Solar power is helping Asia get electricity to people, particularly in rural and remote areas, but it has fallen behind in its use as a clean energy cooking source.

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ISLAMABAD: Former Senate chairman Mian Raza Rabbani on Friday said that the National Council for Minorities nominated through a notification after a considerable delay was illegal because it had been formed in violation of a judgement announced by the then Chief Justice of Pakistan Tasaduq Hussain Jilani on June 19, 2014.

“The said act of violating the judgement of the Supreme Court is condemned,” the PPP leader said in a statement.

Mr Rabbani said that the Supreme Court’s judgement had come in the wake of a bomb blast in a Peshawar church in 2013 which left over a hundred members of the Christian community dead.

In a suo motu case, the then Chief Justice of Pakistan, in paragraph 37(iv) of the judgement, had ordered the setting up of the National Council for Minorities to monitor practical realisation of the rights and safeguards of members of minority communities provided in the 1973 Constitution.

The council was supposed to be mandated to frame policy recommendations for safeguarding and protecting minorities’ rights by the federal and provincial governments.

The judgement clearly mentioned eight steps that the governments were to take, including (i) reservation of quota in services, (ii) a special police force to protect places of worship, (iii) steps to discourage hate speeches, and (iv) revision of school curriculum to promote cultural and religious tolerance.

The PPP leader said that none of the steps had been taken so far by the governments.

He said that the federal government had, after the approval of the cabinet, constituted the council through a notification and the body had become controversial even before its birth and as a result the religious affairs ministry had to amend its summary.

The PPP leader said that the council should be constituted through an act of parliament.

Mr Rabbani said that the importance given to safeguarding the rights of the minorities in the country was evident from the fact that the white colour portion of the national flag represented minorities and in over 20 Articles of the Constitution, 1973, the rights of minorities had been guaranteed.

Published in Dawn, May 9th, 2020

In 2005, the energy sector emitted 88% of Uzbekistan’s 200 million tons of carbon dioxide equivalent greenhouse gas emissions. With the high solar irradiance and abundant land for solar development of Uzbekistan, solar energy was deemed as the most suitable resource that could quickly bridge the energy supply–demand gap, diversify the generation mix, and reduce emissions.

Approved project 53270-001 in Viet Nam.

Approved project 54035-001 in India.

STAMFORD, CT – November 7, 2019 – HARMAN – a wholly-owned subsidiary of Samsung Electronics Co., Ltd., focused on connected technologies for automotive, consumer and enterprise markets, together with the 1,000 Dreams Fund (1DF), today announced the...

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Title: omalizumab (Xolair)
Category: Medications
Created: 5/27/2004 12:00:00 AM
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Category: Medications
Created: 4/19/1999 12:00:00 AM
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Title: Cola May Be Bad to the Bones
Category: Health News
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Category: Health News
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Category: Health News
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Category: Health News
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Overexpression of transcription factor 3 in alveolar soft part sarcoma(ASPS) results in upregulation of cell proliferation pathways. No standard treatment algorithm exists for ASPS; multikinase inhibitors[tyrosine kinase inhibitor (TKI)] and immune checkpoint inhibitors (ICI) have shown clinical benefit. To date, no studies have reported on management strategies or sequencing of therapy. We evaluated ASPS treatment patterns and responses in an experimental therapeutics clinic. Genomic and morphoproteomic analysis was performed to further elucidate novel targets. We retrospectively reviewed patients with ASPS treated on clinical trials. Demographic and clinical next-generation sequencing (NGS) profiles were collected. AACR GENIE database was queried to further evaluate aberrations in ASPS. Morphoproteomic analysis was carried out to better define the biology of ASPS with integration of genomic and proteomic findings. Eleven patients with ASPS were identified; 7 received NGS testing and mutations in CDKN2A (n = 1) and hepatocyte growth factor (n = 1) were present. Ten patients were treated with TKIs with stable disease as best response and 4 patients with ICI (three partial responses). Within GENIE, 20 patients were identified harboring 3 called pathogenic mutations. Tumor mutation burden was low in all samples. Morphoproteomic analysis confirmed the expression of phosphorylated c-Met. In addition, fatty acid synthase and phosphorylated-STAT3 were detected in tumor cell cytoplasm and nuclei. Patients with ASPS have a quiescent genome and derive clinical benefit from VEGF-targeting TKIs. Morphoproteomic analysis has provided both additional correlative pathways and angiogenic mechanisms that are targetable for patients with ASPS. Our study suggests that sequential therapy with TKIs and immune checkpoint inhibitors is a reasonable management strategy.

ABSTRACT

Synthesis and cleavage of the cell wall polymer peptidoglycan (PG) are carefully orchestrated processes and are essential for the growth and survival of bacteria. Yet, the function and importance of many enzymes that act on PG in Mycobacterium tuberculosis remain to be elucidated. We demonstrate that the activity of the N-acetylmuramyl-l-alanine amidase Ami1 is dispensable for cell division in M. tuberculosis in vitro yet contributes to the bacterium’s ability to persist during chronic infection in mice. Furthermore, the d,l-endopeptidase RipA, a predicted essential enzyme, is dispensable for the viability of M. tuberculosis but required for efficient cell division in vitro and in vivo. Depletion of RipA sensitizes M. tuberculosis to rifampin and to cell envelope-targeting antibiotics. Ami1 helps sustain residual cell division in cells lacking RipA, but the partial redundancy provided by Ami1 is not sufficient during infection, as depletion of RipA prevents M. tuberculosis from replicating in macrophages and leads to dramatic killing of the bacteria in mice. Notably, RipA is essential for persistence of M. tuberculosis in mice, suggesting that cell division is required during chronic mouse infection. Despite the multiplicity of enzymes acting on PG with redundant functions, we have identified two PG hydrolases that are important for M. tuberculosis to replicate and persist in the host.

IMPORTANCE Tuberculosis (TB) is a major global heath burden, with 1.6 million people succumbing to the disease every year. The search for new drugs to improve the current chemotherapeutic regimen is crucial to reducing this global health burden. The cell wall polymer peptidoglycan (PG) has emerged as a very successful drug target in bacterial pathogens, as many currently used antibiotics target the synthesis of this macromolecule. However, the multitude of genes encoding PG-synthesizing and PG-modifying enzymes with apparent redundant functions has hindered the identification of novel drug targets in PG synthesis in Mycobacterium tuberculosis. Here, we demonstrate that two PG-cleaving enzymes are important for virulence of M. tuberculosis. In particular, the d,l-endopeptidase RipA represents a potentially attractive drug target, as its depletion results in the clearance of M. tuberculosis from the host and renders the bacteria hypersusceptible to rifampin, a frontline TB drug, and to several cell wall-targeting antibiotics.

ABSTRACT

Bacterial flagella are rotating nanomachines required for motility. Flagellar gene expression and protein secretion are coordinated for efficient flagellar biogenesis. Polar flagellates, unlike peritrichous bacteria, commonly order flagellar rod and hook gene transcription as a separate step after production of the MS ring, C ring, and flagellar type III secretion system (fT3SS) core proteins that form a competent fT3SS. Conserved regulatory mechanisms in diverse polar flagellates to create this polar flagellar transcriptional program have not been thoroughly assimilated. Using in silico and genetic analyses and our previous findings in Campylobacter jejuni as a foundation, we observed a large subset of Gram-negative bacteria with the FlhF/FlhG regulatory system for polar flagellation to possess flagellum-associated two-component signal transduction systems (TCSs). We present data supporting a general theme in polar flagellates whereby MS ring, rotor, and fT3SS proteins contribute to a regulatory checkpoint during polar flagellar biogenesis. We demonstrate that Vibrio cholerae and Pseudomonas aeruginosa require the formation of this regulatory checkpoint for the TCSs to directly activate subsequent rod and hook gene transcription, which are hallmarks of the polar flagellar transcriptional program. By reprogramming transcription in V. cholerae to more closely follow the peritrichous flagellar transcriptional program, we discovered a link between the polar flagellar transcription program and the activity of FlhF/FlhG flagellar biogenesis regulators in which the transcriptional program allows polar flagellates to continue to produce flagella for motility when FlhF or FlhG activity may be altered. Our findings integrate flagellar transcriptional and biogenesis regulatory processes involved in polar flagellation in many species.

IMPORTANCE Relative to peritrichous bacteria, polar flagellates possess regulatory systems that order flagellar gene transcription differently and produce flagella in specific numbers only at poles. How transcriptional and flagellar biogenesis regulatory systems are interlinked to promote the correct synthesis of polar flagella in diverse species has largely been unexplored. We found evidence for many Gram-negative polar flagellates encoding two-component signal transduction systems with activity linked to the formation of flagellar type III secretion systems to enable production of flagellar rod and hook proteins at a discrete, subsequent stage during flagellar assembly. This polar flagellar transcriptional program assists, in some manner, the FlhF/FlhG flagellar biogenesis regulatory system, which forms specific flagellation patterns in polar flagellates in maintaining flagellation and motility when activity of FlhF or FlhG might be altered. Our work provides insight into the multiple regulatory processes required for polar flagellation.

ABSTRACT

OleA, a member of the thiolase superfamily, is known to catalyze the Claisen condensation of long-chain acyl coenzyme A (acyl-CoA) substrates, initiating metabolic pathways in bacteria for the production of membrane lipids and β-lactone natural products. OleA homologs are found in diverse bacterial phyla, but to date, only one homodimeric OleA has been successfully purified to homogeneity and characterized in vitro. A major impediment for the identification of new OleA enzymes has been protein instability and time-consuming in vitro assays. Here, we developed a bioinformatic pipeline to identify OleA homologs and a new rapid assay to screen OleA enzyme activity in vivo and map their taxonomic diversity. The screen is based on the discovery that OleA displayed surprisingly high rates of p-nitrophenyl ester hydrolysis, an activity not shared by other thiolases, including FabH. The high rates allowed activity to be determined in vitro and with heterologously expressed OleA in vivo via the release of the yellow p-nitrophenol product. Seventy-four putative oleA genes identified in the genomes of diverse bacteria were heterologously expressed in Escherichia coli, and 25 showed activity with p-nitrophenyl esters. The OleA proteins tested were encoded in variable genomic contexts from seven different phyla and are predicted to function in distinct membrane lipid and β-lactone natural product metabolic pathways. This study highlights the diversity of unstudied OleA proteins and presents a rapid method for their identification and characterization.

IMPORTANCE Microbially produced β-lactones are found in antibiotic, antitumor, and antiobesity drugs. Long-chain olefinic membrane hydrocarbons have potential utility as fuels and specialty chemicals. The metabolic pathway to both end products share bacterial enzymes denoted as OleA, OleC, and OleD that transform acyl-CoA cellular intermediates into β-lactones. Bacteria producing membrane hydrocarbons via the Ole pathway additionally express a β-lactone decarboxylase, OleB. Both β-lactone and olefin biosynthesis pathways are initiated by OleA enzymes that define the overall structure of the final product. There is currently very limited information on OleA enzymes apart from the single representative from Xanthomonas campestris. In this study, bioinformatic analysis identified hundreds of new, putative OleA proteins, 74 proteins were screened via a rapid whole-cell method, leading to the identification of 25 stably expressed OleA proteins representing seven bacteria phyla.

ABSTRACT

Adjuvants can be used to potentiate the function of antibiotics whose efficacy has been reduced by acquired or intrinsic resistance. In the present study, we discovered that human milk oligosaccharides (HMOs) sensitize strains of group B Streptococcus (GBS) to trimethoprim (TMP), an antibiotic to which GBS is intrinsically resistant. Reductions in the MIC of TMP reached as high as 512-fold across a diverse panel of isolates. To better understand HMOs’ mechanism of action, we characterized the metabolic response of GBS to HMO treatment using ultrahigh-performance liquid chromatography–high-resolution tandem mass spectrometry (UPLC-HRMS/MS) analysis. These data showed that when challenged by HMOs, GBS undergoes significant perturbations in metabolic pathways related to the biosynthesis and incorporation of macromolecules involved in membrane construction. This study represents reports the metabolic characterization of a cell that is perturbed by HMOs.

IMPORTANCE Group B Streptococcus is an important human pathogen that causes serious infections during pregnancy which can lead to chorioamnionitis, funisitis, premature rupture of gestational membranes, preterm birth, neonatal sepsis, and death. GBS is evolving antimicrobial resistance mechanisms, and the work presented in this paper provides evidence that prebiotics such as human milk oligosaccharides can act as adjuvants to restore the utility of antibiotics.

ABSTRACT

A microfluidic system coupled with fluorescence microscopy is a powerful approach for quantitative analysis of bacterial growth. Here, we measure parameters of growth and dynamic localization of the cell division initiation protein FtsZ in Bacillus subtilis. Consistent with previous reports, we found that after division, FtsZ rings remain at the cell poles, and polar FtsZ ring disassembly coincides with rapid Z-ring accumulation at the midcell. In cells mutated for minD, however, the polar FtsZ rings persist indefinitely, suggesting that the primary function of the Min system is in Z-ring disassembly. The inability to recycle FtsZ monomers in the minD mutant results in the simultaneous maintenance of multiple Z-rings that are restricted by competition for newly synthesized FtsZ. Although the parameters of FtsZ dynamics change in the minD mutant, the overall cell division time remains the same, albeit with elongated cells necessary to accumulate a critical threshold amount of FtsZ for promoting medial division. Finally, the minD mutant characteristically produces minicells composed of polar peptidoglycan shown to be inert for remodeling in the wild type. Polar peptidoglycan, however, loses its inert character in the minD mutant, suggesting that the Min system not only is important for recycling FtsZ but also may have a secondary role in the spatiotemporal regulation of peptidoglycan remodeling.

IMPORTANCE Many bacteria grow and divide by binary fission in which a mother cell divides into two identical daughter cells. To produce two equally sized daughters, the division machinery, guided by FtsZ, must dynamically localize to the midcell each cell cycle. Here, we quantitatively analyzed FtsZ dynamics during growth and found that the Min system of Bacillus subtilis is essential to disassemble FtsZ rings after division. Moreover, a failure to efficiently recycle FtsZ results in an increase in cell size. Finally, we show that the Min system has an additional role in inhibiting cell wall turnover and contributes to the "inert" property of cell walls at the poles.

ABSTRACT

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often rewired such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this rewired background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations as PAO1, and in E90, RhlR plays a significant role in mediating cytotoxicity in a three-dimensional lung epithelium cell model. These data illuminate a rewired LasR-independent RhlR regulon in chronic infection isolates and suggest further investigation of RhlR as a possible target for therapeutic development in chronic infections.

IMPORTANCE Pseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Many isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR in chronic P. aeruginosa infections.

ABSTRACT

The Escherichia coli microcin C (McC) and related compounds are potent Trojan horse peptide-nucleotide antibiotics. The peptide part facilitates transport into sensitive cells. Inside the cell, the peptide part is degraded by nonspecific peptidases releasing an aspartamide-adenylate containing a phosphoramide bond. This nonhydrolyzable compound inhibits aspartyl-tRNA synthetase. In addition to the efficient export of McC outside the producing cells, special mechanisms have evolved to avoid self-toxicity caused by the degradation of the peptide part inside the producers. Here, we report that histidine-triad (HIT) hydrolases encoded in biosynthetic clusters of some McC homologs or by standalone genes confer resistance to McC-like compounds by hydrolyzing the phosphoramide bond in toxic aspartamide-adenosine, rendering them inactive.

IMPORTANCE Uncovering the mechanisms of resistance is a required step for countering the looming antibiotic resistance crisis. In this communication, we show how universally conserved histidine-triad hydrolases provide resistance to microcin C, a potent inhibitor of bacterial protein synthesis.

ABSTRACT

Obesity and associated metabolic disorders are worldwide public health issues. The gut microbiota plays a key role in the pathophysiology of diet-induced obesity. Glycerol monolaurate (GML) is a widely consumed food emulsifier with antibacterial properties. Here, we explore the anti-obesity effect of GML (1,600 mg/kg of body weight) in high-fat diet (HFD)-fed mice. HFD-fed mice were treated with 1,600 mg/kg GML. Integrated microbiome, metabolome, and transcriptome analyses were used to systematically investigate the metabolic effects of GML, and antibiotic treatment was used to assess the effects of GML on the gut microbiota. Our data indicated that GML significantly reduced body weight and visceral fat deposition, improved hyperlipidemia and hepatic lipid metabolism, and ameliorated glucose homeostasis and inflammation in HFD-fed mice. Importantly, GML modulated HFD-induced gut microbiota dysbiosis and selectively increased the abundance of Bifidobacterium pseudolongum. Antibiotic treatment abolished all the GML-mediated metabolic improvements. A multiomics (microbiome, metabolome, and transcriptome) association study showed that GML significantly modulated glycerophospholipid metabolism, and the abundance of Bifidobacterium pseudolongum strongly correlated with the metabolites and genes that participated in glycerophospholipid metabolism. Our results indicated that GML may be provided for obesity prevention by targeting the gut microbiota and regulating glycerophospholipid metabolism.

Phosphatidylcholine and phosphatidylethanolamine are two major phospholipid classes in eukaryotes. Each biosynthesis pathway starts with the phosphorylation of choline (Cho) or ethanolamine (Etn) catalyzed by either choline or ethanolamine kinase (CEK). Arabidopsis contains four CEK isoforms, but their isozyme-specific roles in metabolism and development are poorly described. Here, we showed that these four CEKs have distinct substrate specificities in vitro. While CEK1 and CEK2 showed substrate preference for Cho over Etn, CEK3 and CEK4 had clear substrate specificity for Cho and Etn, respectively. In vivo, CEK1, CEK2, and CEK3 exhibited kinase activity for Cho but not Etn, although the latter two isoforms showed rather minor contributions to total Cho kinase activity in both shoots and roots. The knockout mutants of CEK2 and CEK3 both affected root growth, and these isoforms had nonoverlapping cell-type-specific expression patterns in the root meristematic zone. In-depth phenotype analysis, as well as chemical and genetic complementation, revealed that CEK3, a Cho-specific kinase, is involved in cell elongation during root development. Phylogenetic analysis of CEK orthologs in Brassicaceae species showed evolutionary divergence between Etn kinases and Cho kinases. Collectively, our results demonstrate the distinct roles of the four CEK isoforms in Cho/Etn metabolism and plant development.

Terpene volatiles are found in many important fruit crops, but their relationship to flavor is poorly understood. Here, we demonstrate using sensory descriptive and discriminant analysis that 1,8-cineole contributes a key floral/eucalyptus note to the aroma of ripe 'Hort16A’ kiwifruit (Actinidia chinensis). Two quantitative trait loci (QTLs) for 1,8-cineole production were identified on linkage groups 27 and 29a in a segregating A. chinensis population, with the QTL on LG29a colocating with a complex cluster of putative terpene synthase (TPS)-encoding genes. Transient expression in Nicotiana benthamiana and analysis of recombinant proteins expressed in Escherichia coli showed four genes in the cluster (AcTPS1a–AcTPS1d) encoded functional TPS enzymes, which produced predominantly sabinene, 1,8-cineole, geraniol, and springene, respectively. The terpene profile produced by AcTPS1b closely resembled the terpenes detected in red-fleshed A. chinensis. AcTPS1b expression correlated with 1,8-cineole content in developing/ripening fruit and also showed a positive correlation with 1,8-cineole content in the mapping population, indicating the basis for segregation is an expression QTL. Transient overexpression of AcTPS1b in Actinidia eriantha fruit confirmed this gene produced 1,8-cineole in Actinidia. Structure-function analysis showed AcTPS1a and AcTPS1b are natural variants at key TPS catalytic site residues previously shown to change enzyme specificity in vitro. Together, our results indicate that AcTPS1b is a key gene for production of the signature flavor terpene 1,8-cineole in ripe kiwifruit. Using a sensory-directed strategy for compound identification provides a rational approach for applying marker-aided selection to improving flavor in kiwifruit as well as other fruits.

Key Points

Legionella pneumophila, the etiological agent of Legionnaires’ disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.

For cell entry, vaccinia virus requires fusion with the host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that the viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, we performed experimental evolutionary analyses by repeatedly passaging vaccinia virus in HeLa cells overexpressing the A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that they had accumulated a unique G9R open reading frame (ORF) mutation, resulting in a single His44Tyr amino acid change. We engineered a recombinant vaccinia virus to express the G9^H44Y mutant protein, and it readily infected HeLa-A56/K2 cells. Moreover, similar to the A56 virus, the G9^H44Y mutant virus on HeLa cells had a cell fusion phenotype, indicating that G9^H44Y-mediated membrane fusion was less prone to inhibition by A56/K2. Coimmunoprecipitation experiments demonstrated that the G9^H44Y protein bound to A56/K2 at neutral pH, suggesting that the H44Y mutation did not eliminate the binding of G9 to A56/K2. Interestingly, upon acid treatment to inactivate A56/K2-mediated fusion inhibition, the G9^H44Y mutant virus induced robust cell-cell fusion at pH 6, unlike the pH 4.7 required for control and revertant vaccinia viruses. Thus, A56/K2 fusion suppression mainly targets the G9 protein. Moreover, the G9^H44Y mutant protein escapes A56/K2-mediated membrane fusion inhibition most likely because it mimics an acid-induced intermediate conformation more prone to membrane fusion.

IMPORTANCE It remains unclear how the multiprotein entry fusion complex of vaccinia virus mediates membrane fusion. Moreover, vaccinia virus contains fusion suppressor proteins to prevent the aberrant activation of this multiprotein complex. Here, we used experimental evolution to identify adaptive mutant viruses that overcome membrane fusion inhibition mediated by the A56/K2 protein complex. We show that the H44Y mutation of the G9 protein is sufficient to overcome A56/K2-mediated membrane fusion inhibition. Treatment of virus-infected cells at different pHs indicated that the H44Y mutation lowers the threshold of fusion inhibition by A56/K2. Our study provides evidence that A56/K2 inhibits the viral fusion complex via the latter’s G9 subcomponent. Although the G9^H44Y mutant protein still binds to A56/K2 at neutral pH, it is less dependent on low pH for fusion activation, implying that it may adopt a subtle conformational change that mimics a structural intermediate induced by low pH.

The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo...

ABSTRACT

Insights into the interaction between phages and their bacterial hosts are crucial for the development of phage therapy. However, only one study has investigated global gene expression of Clostridioides (formerly Clostridium) difficile carrying prophage, and transcriptional reprogramming during lytic infection has not been studied. Here, we presented the isolation, propagation, and characterization of a newly discovered 35,109-bp phage, JD032, and investigated the global transcriptomes of both JD032 and C. difficile ribotype 078 (RT078) strain TW11 during JD032 infection. Transcriptome sequencing (RNA-seq) revealed the progressive replacement of bacterial host mRNA with phage transcripts. The expressed genes of JD032 were clustered into early, middle, and late temporal categories that were functionally similar. Specifically, a gene (JD032_orf016) involved in the lysis-lysogeny decision was identified as an early expression gene. Only 17.7% (668/3,781) of the host genes were differentially expressed, and more genes were downregulated than upregulated. The expression of genes involved in host macromolecular synthesis (DNA/RNA/proteins) was altered by JD032 at the level of transcription. In particular, the expression of the ropA operon was downregulated. Most noteworthy is that the gene expression of some antiphage systems, including CRISPR-Cas, restriction-modification, and toxin-antitoxin systems, was suppressed by JD032 during infection. In addition, bacterial sporulation, adhesion, and virulence factor genes were significantly downregulated. This study provides the first description of the interaction between anaerobic spore-forming bacteria and phages during lytic infection and highlights new aspects of C. difficile phage-host interactions.

IMPORTANCE C. difficile is one of the most clinically significant intestinal pathogens. Although phages have been shown to effectively control C. difficile infection, the host responses to phage predation have not been fully studied. In this study, we reported the isolation and characterization of a new phage, JD032, and analyzed the global transcriptomic changes in the hypervirulent RT078 C. difficile strain, TW11, during phage JD032 infection. We found that bacterial host mRNA was progressively replaced with phage transcripts, three temporal categories of JD032 gene expression, the extensive interplay between phage-bacterium, antiphage-like responses of the host and phage evasion, and decreased expression of sporulation- and virulence-related genes of the host after phage infection. These findings confirmed the complexity of interactions between C. difficile and phages and suggest that phages undergoing a lytic cycle may also cause different phenotypes in hosts, similar to prophages, which may inspire phage therapy for the control of C. difficile.

Nadia Vicenzi, Alejandro Laspiur, Paola L. Sassi, Ruben Massarelli, John Krenz, and Nora R. Ibargüengoytia

In ectotherms, temperature exerts a strong influence on the performance of physiological and ecological traits. One approach to understand the impact of rising temperatures on animals and their ability to cope with climate change is to quantify variation in thermal-sensitive traits. Here, we examined the thermal biology, the temperature dependence and the thermal plasticity of bite force (endurance and magnitude) in Diplolaemus leopardinus, an aggressive and territorial lizard, endemic to Mendoza province, Argentina. Our results indicated that this lizard behaves like a moderate thermoregulator which uses the rocks of its environment as the main heat source. Bite endurance was not influenced by head morphometry and body temperature, whereas bite force was influenced by head length and jaw length, and exhibited thermal dependence. Before thermal acclimation treatments, the maximum bite force for D. leopardinus occured at the lowest body temperature and fell sharply with increasing body temperature. After acclimation treatments, lizards acclimated at higher temperatures exhibited greater bite force. Bite force showed phenotypic plasticity, which reveals that leopard iguanas are able to maintain (and even improve) their bite force under a rising-temperature scenario.

Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. We hypothesized that epithelial remodeling during diseases characterized by cilial loss and squamous metaplasia may alter PAR-2 polarization. Here, using a fluorescent arrestin assay, we confirmed that the common fungal airway pathogen Aspergillus fumigatus activates heterologously-expressed PAR-2. Endogenous PAR-2 activation in submerged airway RPMI 2650 or NCI–H520 squamous cells increased intracellular calcium levels and granulocyte macrophage–colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-6 secretion. RPMI 2650 cells cultured at an air–liquid interface (ALI) responded to apically or basolaterally applied PAR-2 agonists. However, well-differentiated primary nasal epithelial ALIs responded only to basolateral PAR-2 stimulation, indicated by calcium elevation, increased cilia beat frequency, and increased fluid and cytokine secretion. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states. These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. Imaging nasal polyps and control middle turbinate explants, we found that nasal polyps, but not turbinates, exhibit apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyps and turbinates maintained basolateral PAR-2 polarization, suggesting that the calcium responses originated from nonciliated cells. Altered PAR-2 polarization in disease-remodeled epithelia may enhance apical responses and increase sensitivity to inhaled proteases.

Arrestin-1 is the arrestin family member responsible for inactivation of the G protein–coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1–enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.