Members of the Eu Sou Eu group are giving a hand to residents of Rio de Janeiro's favelas, supplying the needy with food and face masks

Introducing Hong Kong’s four-step fining framework… On 29 April 2020, Justice Godfrey Lam, President of the Competition Tribunal, handed down judgment in relation to the fines to be imposed on the parties in the W. Hing and Others case....

PICURIS PUEBLO, N.M. (AP) — On a dusty plaza in a Native American village that dates back nearly a millennium, a steady trickle of vehicles inched through a pop-up coronavirus testing site. From the bed of a pickup truck...

Seven people were killed when protesters angry over what they see as unfair food aid distribution during the coronavirus pandemic clashed with police in Afghanistan's western Ghor province on Saturday, according to a local member of parliament. Fourteen more were wounded during the protest - sparked by growing unhappiness at the distribution...

Turkey’s state-run aid agency on May 8 distributed food packages and hygiene kits among 180 refugee families in Tunisia.

Our client briefing, “The final AIFMD cross-border fund distribution package: mapping the changes” published on 22 July 2019, provided a summary of the main changes introduced by Regulation (EU) 2019/1156 on facilitating cross-border d...

Fourteen more were wounded during the protest - sparked by growing unhappiness at the distribution allegedly favouring people with political connections, said Gulzaman Nayeb, a local lawmaker.

With the aim of expressing appreciation for the medical staff from around globe, the Iran's National Instruments Orchestra performed ";The Avicenna Suite"; by maestro Farhad Fakhreddini. The work has been recorded and edited by cell phone at home.

TAKHTBHAI: Food packages were distributed among 100 deserving families at the Government Girls Degree College here on Friday while ensuring social distancing.Additional Deputy Commissioner Mardan Nek Mohammad Khan was the chief guest. The people receiving the food showed discipline and did not...

Prague Daily Monitor

Police from the National Center Against Organized Crime suspect an employee from the General Financial Directorate of agreeing to a ten million crown bribe, five million of which he had personally taken possession of. The spokesperson for the General Financial Directorate Zuzana Masatova said "the employee has been removed from service."

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CARICOM-CUBA SUMMIT Toward the indispensable political, economic and social integration of Latin America and the Caribbean Key remarks by President Raúl Castro opening the Fifth CARICOM-Cuba Summit in Havana, December 8, 2014

Kanika Kapoor posted an adorable picture of her kids in a heartfelt post

NEW DELHI: The National Green Tribunal on Friday issued notices to South Korean company LG Polymers, Union Environment Ministry, Central Pollution Control Board and others regarding the Vizag gas...

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NEW DELHI: The National Green Tribunal on Friday issued notices to South Korean company LG Polymers, Union Environment Ministry, Central Pollution Control Board and others regarding the Vizag gas leak incident.The NGT has directed LG Polymers, India, at whose plant the gas leakage occurred, to...

tNEW DELHI: The National Green Tribunal on Friday issued notices to South Korean company LG Polymers, Union Environment Ministry, Central Pollution Control Board and others regarding the Vizag gas leak incident.The NGT has directed LG Polymers, India, at whose plant the gas leakage occurred, to...

Rawalpindi : Pakistan Post has distributed Rs21.5 billion to more than 6.5 lakh military pensioners and family pensioners in two weeks in the second phase of pension distribution to pensioners at their doorsteps, which is a unique service of its kind and such huge sums of money. Rawalpindi Circle...

Approved project 50193-003 in India.

NEW DELHI: Builders in Noida are unable to hand over more than 30,000 completed homes to their buyers, because of a National Green Tribunal order forbidding the Noida Authority from giving completion certificates to projects within a 10-kilometre radius of the Okhla bird sanctuary. Developers in Noida say they have in the past seven months since the tribunal’s order lost close to Rs 1,000 crore as they have been holding on the flats, paying interest on their loans as well as penalty to home buyers. “Everyone is bleeding,” said Vineet Gupta, director of the Ajnara group, which has around 1,500 apartments waiting to be handed over to buyers. Projects of […]

It's not every day that the face of a chief epidemiologist is inked as a tattoo. But then it's not every country that has tackled the coronavirus pandemic like Sweden.

A young boy chooses a nurse as the superhero he wants to play with over Batman and Spiderman in a new artwork by Banksy that encapsulates the gratitude Britons have felt toward the country's National Health Service during the coronavirus crisis.

Hurricane Irma’s deadly tear through the Caribbean will hobble the region’s multi-billion dollar tourism industry for months, just as hotels, airlines, and cruises were gearing up for the region’s peak winter season.

Title: Is Thyroid Hormone Dangerously Overprescribed in Older Patients?
Category: Health News
Created: 4/1/2020 12:00:00 AM
Last Editorial Review: 4/2/2020 12:00:00 AM

Title: VA Doctors Prescribing Unnecessary Antibiotics, Study Says
Category: Health News
Created: 4/26/2019 12:00:00 AM
Last Editorial Review: 4/29/2019 12:00:00 AM

Title: Doctors Describe First Drone Delivery of Diabetes Meds to Patient
Category: Health News
Created: 3/30/2020 12:00:00 AM
Last Editorial Review: 3/31/2020 12:00:00 AM

Title: AHA News: These Stroke Survivors May Not Be Prescribed Enough Blood Pressure Meds
Category: Health News
Created: 2/21/2020 12:00:00 AM
Last Editorial Review: 2/24/2020 12:00:00 AM

Title: Kisqali (ribociclib)
Category: Medications
Created: 4/22/2020 12:00:00 AM
Last Editorial Review: 4/22/2020 12:00:00 AM

Long noncoding RNAs (lncRNA) have been found to play critical roles in tumorigenesis and the development of various cancers, including hepatocellular carcinoma (HCC). Metastasis associated with lung adenocarcinoma transcript-1 (MALAT1) has been identified as an oncogene and prognostic biomarker in HCC. Here, we demonstrated that MALAT1 expression was obviously high in sorafenib-resistant HCC cells. Furthermore, knockdown of MALAT1 increased sorafenib sensitivity in nonresponsive HCC cells, whereas forced expression of MALAT1 conferred sorafenib resistance to responsive HCC cells in vitro. In addition, loss/gain-of-function assays revealed that MALAT1 promoted cell proliferation, migration, and epithelial–mesenchymal transition in HCC cells. Mechanistically, MALAT1 regulated Aurora-A expression by sponging miR-140-5p, thus promoting sorafenib resistance in HCC cells. Moreover, MALAT1 inhibition enhanced the antitumor efficacy of sorafenib in vivo. Clinically, we found that MALAT1 expression was negatively correlated with miR-140-5p expression but positively correlated with Aurora-A expression in patients with HCC and that upregulated MALAT1 was closely correlated with poor survival outcomes in patients with HCC. These findings indicated that MALAT1 may be a novel target for prognosis prediction and therapeutic strategies in patients with HCC treated with sorafenib.

ABSTRACT

Bacteria that encounter antibiotics can efficiently change their physiology to develop resistance. This intrinsic antibiotic resistance is mediated by multiple pathways, including a regulatory system(s) that activates specific genes. In some Streptomyces and Mycobacterium spp., the WblC/WhiB7 transcription factor is required for intrinsic resistance to translation-targeting antibiotics. Wide conservation of WblC/WhiB7 within Actinobacteria indicates a critical role of WblC/WhiB7 in developing resistance to such antibiotics. Here, we identified 312 WblC target genes in Streptomyces coelicolor, a model antibiotic-producing bacterium, using a combined analysis of RNA sequencing and chromatin immunoprecipitation sequencing. Interestingly, WblC controls many genes involved in translation, in addition to previously identified antibiotic resistance genes. Moreover, WblC promotes translation rate during antibiotic stress by altering the ribosome-associated protein composition. Our genome-wide analyses highlight a previously unappreciated antibiotic resistance mechanism that modifies ribosome composition and maintains the translation rate in the presence of sub-MIC levels of antibiotics.

IMPORTANCE The emergence of antibiotic-resistant bacteria is one of the top threats in human health. Therefore, we need to understand how bacteria acquire resistance to antibiotics and continue growth even in the presence of antibiotics. Streptomyces coelicolor, an antibiotic-producing soil bacterium, intrinsically develops resistance to translation-targeting antibiotics. Intrinsic resistance is controlled by the WblC/WhiB7 transcription factor that is highly conserved within Actinobacteria, including Mycobacterium tuberculosis. Here, identification of the WblC/WhiB7 regulon revealed that WblC/WhiB7 controls ribosome maintenance genes and promotes translation in the presence of antibiotics by altering the composition of ribosome-associated proteins. Also, the WblC-mediated ribosomal alteration is indeed required for resistance to translation-targeting antibiotics. This suggests that inactivation of the WblC/WhiB7 regulon could be a potential target to treat antibiotic-resistant mycobacteria.

ABSTRACT

The capacity of Candida albicans to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes ALS3 and ECE1, which are involved in pathogenicity processes such as invasion, iron acquisition, and host cell damage. The global repressor Tup1 and its cofactor Nrg1 are considered to be the main antagonists of hyphal development in C. albicans. However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of ALS3 and ECE1. In contrast to NRG1, overexpression of TUP1 was found to inhibit neither filamentous growth nor transcription of ALS3 and ECE1. In addition, we identified the transcription factor Ahr1 as being required for full expression of both genes. A hyperactive version of Ahr1 bound directly to the promoters of ALS3 and ECE1 and induced their transcription even in the absence of environmental stimuli. This regulation worked even in the absence of the crucial hyphal growth regulators Cph1 and Efg1 but was dependent on the presence of Tup1. Overall, our results show that Ahr1 and Tup1 are key contributors in the complex regulation of virulence-associated genes in the different C. albicans morphologies.

IMPORTANCE Candida albicans is a major human fungal pathogen and the leading cause of systemic Candida infections. In recent years, Als3 and Ece1 were identified as important factors for fungal virulence. Transcription of both corresponding genes is closely associated with hyphal growth. Here, we describe how Tup1, normally a global repressor of gene expression as well as of filamentation, and the transcription factor Ahr1 contribute to full expression of ALS3 and ECE1 in C. albicans hyphae. Both regulators are required for high mRNA amounts of the two genes to ensure functional relevant protein synthesis and localization. These observations identified a new aspect of regulation in the complex transcriptional control of virulence-associated genes in C. albicans.

Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley (Hordeum vulgare) and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in Nicotiana benthamiana that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163–185) is removed, initiates cell death, leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in N. benthamiana, as well as through an in vitro translation-inactivation assay. We found that Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.

Blue-light-induced chloroplast movements play an important role in maximizing light utilization for photosynthesis in plants. Under a weak light condition, chloroplasts accumulate to the cell surface to capture light efficiently (chloroplast accumulation response). Conversely, chloroplasts escape from strong light and move to the side wall to reduce photodamage (chloroplast avoidance response). The blue light receptor phototropin (phot) regulates these chloroplast movements and optimizes leaf photosynthesis by controlling other responses in addition to chloroplast movements. Seed plants such as Arabidopsis (Arabidopsis thaliana) have phot1 and phot2. They redundantly mediate phototropism, stomatal opening, leaf flattening, and the chloroplast accumulation response. However, the chloroplast avoidance response is induced by strong blue light and regulated primarily by phot2. Phots are localized mainly on the plasma membrane. However, a substantial amount of phot2 resides on the chloroplast outer envelope. Therefore, differentially localized phot2 might have different functions. To determine the functions of plasma membrane- and chloroplast envelope-localized phot2, we tethered it to these structures with their respective targeting signals. Plasma membrane-localized phot2 regulated phototropism, leaf flattening, stomatal opening, and chloroplast movements. Chloroplast envelope-localized phot2 failed to mediate phototropism, leaf flattening, and the chloroplast accumulation response but partially regulated the chloroplast avoidance response and stomatal opening. Based on the present and previous findings, we propose that phot2 localized at the interface between the plasma membrane and the chloroplasts is required for the chloroplast avoidance response and possibly for stomatal opening as well.

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa. However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa. However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.

Ducks usually show little or no clinical signs following highly pathogenic avian influenza virus infection. In order to analyze whether the microbiota could contribute to the control of influenza virus replication in ducks, we used a broad-spectrum oral antibiotic treatment to deplete the microbiota before infection with a highly pathogenic H5N9 avian influenza virus. Antibiotic-treated ducks and nontreated control ducks did not show any clinical signs following H5N9 virus infection. We did not detect any significant difference in virus titers neither in the respiratory tract nor in the brain nor spleen. However, we found that antibiotic-treated H5N9 virus-infected ducks had significantly increased intestinal virus excretion at days 3 and 5 postinfection. This was associated with a significantly decreased antiviral immune response in the intestine of antibiotic-treated ducks. Our findings highlight the importance of an intact microbiota for an efficient control of avian influenza virus replication in ducks.

IMPORTANCE Ducks are frequently infected with avian influenza viruses belonging to multiple subtypes. They represent an important reservoir species of avian influenza viruses, which can occasionally be transmitted to other bird species or mammals, including humans. Ducks thus have a central role in the epidemiology of influenza virus infection. Importantly, ducks usually show little or no clinical signs even following infection with a highly pathogenic avian influenza virus. We provide evidence that the microbiota contributes to the control of influenza virus replication in ducks by modulating the antiviral immune response. Ducks are able to control influenza virus replication more efficiently when they have an intact intestinal microbiota. Therefore, maintaining a healthy microbiota by limiting perturbations to its composition should contribute to the prevention of avian influenza virus spread from the duck reservoir.

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.

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.

Andreas Nord, Arne Hegemann, and Lars P. Folkow

Animals in seasonal environments must prudently manage energy expenditure to survive the winter. This may be achieved through reductions in the allocation of energy for various purposes (e.g. thermoregulation, locomotion, etc.). We studied whether such trade-offs also include suppression of the innate immune response, by subjecting captive male Svalbard ptarmigan (Lagopus muta hyperborea) to bacterial lipopolysaccharide (LPS) during exposure to either mild temperature (0°C) or cold snaps (acute exposure to –20°C), in constant winter darkness when birds were in energy-conserving mode, and in constant daylight in spring. The innate immune response was mostly unaffected by temperature. However, energy expenditure was below baseline when birds were immune challenged in winter, but significantly above baseline in spring. This suggests that the energetic component of the innate immune response was reduced in winter, possibly contributing to energy conservation. Immunological parameters decreased (agglutination, lysis, bacteriostatic capacity) or did not change (haptoglobin/PIT54) after the challenge, and behavioural modifications (anorexia, mass loss) were lengthy (9 days). While we did not study the mechanisms explaining these weak, or slow, responses, it is tempting to speculate they may reflect the consequences of having evolved in an environment where pathogen transmission rate is presumably low for most of the year. This is an important consideration if climate change and increased exploitation of the Arctic would alter pathogen communities at a pace outwith counter-adaption in wildlife.

Meike Stumpp, Inga Petersen, Femke Thoben, Jia-Jiun Yan, Matthias Leippe, and Marian Y. Hu

Larval stages of the abulacraria superphylum including echinoderms and hemichordates have highly alkaline midguts. To date the reason for the evolution of such extreme pH conditions in the gut of these organisms remains unknown. Here, we test the hypothesis that analogous to the acidic stomachs of vertebrates, these alkaline conditions may represent a first defensive barrier to protect from environmental pathogens.

pH-optimum curves for five different species of marine bacteria demonstrated a rapid decrease in proliferation rates by 50-60% between pH 8.5 and 9.5. Using the marine bacterium Vibrio diazotrophicus which elicits a coordinated immune response in the sea urchin larva of Strongylocentrotus purpuratus, we studied the physiological responses of the midgut pH regulatory machinery to this pathogen. Gastroscopic microelectrode measurements demonstrate a stimulation of midgut alkalization upon infection with V. diazotrophicus accompanied by an upregulation of acid-base transporter transcripts of the midgut. Pharmacological inhibition of midgut alkalization resulted in an increased mortality rate of larvae during Vibrio infection. Reductions in seawater pH resembling ocean acidification (OA) conditions lead to moderate reductions in midgut alkalization. However, these reductions in midgut pH do not affect the immune response and resilience of sea urchin larvae to a Vibrio infection under OA conditions.

Our study addressed the evolutionary benefits of the alkaline midgut of ambulacraria larval stages. The data indicate that alkaline conditions in the gut may serve as a first defensive barrier against environmental pathogens and that this mechanism can compensate for changes in seawater pH.

Functions of eukaryotic mRNAs are characterized by intramolecular interactions between their ends. We have addressed the question whether 5' and 3' ends meet by diffusion-controlled encounter "through solution" or by a mechanism involving the RNA backbone. For this purpose, we used a translation system derived from Drosophila embryos that displays two types of 5'–3' interactions: Cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug recognition elements (SREs) in the 3' UTR. Chimeric RNAs were made consisting of one RNA molecule carrying a luciferase coding sequence and a second molecule containing SREs and a poly(A) tail; the two were connected via a protein linker. The poly(A) tail stimulated translation of such chimeras even when disruption of the RNA backbone was combined with an inversion of the 5'–3' polarity between the open reading frame and poly(A) segment. Stimulation by the poly(A) tail also decreased with increasing RNA length. Both observations suggest that contacts between the poly(A) tail and the 5' end are established through solution, independently of the RNA backbone. In the same chimeric constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the backbone is not involved in the repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting that the contact between the SREs in the 3' UTR and the 5' end of the RNA might be established in a manner that differs from the contact between the poly(A) tail and the cap.

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K_1/2, while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae, which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches.

BACKGROUND:Electrical impedance tomography (EIT) is a noninvasive, portable lung imaging technique that provides functional distribution of ventilation. We aimed to describe the relationship between the distribution of ventilation by mode of ventilation and level of oxygenation impairment in children who are critically ill. We also aimed to describe the safety of EIT application.METHODS:A prospective observational study of EIT images obtained from subjects in the pediatric ICU. Images were categorized by whether the subjects were on intermittent mandatory ventilation (IMV), continuous spontaneous ventilation, or no positive-pressure ventilation. Images were categorized by the level of oxygenation impairment when using SpO2/FIO2. Distribution of ventilation is described by the center of ventilation.RESULTS:Sixty-four images were obtained from 25 subjects. Forty-two images obtained during IMV with a mean ± SD center of ventilation of 55 ± 6%, 14 images during continuous spontaneous ventilation with a mean ± SD center of ventilation of 48.1 ± 11%, and 8 images during no positive-pressure ventilation with a mean ± SD center of ventilation of 47.5 ± 10%. Seventeen images obtained from subjects with moderate oxygenation impairment with a mean ± SD center of ventilation of 59.3 ± 1.9%, 12 with mild oxygenation impairment with a mean ± SD center of ventilation of 52.6 ± 2.3%, and 4 without oxygenation impairment with a mean ± SD center of ventilation of 48.3 ± 4%. There was more ventral distribution of ventilation with IMV versus continuous spontaneous ventilation (P = .009), with IMV versus no positive-pressure ventilation (P = .01) cohorts, and with moderate oxygenation impairment versus cohorts without oxygenation impairment (P = .009). There were no adverse events related to the placement and use of EIT in our study.CONCLUSIONS:Children who had worse oxygen impairment or who received controlled modes of ventilation had more ventral distribution of ventilation than those without oxygen impairment or the subjects who were spontaneously breathing. The ability of EIT to detect changes in the distribution of ventilation in real time may allow for distribution-targeted mechanical ventilation strategies to be deployed proactively; however, future studies are needed to determine the effectiveness of such a strategy.

The kidneys play an important role in many processes, including urine formation, water conservation, acid-base equilibrium, and elimination of waste. The anatomic and functional development of the kidney has different maturation time points in humans versus animals, with critical differences between species in maturation before and after birth. Absorption, distribution, metabolism, and excretion (ADME) of drugs vary depending on age and maturation, which will lead to differences in toxicity and efficacy. When neonate/juvenile laboratory animal studies are designed, a thorough knowledge of the differences in kidney development between newborns/children and laboratory animals is essential. The human and laboratory animal data must be combined to obtain a more complete picture of the development in the kidneys around the neonatal period and the complexity of ADME in newborns and children. This review examines the ontogeny and cross-species differences in ADME processes in the developing kidney in preterm and term laboratory animals and children. It provides an overview of insights into ADME functionality in the kidney by identifying what is currently known and which gaps still exist. Currently important renal function properties such as glomerular filtration rate, renal blood flow, and ability to concentrate are generally well known, while detailed knowledge about transporter and metabolism maturation is growing but is still lacking. Preclinical data in those properties is limited to rodents and generally covers only the expression levels of transporter or enzyme-encoding genes. More knowledge on a functional level is needed to predict the kinetics and toxicity in neonate/juvenile toxicity and efficacy studies.

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505–527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515–519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

Genome-wide association studies have implicated thousands of noncoding variants across common human phenotypes. However, they cannot directly inform the cellular context in which disease-associated variants act. Here, we use open chromatin profiles from discrete mouse cell populations to address this challenge. We applied stratified linkage disequilibrium score regression and evaluated heritability enrichment in 64 genome-wide association studies, emphasizing schizophrenia. We provide evidence that mouse-derived human open chromatin profiles can serve as powerful proxies for difficult to obtain human cell populations, facilitating the illumination of common disease heritability enrichment across an array of human phenotypes. We demonstrate that signatures from discrete subpopulations of cortical excitatory and inhibitory neurons are significantly enriched for schizophrenia heritability with maximal enrichment in cortical layer V excitatory neurons. We also show that differences between schizophrenia and bipolar disorder are concentrated in excitatory neurons in cortical layers II-III, IV, and V, as well as the dentate gyrus. Finally, we leverage these data to fine-map variants in 177 schizophrenia loci nominating variants in 104/177. We integrate these data with transcription factor binding site, chromatin interaction, and validated enhancer data, placing variants in the cellular context where they may modulate risk.

Lisa Müller, Katrin Rietscher, Rene Keil, Marvin Neuholz, and Mechthild Hatzfeld

Desmosome remodeling is crucial for epidermal regeneration, differentiation and wound healing. It is mediated by adapting the composition, and by post-translational modifications, of constituent proteins. We have previously demonstrated in mouse suprabasal keratinocytes that plakophilin (PKP) 1 mediates strong adhesion, which is negatively regulated by insulin-like growth factor 1 (IGF1) signaling. The importance of PKP3 for epidermal adhesion is incompletely understood. Here, we identify a major role of epidermal growth factor (EGF), but not IGF1, signaling in PKP3 recruitment to the plasma membrane to facilitate desmosome assembly. We find that ribosomal S6 kinases (RSKs) associate with and phosphorylate PKP3, which promotes PKP3 association with desmosomes downstream of the EGF receptor. Knockdown of RSKs as well as mutation of an RSK phosphorylation site in PKP3 interfered with desmosome formation, maturation and adhesion. Our findings implicate a coordinate action of distinct growth factors in the control of adhesive properties of desmosomes through modulation of PKPs in a context-dependent manner.

Radiohybrid PSMA (rhPSMA) ligands, a new class of theranostic prostate-specific membrane antigen (PSMA)–targeting agents, feature fast ¹⁸F synthesis and utility for labeling with radiometals. Here, we assessed the biodistribution and image quality of ¹⁸F-rhPSMA-7 to determine the best imaging time point for patients with prostate cancer. Methods: In total, 202 prostate cancer patients who underwent a clinically indicated ¹⁸F-rhPSMA-7 PET/CT were retrospectively analyzed, and 12 groups based on the administered activity and uptake time of PET scanning were created: 3 administered activities (low, 222–296 MBq; moderate, 297–370 MBq; and high, 371–444 MBq) and 4 uptake time points (short, 50–70 min; intermediate, 71–90 min; long, 91–110 min; and extra long, ≥111 min). For quantitative analyses, SUV_mean and organ- or tumor-to-background ratio were determined for background, healthy organs, and 3 representative tumor lesions. Qualitative analyses assessed overall image quality, nonspecific blood-pool activity, and background uptake in bone or marrow using 3- or 4-point scales. Results: In quantitative analyses, SUV_mean showed a significant decrease in the blood pool and lungs and an increase in the kidneys, bladder, and bones as the uptake time increased. SUV_mean showed a trend to increase in the blood pool and bones as the administered activity increased. However, no significant differences were found in 377 tumor lesions with respect to the administered activity or uptake time. In qualitative analyses, the overall image quality was stable along with the uptake time, but the proportion rated to have good image quality decreased as the administered activity increased. All other qualitative image parameters showed no significant differences for the administered activities, but they showed significant trends with increasing uptake time: less nonspecific blood activity, more frequent background uptake in the bone marrow, and increased negative impact on clinical decision making. Conclusion: The biodistribution of ¹⁸F-rhPSMA-7 was similar to that of established PSMA ligands, and tumor uptake of ¹⁸F-rhPSMA-7 was stable across the administered activities and uptake times. An early imaging time point (50–70 min) is recommended for ¹⁸F-rhPSMA-7 PET/CT to achieve the highest overall image quality.

ABSTRACT

The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, β-lactamase (βlac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (⁷⁶⁷DKE⁷⁶⁹) connecting α15 and α16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K⁷⁶⁸ was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation.

IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, β-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (βlac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K⁷⁶⁸ in LC translocation. K⁷⁶⁸ was located between α15 and α16 (termed the cis-loop). Cellular assays showed that K⁷⁶⁸ did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K⁷⁶⁸ is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.

Escherichia coli ribosomal protein (r-protein) L4 has extraribosomal biological functions. Previously, we described L4 as inhibiting RNase E activity through protein-protein interactions. Here, we report that from stabilized transcripts regulated by L4-RNase E, mRNA levels of tnaA (encoding tryptophanase from the tnaCAB operon) increased upon ectopic L4 expression, whereas TnaA protein levels decreased. However, at nonpermissive temperatures (to inactivate RNase E), tnaA mRNA and protein levels both increased in an rne temperature-sensitive [rne(Ts)] mutant strain. Thus, L4 protein fine-tunes TnaA protein levels independently of its inhibition of RNase E. We demonstrate that ectopically expressed L4 binds with transcribed spacer RNA between tnaC and tnaA and downregulates TnaA translation. We found that deletion of the 5' or 3' half of the spacer compared to the wild type resulted in a similar reduction in TnaA translation in the presence of L4. In vitro binding of L4 to the tnaC-tnaA transcribed spacer RNA results in changes to its secondary structure. We reveal that during early stationary-phase bacterial growth, steady-state levels of tnaA mRNA increased but TnaA protein levels decreased. We further confirm that endogenous L4 binds to tnaC-tnaA transcribed spacer RNA in cells at early stationary phase. Our results reveal the novel function of L4 in fine-tuning TnaA protein levels during cell growth and demonstrate that r-protein L4 acts as a translation regulator outside the ribosome and its own operon.

IMPORTANCE Some ribosomal proteins have extraribosomal functions in addition to ribosome translation function. The extraribosomal functions of several r-proteins control operon expression by binding to own-operon transcripts. Previously, we discovered a posttranscriptional, RNase E-dependent regulatory role for r-protein L4 in the stabilization of stress-responsive transcripts. Here, we found an additional extraribosomal function for L4 in regulating the tna operon by L4-intergenic spacer mRNA interactions. L4 binds to the transcribed spacer RNA between tnaC and tnaA and alters the structural conformation of the spacer RNA, thereby reducing the translation of TnaA. Our study establishes a previously unknown L4-mediated mechanism for regulating gene expression, suggesting that bacterial cells have multiple strategies for controlling levels of tryptophanase in response to varied cell growth conditions.

The capacity of Listeria monocytogenes to adapt to environmental changes is facilitated by a large number of regulatory proteins encoded by its genome. Among these proteins are the uncharacterized LysR-type transcriptional regulators (LTTRs). LTTRs can work as positive and/or negative transcription regulators at both local and global genetic levels. Previously, our group determined by comparative genome analysis that one member of the LTTRs (NCBI accession no. WP_003734782) was present in pathogenic strains but absent from nonpathogenic strains. The goal of the present study was to assess the importance of this transcription factor in the virulence of L. monocytogenes strain F2365 and to identify its regulons. An L. monocytogenes strain lacking lysR (the F2365lysR strain) displayed significant reductions in cell invasion of and adhesion to Caco-2 cells. In plaque assays, the deletion of lysR resulted in a 42.86% decrease in plaque number and a 13.48% decrease in average plaque size. Furthermore, the deletion of lysR also attenuated the virulence of L. monocytogenes in mice following oral and intraperitoneal inoculation. The analysis of transcriptomics revealed that the transcript levels of 139 genes were upregulated, while 113 genes were downregulated in the F2365lysR strain compared to levels in the wild-type bacteria. lysR-repressed genes included ABC transporters, important for starch and sucrose metabolism as well as glycerolipid metabolism, flagellar assembly, quorum sensing, and glycolysis/gluconeogenesis. Conversely, lysR activated the expression of genes related to fructose and mannose metabolism, cationic antimicrobial peptide (CAMP) resistance, and beta-lactam resistance. These data suggested that lysR contributed to L. monocytogenes virulence by broad impact on multiple pathways of gene expression.

IMPORTANCE Listeria monocytogenes is the causative agent of listeriosis, an infectious and fatal disease of animals and humans. In this study, we have shown that lysR contributes to Listeria pathogenesis and replication in cell lines. We also highlight the importance of lysR in regulating the transcription of genes involved in different pathways that might be essential for the growth and persistence of L. monocytogenes in the host or under nutrient limitation. Better understanding L. monocytogenes pathogenesis and the role of various virulence factors is necessary for further development of prevention and control strategies.