purR mutations can be attenuated with the addition of purine biosynthesis mutations, implicating the necessity for de novo purine biosynthesis in this phenotype and indicating that S. aureus in the mammalian host experiences purine limitation. Using cell culture, we showed that while purR mutants are not altered in epithelial cell binding, compared to that of wild-type (WT) S. aureus, purR mutants have enhanced invasion of these nonprofessional phagocytes, consistent with the requirement of FnBPs for invasion of these cells. This correlates with purR mutants having increased transcription of fnb genes, resulting in higher levels of surface-exposed FnBPs to promote invasion. These data provide important contributions to our understanding of how the pathogenesis of S. aureus is affected by sensing of purine levels during infection of the mammalian host.

Upon infection, the highly structured 5' untranslated region (5' UTR) of picornavirus is involved in viral protein translation and RNA synthesis. As a critical element in the 5' UTR, the internal ribosome entry site (IRES) binds to various cellular proteins to function in the processes of picornavirus replication. Foot-and-mouth disease virus (FMDV) is an important member in the family Picornaviridae, and its 5' UTR contains a functional IRES element. In this study, the cellular heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as an IRES-binding protein for FMDV by biotinylated RNA pulldown assays, mass spectrometry (MS) analysis, and determination of hnRNP L-IRES interaction regions. Further, we found that hnRNP L inhibited the growth of FMDV through binding to the viral IRES and that the inhibitory effect of hnRNP L on FMDV growth was not due to FMDV IRES-mediated translation, but to influence on viral RNA synthesis. Finally, hnRNP L was demonstrated to coimmunoprecipitate with RNA-dependent RNA polymerase (3D^pol) in an FMDV RNA-dependent manner in the infected cells. Thus, our results suggest that hnRNP L, as a critical IRES-binding protein, negatively regulates FMDV replication by inhibiting viral RNA synthesis, possibly by remaining in the replication complex.

IMPORTANCE Picornaviruses, as a large family of human and animal pathogens, cause a bewildering array of disease syndromes. Many host factors are implicated in the pathogenesis of these viruses, and some proteins interact with the viral IRES elements to affect function. Here, we report for the first time that cellular hnRNP L specifically interacts with the IRES of the picornavirus FMDV and negatively regulates FMDV replication through inhibiting viral RNA synthesis. Further, our results showed that hnRNP L coimmunoprecipitates with FMDV 3D^pol in a viral RNA-dependent manner, suggesting that it may remain in the replication complex to function. The data presented here would facilitate further understanding of virus-host interactions and the pathogenesis of picornavirus infections.

Respiratory syncytial virus (RSV) is an enveloped RNA virus which is responsible for approximately 80% of lower respiratory tract infections in children. Current lines of evidence have supported the functional involvement of long noncoding RNA (lncRNA) in many viral infectious diseases. However, the overall biological effect and clinical role of lncRNAs in RSV infection remain unclear. In this study, lncRNAs related to respiratory virus infection were obtained from the lncRNA database, and we collected 144 clinical sputum specimens to identify lncRNAs related to RSV infection. Quantitative PCR (qPCR) detection indicated that the expression of lncRNA negative regulator of antiviral response (NRAV) in RSV-positive patients was significantly lower than that in uninfected patients, but lncRNA psoriasis-associated non-protein coding RNA induced by stress (PRINS), nuclear paraspeckle assembly transcript 1 (NEAT1), and Nettoie Salmonella pas Theiler’s (NeST) showed no difference in vivo and in vitro. Meanwhile, overexpression of NRAV promoted RSV proliferation in A549 and BEAS-2B cells, and vice versa, indicating that the downregulation of NRAV was part of the host antiviral defense. RNA fluorescent in situ hybridization (FISH) confirmed that NRAV was mainly located in the cytoplasm. Through RNA sequencing, we found that Rab5c, which is a vesicle transporting protein, showed the same change trend as NRAV. Subsequent investigation revealed that NRAV was able to favor RSV production indirectly by sponging microRNA miR-509-3p so as to release Rab5c and facilitate vesicle transportation. The study provides a new insight into virus-host interaction through noncoding RNA, which may contribute to exploring potential antivirus targets for respiratory virus.

IMPORTANCE The mechanism of interaction between RSV and host noncoding RNAs is not fully understood. In this study, we found that the expression of long noncoding RNA (lncRNA) negative regulator of antiviral response (NRAV) was reduced in RSV-infected patients, and overexpression of NRAV facilitated RSV production in vitro, suggesting that the reduction of NRAV in RSV infection was part of the host antiviral response. We also found that NRAV competed with vesicle protein Rab5c for microRNA miR509-3p in cytoplasm to promote RSV vesicle transport and accelerate RSV proliferation, thereby improving our understanding of the pathogenic mechanism of RSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is a serious viral disease affecting the global swine industry. Its causative agent, PRRS virus (PRRSV), is an enveloped virus, and therefore membrane fusion between its envelope and host cell target membrane is critical for viral infection. Though much research has focused on PRRSV infection, the detailed mechanisms involved in its membrane fusion remain to be elucidated. In the present study, we performed confocal microscopy in combination with a constitutively active (CA) or dominant negative (DN) mutant, specific inhibitors, and small interfering RNAs (siRNAs), as well as multiple other approaches, to explore PRRSV membrane fusion. We first observed that PRRSV membrane fusion occurred in Rab11-recycling endosomes during early infection using labeled virions and subcellular markers. We further demonstrated that low pH and cathepsin E in Rab11-recycling endosomes are critical for PRRSV membrane fusion. Moreover, PRRSV glycoprotein 5 (GP5) is identified as being cleaved by cathepsin E during this process. Taken together, our findings provide in-depth information regarding PRRSV pathogenesis, which support a novel basis for the development of antiviral drugs and vaccines.

IMPORTANCE PRRS, caused by PRRSV, is an economically critical factor in pig farming worldwide. As PRRSV is a lipid membrane-wrapped virus, merging of the PRRSV envelope with the host cell membrane is indispensable for viral infection. However, there is a lack of knowledge on its membrane fusion. Here, we first explored when and where PRRSV membrane fusion occurs. Furthermore, we determined which host cell factors were involved in the process. Importantly, PRRSV GP5 is shown to be cleaved by cathepsin E during membrane fusion. Our work not only provides information on PRRSV membrane fusion for the first time but also deepens our understanding of the molecular mechanisms of PRRSV infection, which provides a foundation for future applications in the prevention and control of PRRS.

The entry/fusion complex (EFC) consists of 11 conserved proteins embedded in the membrane envelope of mature poxvirus particles. Poxviruses also encode proteins that localize in cell membranes and negatively regulate superinfection and syncytium formation. The vaccinia virus (VACV) A56/K2 fusion regulatory complex associates with the G9/A16 EFC subcomplex, but functional support for the importance of this interaction was lacking. Here, we describe serially passaging VACV in nonpermissive cells expressing A56/K2 as an unbiased approach to isolate and analyze escape mutants. Viruses forming large plaques in A56/K2 cells increased in successive rounds of infection, indicating the occurrence and enrichment of adaptive mutations. Sequencing of genomes of passaged and cloned viruses revealed mutations near the N terminus of the G9 open reading frame but none in A16 or other genes. The most frequent mutation was His to Tyr at amino acid 44; additional escape mutants had a His-to-Arg mutation at amino acid 44 or a duplication of amino acids 26 to 39. An adaptive Tyr-to-Cys substitution at amino acid 42 was discovered using error-prone PCR to generate additional mutations. Myristoylation of G9 was unaffected by the near-N-terminal mutations. The roles of the G9 mutations in enhancing plaque size were validated by homologous recombination. The mutants exhibited enhanced entry and spread in A56/K2 cells and induced syncytia at neutral pH in HeLa cells despite the expression of A56/K2. The data suggest that the mutations perturb the interaction of G9 with A56/K2, although some association was still detected in detergent-treated infected cell lysates.

IMPORTANCE The entry of enveloped viruses is achieved by the fusion of viral and cellular membranes, a critical step in infection that determines host range and provides targets for vaccines and therapeutics. Poxviruses encode an exceptionally large number of proteins comprising the entry/fusion complex (EFC), which enables infection of diverse cells. Vaccinia virus (VACV), the prototype member of the poxvirus family, also encodes the fusion regulatory proteins A56 and K2, which are displayed on the plasma membrane and may be beneficial by preventing reinfection and cell-cell fusion. Previous studies showed that A56/K2 interacts with the G9/A16 EFC subcomplex in detergent-treated cell extracts. Functional evidence for the importance of this interaction was obtained by serially passaging wild-type VACV in cells that are nonpermissive because of A56/K2 expression. VACV mutants with amino acid substitutions or duplications near the N terminus of G9 were enriched because of their ability to overcome the block to entry imposed by A56/K2.

Synesthesia is a neurologic trait in which specific inducers, such as sounds, automatically elicit additional idiosyncratic percepts, such as color (thus “colored hearing”). One explanation for this trait—and the one tested here—is that synesthesia results from unusually weak pruning of cortical synaptic hyperconnectivity during early perceptual development. We tested the...

Deregulation of mitochondrial dynamics leads to the accumulation of oxidative stress and unhealthy mitochondria; consequently, this accumulation contributes to premature aging and alterations in mitochondria linked to metabolic complications. We postulate that restrained mitochondrial ATP synthesis might alleviate age-associated disorders and extend healthspan in mammals. Herein, we prepared a previously...

ABSTRACT

The effect of the rapid accumulation of nonsynonymous mutations on the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not yet known. The 3a protein is unique to SARS-CoV and is essential for disease pathogenesis. Our study aimed at determining the nonsynonymous mutations in the 3a protein in SARS-CoV-2 and determining and characterizing the protein’s structure and spatial orientation in comparison to those of 3a in SARS-CoV. A total of 51 different nonsynonymous amino acid substitutions were detected in the 3a proteins among 2,782 SARS-CoV-2 strains. We observed microclonality within the ORF3a gene tree defined by nonsynonymous mutations separating the isolates into distinct subpopulations. We detected and identified six functional domains (I to VI) in the SARS-CoV-2 3a protein. The functional domains were linked to virulence, infectivity, ion channel formation, and virus release. Our study showed the importance of conserved functional domains across the species barrier and revealed the possible role of the 3a protein in the viral life cycle. Observations reported in this study merit experimental confirmation.

IMPORTANCE At the surge of the coronavirus disease 2019 (COVID-19) pandemic, we detected and identified six functional domains (I to VI) in the SARS-CoV-2 3a protein. Our analysis showed that the functional domains were linked to virulence, infectivity, ion channel formation, and virus release in SARS-CoV-2 3a. Our study also revealed the functional importance of conserved domains across the species barrier. Observations reported in this study merit experimental confirmation.

VOLUME 295 (2020) PAGES 4079–4092There was an error in the abstract. “The pyridinium cation hampers uptake of OPs into the central nervous system (CNS)” should read as “The pyridinium cation hampers uptake into the central nervous system (CNS).”

SUMOylation is a posttranslational modification (PTM) at a lysine residue and is crucial for the proper functions of many proteins, particularly of transcription factors, in various biological processes. Zinc finger homeobox 3 (ZFHX3), also known as AT motif-binding factor 1 (ATBF1), is a large transcription factor that is active in multiple pathological processes, including atrial fibrillation and carcinogenesis, and in circadian regulation and development. We have previously demonstrated that ZFHX3 is SUMOylated at three or more lysine residues. Here, we investigated which enzymes regulate ZFHX3 SUMOylation and whether SUMOylation modulates ZFHX3 stability and function. We found that SUMO1, SUMO2, and SUMO3 each are conjugated to ZFHX3. Multiple lysine residues in ZFHX3 were SUMOylated, but Lys-2806 was the major SUMOylation site, and we also found that it is highly conserved among ZFHX3 orthologs from different animal species. Using molecular analyses, we identified the enzymes that mediate ZFHX3 SUMOylation; these included SUMO1-activating enzyme subunit 1 (SAE1), an E1-activating enzyme; SUMO-conjugating enzyme UBC9 (UBC9), an E2-conjugating enzyme; and protein inhibitor of activated STAT2 (PIAS2), an E3 ligase. Multiple analyses established that both SUMO-specific peptidase 1 (SENP1) and SENP2 deSUMOylate ZFHX3. SUMOylation at Lys-2806 enhanced ZFHX3 stability by interfering with its ubiquitination and proteasomal degradation. Functionally, Lys-2806 SUMOylation enabled ZFHX3-mediated cell proliferation and xenograft tumor growth of the MDA-MB-231 breast cancer cell line. These findings reveal the enzymes involved in, and the functional consequences of, ZFHX3 SUMOylation, insights that may help shed light on ZFHX3's roles in various cellular and pathophysiological processes.

Assembled α-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of α-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of α-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T α-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD, and six cases of multiple system atrophy (MSA) for their ability to induce α-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain Sarkosyl-insoluble α-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled α-synuclein in these different samples. We conclude that α-synuclein filaments are the main driving force for amplification and propagation of pathology in synucleinopathies.

Multidrug resistance (MDR) in cancer arises from cross-resistance to structurally- and functionally-divergent chemotherapeutic drugs. In particular, MDR is characterized by increased expression and activity of ATP-binding cassette (ABC) superfamily transporters. Sphingolipids are substrates of ABC proteins in cell signaling, membrane biosynthesis, and inflammation, for example, and their products can favor cancer progression. Glucosylceramide (GlcCer) is a ubiquitous glycosphingolipid (GSL) generated by glucosylceramide synthase, a key regulatory enzyme encoded by the UDP-glucose ceramide glucosyltransferase (UGCG) gene. Stressed cells increase de novo biosynthesis of ceramides, which return to sub-toxic levels after UGCG mediates incorporation into GlcCer. Given that cancer cells seem to mobilize UGCG and have increased GSL content for ceramide clearance, which ultimately contributes to chemotherapy failure, here we investigated how inhibition of GSL biosynthesis affects the MDR phenotype of chronic myeloid leukemias. We found that MDR is associated with higher UGCG expression and with a complex GSL profile. UGCG inhibition with the ceramide analog d-threo-1-(3,4,-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4) greatly reduced GSL and monosialotetrahexosylganglioside levels, and co-treatment with standard chemotherapeutics sensitized cells to mitochondrial membrane potential loss and apoptosis. ABC subfamily B member 1 (ABCB1) expression was reduced, and ABCC-mediated efflux activity was modulated by competition with nonglycosylated ceramides. Consistently, inhibition of ABCC-mediated transport reduced the efflux of exogenous C6-ceramide. Overall, UGCG inhibition impaired the malignant glycophenotype of MDR leukemias, which typically overcomes drug resistance through distinct mechanisms. This work sheds light on the involvement of GSL in chemotherapy failure, and its findings suggest that targeted GSL modulation could help manage MDR leukemias.

The arrangement of functionally-related genes in operons is a fundamental element of how genetic information is organized in prokaryotes. This organization ensures coordinated gene expression by co-transcription. Often, however, alternative genetic responses to specific stress conditions demand the discoordination of operon expression. During cold temperature stress, accumulation of the gene encoding the sole Asp–Glu–Ala–Asp (DEAD)-box RNA helicase in Synechocystis sp. PCC 6803, crhR (slr0083), increases 15-fold. Here, we show that crhR is expressed from a dicistronic operon with the methylthiotransferase rimO/miaB (slr0082) gene, followed by rapid processing of the operon transcript into two monocistronic mRNAs. This cleavage event is required for and results in destabilization of the rimO transcript. Results from secondary structure modeling and analysis of RNase E cleavage of the rimO–crhR transcript in vitro suggested that CrhR plays a role in enhancing the rate of the processing in an auto-regulatory manner. Moreover, two putative small RNAs are generated from additional processing, degradation, or both of the rimO transcript. These results suggest a role for the bacterial RNA helicase CrhR in RNase E-dependent mRNA processing in Synechocystis and expand the known range of organisms possessing small RNAs derived from processing of mRNA transcripts.

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene–Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis, which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branchpoint enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering, and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkably, generating a nonallosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.

Interpretation of a 3D seismic survey located on the western margin of the Northern North Sea Basin demonstrates how the propagation, overlap and linkage of two north–south-striking, en echelon normal fault segments exerted a powerful influence over prospective subtle stratigraphic traps. The relay ramp that formed between the segments appears to have focused sediment dispersal, controlled reservoir distribution and aided post-depositional petroleum migration. Integration of electrical well log data, root-mean-square (RMS)-amplitude analysis and biostratigraphy with seismic interpretation demonstrates that a series of elongate, linear, sand-prone (reservoir) channel complexes characterize the depositional slopes generated by fault growth. The combination of synsedimentary rotation of bedding due to fault propagation and associated footwall uplift led to erosion and truncation of a laterally extensive, older channelized system (Lower Sequence), the downdip parts of which extend beyond the relay ramp. Its subsequent drape by transgressive shales created the subtle stratigraphic trap that now hosts the Cladhan Field, with charge occurring because the sandstones belonging to the Lower Sequence extend as far as the active kitchen in the neighbouring (hanging-wall) depocentre situated downdip and to the east. In contrast, the exploration of a younger, Upper Sequence of sandstones has proven to be disappointing due to their more restricted distribution, lack of access to charge, and occurrence of faults that offset and breach the thin end of the stratigraphic wedge. The implication is that partially breached relay ramps not only provide a preferential site for syn-rift clastic reservoirs to develop but also form important migration pathways through which oil passed from a petroleum kitchen into a trap.

Thematic collection: This article is part of the Under-explored plays and frontier basins of the UK continental shelf collection available at: http://www.lyellcollection.org/cc/under-explored-plays-and-frontier-basins-of-the-uk-continental-shelf

Neutralizing antibodies specific for respiratory syncytial virus (RSV) represent a major protective mechanism against RSV infection, as demonstrated by the efficacy of the immune-prophylactic monoclonal antibody palivizumab in preventing RSV-associated lower respiratory tract infections in premature infants. Accordingly, the RSV neutralization assay has become a key functional method to assess the neutralizing activity of serum antibodies in preclinical animal models, epidemiology studies, and clinical trials. In this study, we qualified a 24-h, fluorescent focus-based microneutralization (RSVA FFA-MN) method that requires no medium exchange or pre- or postinfection processing to detect green fluorescent protein-expressing RSV strain A2 (RSVA-GFP)-infected cells, using a high-content imaging system for automated image acquisition and focus enumeration. The RSVA FFA-MN method was shown to be sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1:10, or 3.32 log₂; linear over a range of 4.27 to 9.65 log₂ 50% inhibitory concentration (IC₅₀); and precise, with intra- and interassay coefficients of variation of <21%. This precision allowed the choice of a statistically justified 3-fold-rise seroresponse cutoff criterion. The repeatability and robustness of this method were demonstrated by including a pooled human serum sample in every assay as a positive control (PC). Over 3 years of testing between two laboratories, this PC generated data falling within 2.5 standard deviations of the mean 98.7% of the time (n = 1,720). This high-throughput and reliable RSV microneutralization assay has proven useful for testing sera from preclinical vaccine candidate evaluation studies, epidemiology studies, and both pediatric and adult vaccine clinical trials.

At the time of this writing, healthcare systems are facing worldwide the pandemic of the coronavirus severe acute respiratory coronavirus 2 (SARS-COV-2) and its associated disease, named cronavirus disease 19 (COVID-19). This virus is a new human pathogen, and currently, there are no specific treatment options.¹ COVID-19 mostly affects the respiratory system, ranging from mild flu-like symptoms to severe pneumonia, but extrarespiratory multisystemic involvement has also been reported.² Li et al.³ recently described the neuroinvasive potential of COVID-19, but, to our knowledge, no case of acute dysimmune neuropathy has been described so far. Here, the authors report the case of an acute and severe peripheral nervous system disorder possibly related to COVID-19 infection.

In the article "Neurologic syndromes related to anti-GAD65: Clinical and serologic response to treatment" by Muñoz-Lopetegi et al.,¹ published online March 2, 2020, the y-axis label for figure 5’s right graph should be "CSF anti-GAD65 concentration (IU/mL)." The editorial office regrets the error.

Alemtuzumab is approved for the treatment of relapsing-remitting MS and is used off-label for patients with chronic lymphocytic leukemia and as induction and antirejection therapy in kidney transplant recipients.¹ Guillain-Barré syndrome (GBS) or chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) complicating alemtuzumab treatment was reported in 9 patients with hematologic malignancy or MS.^1–3 The risk of GBS or CIDP in solid organ transplant recipients treated with alemtuzumab is unknown.

Stiff person syndrome (SPS) is a rare neurologic disorder characterized by progressively worsening rigidity and spasms of the axial and limb muscles. Dyspnea has been recently recognized as a common symptom in SPS,¹ and life-threatening respiratory crises have been occasionally reported and suspected to be responsible for sudden death in these patients.^2,3 The pathophysiologic mechanisms of these respiratory manifestations remain unclear. Some authors have hypothesized that rigidity and/or spasm of the muscles of the trunk could prevent normal rib cage movements and excursion of the diaphragm.¹

The new decade of the 21^st century (2020) started with the emergence of a novel coronavirus known as SARS-CoV-2 that caused an epidemic of coronavirus disease (COVID-19) in Wuhan, China. It is the third highly pathogenic and transmissible coronavirus after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in humans. The source of origin, transmission to humans, and mechanisms associated with the pathogenicity of SARS-CoV-2 are not yet clear, however, its resemblance to SARS-CoV and several other bat coronaviruses was recently confirmed through genome sequencing-related studies. The development of therapeutic strategies is necessary in order to prevent further epidemics and cure infections. In this review, we summarize current information about the emergence, origin, diversity, and epidemiology of three pathogenic coronaviruses with a specific focus on the current outbreak in Wuhan, China. Furthermore, we discuss the clinical features and potential therapeutic options that may be effective against SARS-CoV-2.

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

Cinnamaldehyde (Cin), a bioactive cinnamon essential oil from traditional Chinese medicine herb Cinnamomum cassia, has been reported to have multipharmacological activities including anti-inflammation. However, its role and molecular mechanism of anti-inflammatory activity in musculoskeletal tissues remains unclear. Here, we first investigated the effects and molecular mechanisms of Cin in human synoviocyte cells. Then in vivo therapeutic effect of Cin on collagen-induced arthritis (CIA) also studied. Cell Counting Kit ‎CCK-8 assay was performed to evaluate the cell cytotoxicity. Proinflammatory cytokine expression was evaluated using quantitative polymerase chain reaction and ELISA. Protein expression was measured by western blotting. The in vivo effect of Cin (75 mg/kg per day) was evaluated in rats with CIA by gavage administration. Disease progression was assessed by clinical scoring, radiographic, and histologic examinations. Cin significantly inhibited interleukin (IL)-1β–induced IL-6, IL-8, and tumor necrosis factor-α release from human synoviocyte cells. The molecular analysis revealed that Cin impaired IL-6–induced activation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), and STAT3 signaling pathway by inhibiting the phosphorylation of JAK2, STAT1, and STAT3, without affecting NF-B pathway. Cin reduced collagen-induced swollen paw volume of arthritic rats. The anti-inflammation effects of Cin were associated with decreased severity of arthritis, joint swelling, and reduced bone erosion and destruction. Furthermore, serum IL-6 level was decreased when Cin administered therapeutically to CIA rats. Cin suppresses IL-1β–induced inflammation in synoviocytes through the JAK/STAT pathway and alleviated collagen-induced arthritis in rats. These data indicated that Cin might be a potential traditional Chinese medicine–derived, disease-modifying, antirheumatic herbal drug.

Glucagon-like peptide-2 (GLP-2) agonists have therapeutic potential in clinical indications in which the integrity or absorptive function of the intestinal mucosa is compromised, such as in short bowel syndrome (SBS). Native hGLP-2, a 33–amino acid peptide secreted from the small intestine, contributes to nutritional absorption but has a very short half-life because of enzymatic cleavage and renal clearance and thus is of limited therapeutic value. The GLP-2 analog teduglutide (Revestive/Gattex; Shire Inc.) has been approved for use in SBS since 2012 but has a once-daily injection regimen. Pharmacokinetic (PK) and pharmacodynamic studies confirm that apraglutide, a novel GLP-2 analog, has very low clearance, long elimination half-life, and high plasma protein binding compared with GLP-2 analogs teduglutide and glepaglutide. Apraglutide and teduglutide retain potency and selectivity at the GLP-2 receptor comparable to native hGLP-2, whereas glepaglutide was less potent and less selective. In rat intravenous PK studies, hGLP-2, teduglutide, glepaglutide, and apraglutide had clearances of 25, 9.9, 2.8, and 0.27 ml/kg per minute, respectively, and elimination half-lives of 6.4, 19, 16, and 159 minutes, respectively. The unique PK profile of apraglutide administered via intravenous and subcutaneous routes was confirmed in monkey and minipig and translated into significantly greater in vivo pharmacodynamic activity, measured as small intestinal growth in rats. Apraglutide showed greater intestinotrophic activity than the other peptides when administered at less-frequent dosing intervals because of its prolonged half-life. We postulate that apraglutide offers several advantages over existing GLP-2 analogs and is an excellent candidate for the treatment of gastrointestinal diseases, such as SBS.

Decreased release of palmitic acid methyl ester (PAME), a vasodilator, from perivascular adipose tissue (PVAT) might contribute to hypertension pathogenesis. However, the PAME biosynthetic pathway remains unclear. In this study, we hypothesized that PAME is biosynthesized from palmitic acid (PA) via human catechol-O-methyltransferase (COMT) catalysis and that decreased PAME biosynthesis plays a role in hypertension pathogenesis. We compared PAME biosynthesis between age-matched normotensive Wistar Kyoto (WKY) rats and hypertensive spontaneously hypertensive rats (SHRs) and investigated the effects of losartan treatment on PAME biosynthesis. Computational molecular modeling indicated that PA binds well at the active site of COMT. Furthermore, in in vitro enzymatic assays in the presence of COMT and S-5'-adenosyl-L-methionine (AdoMet), the stable isotope [¹³C₁₆]-PA was methylated to form [¹³C₁₆]-PAME in incubation medium or the Krebs–Henseleit solution containing 3T3-L1 adipocytes or rat PVAT. The adipocytes and PVATs expressed membrane-bound (MB)-COMT and soluble (S)-COMT proteins. [¹³C₁₆]-PA methylation to form [¹³C₁₆]-PAME in 3T3-L1 adipocytes and rat PVAT was blocked by various COMT inhibitors, such as S-(5'-adenosyl)-L-homocysteine, adenosine-2',3'-dialdehyde, and tolcapone. MB- and S-COMT levels in PVATs of established SHRs were significantly lower than those in PVATs of age-matched normotensive WKY rats, with decreased [¹³C₁₆]-PA methylation to form [¹³C₁₆]-PAME. This decrease was reversed by losartan, an angiotensin II (Ang II) type 1 receptor antagonist. Therefore, PAME biosynthesis in rat PVAT is dependent on AdoMet, catalyzed by COMT, and decreased in SHRs, further supporting the role of PVAT/PAME in hypertension pathogenesis. Moreover, the antihypertensive effect of losartan might be due partly to its increased PAME biosynthesis.

Recent studies have strived to find an association between rapid antidepressant effects and a specific subset of pharmacological targets and molecular pathways. Here, we propose a broader hypothesis of encoding, consolidation, and renormalization in depression (ENCORE-D), which suggests that, fundamentally, rapid and sustained antidepressant effects rely on intrinsic homeostatic mechanisms evoked as a response to the acute pharmacological or physiologic effects triggered by the treatment. We review evidence that supports the notion that various treatments with a rapid onset of action, such as ketamine, electroconvulsive therapy, and sleep deprivation, share the ability to acutely excite cortical networks, which increases synaptic potentiation, alters patterns of functional connectivity, and ameliorates depressive symptoms. We proceed to examine how the initial effects are short-lived and, as such, require both consolidation during wake and maintenance throughout sleep to remain sustained. Here, we incorporate elements from the synaptic homeostasis hypothesis and theorize that the fundamental mechanisms of synaptic plasticity and sleep, particularly the homeostatic emergence of slow-wave electroencephalogram activity and the renormalization of synaptic strength, are at the center of sustained antidepressant effects. We conclude by discussing the various implications of the ENCORE-D hypothesis and offer several considerations for future experimental and clinical research.

⁶⁸Ga-labeled urea-based inhibitors of the prostate-specific membrane antigen (PSMA), such as ⁶⁸Ga-PSMA-11, are promising small molecules for targeting prostate cancer (PCa). Although this radiopharmaceutical was produced mostly by means of manual synthesis and automated synthesis modules, a sterile cold kit was recently introduced. The aim of our study was to evaluate the image quality of ⁶⁸Ga-PSMA-11 PET/CT (PSMA-PET) in a population of PCa patients after the injection of comparable activities of ⁶⁸Ga-PSMA-11 obtained with the 2 different synthetic procedures. A secondary aim was to identify secondary factors that may have an impact on image quality and, thus, final interpretation. Methods: Two different groups of 100 consecutive PCa patients who underwent PSMA-PET were included in the study. The first group of patients was imaged with ⁶⁸Ga-PSMA-11 obtained using synthesis modules, whereas the second group’s tracer activity was synthesized using a sterile cold kit. All PET images were independently reviewed by 2 nuclear medicine diagnosticians with at least 2 y of experience in PSMA-based imaging and unaware of the patients’ clinical history. The 2 reviewers independently rated the quality of each PSMA-PET scan using a 3-point Likert-type scale. In cases of discordance, the operators together reviewed the images and reached a consensus. Performance was evaluated on the basis of the expected biodistribution, lesion detection rate, and physiologic background uptake. Results: Overall, 104 of 200 (52%) PSMA-PET scans were positive for PCa-related findings. No significant differences in image quality between cold kits and synthesis modules were found (P = 0.13), although a higher proportion of images was rated as excellent by the observers for kits than for modules (45% vs. 34%). Furthermore, after image quality had been dichotomized as excellent or not excellent, multivariate regression analysis found several factors to be significantly associated with a not-excellent quality: an increase in patient age (+5 y: odds ratio [OR], 1.40; 95% confidence interval [CI], 1.12–1.75), an increase in patient weight (+5 kg: OR, 1.89; 95% CI, 1.53–2.32), an increase in ⁶⁸Ga-PSMA-11 uptake time (+10 min: OR, 1.45; 95% CI, 1.08–1.96), and a decrease in injected activity (–10 MBq: OR, 1.28; 95% CI, 1.07–1.52). Conclusion: No significant differences were identified between the 2 groups of patients undergoing PSMA-PET; therefore, we were not able to ascertain any significant influences of tracer production methodology on final scan quality. However, increased patient age, increased patient weight, decreased injected activity, and increased ⁶⁸Ga-PSMA-11 uptake time were significantly associated with an overall poorer image quality.

OBJECTIVE

Type 2 diabetes is characterized by increased death rate. In order to tackle this dramatic event, it becomes essential to discover novel biomarkers capable of identifying high-risk patients to be exposed to more aggressive preventive and treatment strategies. hs-CRP and serum amyloid P component (SAP) are two acute-phase inflammation proteins, which interact physically and share structural and functional features. We investigated their combined role in associating with and improving prediction of mortality in type 2 diabetes.

BackgroundPolycystic ovary syndrome (PCOS) is a common lifelong metabolic condition with serious associated comorbidities. Evidence points to a delay in diagnosis and inconsistency in the information provided to women with PCOS.AimTo capture women’s experiences of how PCOS is diagnosed and managed in UK general practice.Design and settingThis was a mixed-methods study with an online questionnaire survey and semi-structured telephone interviews with a subset of responders.MethodAn online survey to elicit women’s experiences of general practice PCOS care was promoted by charities and BBC Radio Leicester. The survey was accessible online between January 2018 and November 2018. A subset of responders undertook a semi-structured telephone interview to provide more in-depth data.ResultsA total of 323 women completed the survey (average age 35.4 years) and semi-structured interviews were conducted with 11 women. There were five key themes identified through the survey responses. Participants described a variable lag time from presentation to PCOS diagnosis, with a median of 6–12 months. Many had experienced mental health problems associated with their PCOS symptoms, but had not discussed these with the GP. Many were unable to recall any discussion about associated comorbidities with the GP. Some differences were identified between the experiences of women from white British backgrounds and those from other ethnic backgrounds.ConclusionFrom the experiences of the women in this study, it appears that PCOS in general practice is not viewed as a long-term condition with an increased risk of comorbidities including mental health problems. Further research should explore GPs’ awareness of comorbidities and the differences in PCOS care experienced by women from different ethnic backgrounds.

PURPOSE

Operational failures are system-level errors in the supply of information, equipment, and materials to health care personnel. We aimed to review and synthesize the research literature to determine how operational failures in primary care affect the work of primary care physicians.

Oncolytic virotherapy can lead to systemic antitumor immunity, but the therapeutic potential of oncolytic viruses in humans is limited due to their insufficient ability to overcome the immunosuppressive tumor microenvironment (TME). Here, we showed that locoregional oncolytic virotherapy upregulated the expression of PD-L1 in the TME, which was mediated by virus-induced type I and type II IFNs. To explore PD-1/PD-L1 signaling as a direct target in tumor tissue, we developed a novel immunotherapeutic herpes simplex virus (HSV), OVH-aMPD-1, that expressed a single-chain variable fragment (scFv) against PD-1 (aMPD-1 scFv). The virus was designed to locally deliver aMPD-1 scFv in the TME to achieve enhanced antitumor effects. This virus effectively modified the TME by releasing damage-associated molecular patterns, promoting antigen cross-presentation by dendritic cells, and enhancing the infiltration of activated T cells; these alterations resulted in antitumor T-cell activity that led to reduced tumor burdens in a liver cancer model. Compared with OVH, OVH-aMPD-1 promoted the infiltration of myeloid-derived suppressor cells (MDSC), resulting in significantly higher percentages of CD155⁺ granulocytic-MDSCs (G-MDSC) and monocytic-MDSCs (M-MDSC) in tumors. In combination with TIGIT blockade, this virus enhanced tumor-specific immune responses in mice with implanted subcutaneous tumors or invasive tumors. These findings highlighted that intratumoral immunomodulation with an OV expressing aMPD-1 scFv could be an effective stand-alone strategy to treat cancers or drive maximal efficacy of a combination therapy with other immune checkpoint inhibitors.

Vibralactone, a hybrid compound derived from phenols and a prenyl group, is a strong pancreatic lipase inhibitor with a rare fused bicyclic β-lactone skeleton. Recently, a researcher reported a vibralactone derivative (compound C1) that caused inhibition of pancreatic lipase with a half-maximal inhibitory concentration of 14 nM determined by structure-based optimization, suggesting a potential candidate as a new antiobesity treatment. In the present study, we sought to identify the main gene encoding prenyltransferase in Stereum vibrans, which is responsible for the prenylation of phenol leading to vibralactone synthesis. Two RNA silencing transformants of the identified gene (vib-PT) were obtained through Agrobacterium tumefaciens-mediated transformation. Compared to wild-type strains, the transformants showed a decrease in vib-PT expression ranging from 11.0 to 56.0% at 5, 10, and 15 days in reverse transcription-quantitative PCR analysis, along with a reduction in primary vibralactone production of 37 to 64% at 15 and 21 days, respectively, as determined using ultra-high-performance liquid chromatography-mass spectrometry analysis. A soluble and enzymatically active fusion Vib-PT protein was obtained by expressing vib-PT in Escherichia coli, and the enzyme’s optimal reaction conditions and catalytic efficiency (K_m/k_cat) were determined. In vitro experiments established that Vib-PT catalyzed the C-prenylation at C-3 of 4-hydroxy-benzaldehyde and the O-prenylation at the 4-hydroxy of 4-hydroxy-benzenemethanol in the presence of dimethylallyl diphosphate. Moreover, Vib-PT shows promiscuity toward aromatic compounds and prenyl donors.

IMPORTANCE Vibralactone is a lead compound with a novel skeleton structure that shows strong inhibitory activity against pancreatic lipase. Vibralactone is not encoded by the genome directly but rather is synthesized from phenol, followed by prenylation and other enzyme reactions. Here, we used an RNA silencing approach to identify and characterize a prenyltransferase in a basidiomycete species that is responsible for the synthesis of vibralactone. The identified gene, vib-PT, was expressed in Escherichia coli to obtain a soluble and enzymatically active fusion Vib-PT protein. In vitro characterization of the enzyme demonstrated the catalytic mechanism of prenylation and broad substrate range for different aromatic acceptors and prenyl donors. These characteristics highlight the possibility of Vib-PT to generate prenylated derivatives of aromatics and other compounds as improved bioactive agents or potential prodrugs.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia coli fadD mutant JW1794. RpfB activates long-chain fatty acids (n-C_16:0 and n-C_18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes. Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2. In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenes.

IMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes. Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes. Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes. Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

The response to iron limitation of the Gram-positive soil bacterium Corynebacterium glutamicum was analyzed with respect to secreted metabolites, the transcriptome, and the proteome. During growth in glucose minimal medium, iron limitation caused a shift from lactate to pyruvate as the major secreted organic acid complemented by l-alanine and 2-oxoglutarate. Transcriptome and proteome analyses revealed that a pronounced iron starvation response governed by the transcriptional regulators DtxR and RipA was detectable in the late, but not in the early, exponential-growth phase. A link between iron starvation and thiamine pyrophosphate (TPP) biosynthesis was uncovered by the strong upregulation of thiC. As phosphomethylpyrimidine synthase (ThiC) contains an iron-sulfur cluster, limiting activities of the TPP-dependent pyruvate–2-oxoglutarate dehydrogenase supercomplex probably cause the excretion of pyruvate and 2-oxoglutarate. In line with this explanation, thiamine supplementation could strongly diminish the secretion of these acids. The upregulation of thiC and other genes involved in thiamine biosynthesis and transport is presumably due to TPP riboswitches present at the 5' end of the corresponding operons. The results obtained in this study provide new insights into iron homeostasis in C. glutamicum and demonstrate that the metabolic consequences of iron limitation can be due to the iron dependency of coenzyme biosynthesis.

IMPORTANCE Iron is an essential element for most organisms but causes problems due to poor solubility under oxic conditions and due to toxicity by catalyzing the formation of reactive oxygen species (ROS). Therefore, bacteria have evolved complex regulatory networks for iron homeostasis aiming at a sufficient iron supply while minimizing ROS formation. In our study, the responses of the actinobacterium Corynebacterium glutamicum to iron limitation were analyzed, resulting in a detailed view on the processes involved in iron homeostasis in this model organism. In particular, we provide evidence that iron limitation causes TPP deficiency, presumably due to insufficient activity of the iron-dependent phosphomethylpyrimidine synthase (ThiC). TPP deficiency was deduced from the upregulation of genes controlled by a TPP riboswitch and secretion of metabolites caused by insufficient activity of the TPP-dependent enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. To our knowledge, the link between iron starvation and thiamine synthesis has not been elaborated previously.

Cushing syndrome results from chronic excessive exposure to glucocorticoids, impacting virtually every organ system with the most dominant effects on fat metabolism, immune function, and musculoskeletal systems. Endogenous Cushing syndrome is rare, most usually due to excess ACTH secretion from pituitary, and less frequently from ectopic tumors. Other cases result from ACTH-independent adrenal overproduction of cortisol.