Rapid Screening for Entry Inhibitors of Highly Pathogenic Viruses under Low-Level Biocontainment

Emerging viruses including Nipah, Hendra, Lujo, and Junin viruses have enormous potential to spread rapidly. Nipah virus, after emerging as a zoonosis, has also evolved the capacity for human-to-human transmission. Most of the diseases caused by these pathogens are untreatable and require high biocontainment conditions. Universal methods for rapidly identifying and screening candidate antivirals are urgently needed. We have developed a modular antiviral platform strategy that relies on simple bioinformatic and genetic information about each pathogen. Central to this platform is the use of envelope glycoprotein cDNAs to establish multi-cycle replication systems under BSL2 conditions for viral pathogens that normally require BSL3 and BSL4 facilities. We generated monoclonal antibodies against Nipah G by cDNA immunization in rats, and we showed that these antibodies neutralize both Nipah and Hendra live viruses. We then used these effective Henipavirus inhibitors to validate our screening strategy. Our proposed strategy should contribute to the response capability for emerging infectious diseases, providing a way to initiate antiviral development immediately upon identifying novel viruses

A combination of gemcitabine and 5-fluorouracil in advanced pancreatic cancer, a report from the Italian Group for the Study of Digestive Tract Cancer (GISCAD)

In a randomized clinical trial, gemcitabine (GEM) was more effective than 5-fluorouracil (5-FU) in advanced pancreatic cancer patients. GEM and 5-FU have different mechanisms of action and their combination, from a theoretical point of view, could result in a higher activity. To test activity and feasibility of such a combination, a multi-institutional phase II study was initiated in November 1996 by the Italian Group for the study of Digestive Tract Cancer (GISCAD). Primary objectives of this study were to determine the activity in terms of response rate and clinical benefit, while the secondary objective was toxicity. According to the optimal two-stage phase II design, 54 patients were enrolled. Schedule was: GEM 1000 mg m(-2) intravenous (i.v.), and 5-FU 600 mg m(-2) bolus i.v. weekly for 3 weeks out of every 4. All the 54 patients were symptomatic (pain, weight loss, dyspepsia). A clinical benefit was obtained in 28 patients (51\%) (95\% confidence interval (CI) 38-64\%). Two patients achieved a partial response and 34 a stable disease. Median survival for all the patients was 7 months. Side-effects were mild: no gastrointestinal or haematological grade 3-4 toxicity (WHO) were recorded. We observed only six episodes of grade 2 (WHO) leukopenia and seven episodes of thrombocytopenia. Although the non-randomized design of this study suggests caution in the interpretation of these data, in consideration of the low incidence of toxicity and the favourable results obtained in terms of clinical benefit, it may be worthwhile to test more active schedules of 5-FU (continuous infusion) in combination with gemcitabine

Broad-emission-line dominated hydrogen-rich luminous supernovae

Hydrogen-rich Type II supernovae (SNe II) are the most frequently observed class of core-collapse SNe (CCSNe). However, most studies that analyse large samples of SNe II lack events with absolute peak magnitudes brighter than -18.5 mag at rest-frame optical wavelengths. Thanks to modern surveys, the detected number of such luminous SNe II (LSNe II) is growing. There exist several mechanisms that could produce luminous SNe II. The most popular propose either the presence of a central engine (a magnetar gradually spinning down or a black hole accreting fallback material) or the interaction of supernova ejecta with circumstellar material (CSM) that turns kinetic energy into radiation energy. In this work, we study the light curves and spectral series of a small sample of six LSNe II that show peculiarities in their H$\alpha$ profile, to attempt to understand the underlying powering mechanism. We favour an interaction scenario with CSM that is not dense enough to be optically thick to electron scattering on large scales -- thus, no narrow emission lines are observed. This conclusion is based on the observed light curve (higher luminosity, fast decline, blue colours) and spectral features (lack of persistent narrow lines, broad H$\alpha$ emission, lack of H$\alpha$ absorption, weak or nonexistent metal lines) together with comparison to other luminous events available in the literature. We add to the growing evidence that transients powered by ejecta-CSM interaction do not necessarily display persistent narrow emission lines.Comment: 27 pages, 16 figures, accepted for publication in MNRA

Spherical Lactic Acid Bacteria Activate Plasmacytoid Dendritic Cells Immunomodulatory Function via TLR9-Dependent Crosstalk with Myeloid Dendritic Cells

Plasmacytoid dendritic cells (pDC) are a specialized sensor of viral and bacterial nucleic acids and a major producer of IFN-α that promotes host defense by priming both innate and acquired immune responses. Although synthetic Toll-like receptor (TLR) ligands, pathogenic bacteria and viruses activate pDC, there is limited investigation of non-pathogenic microbiota that are in wide industrial dietary use, such as lactic acid bacteria (LAB). In this study, we screened for LAB strains, which induce pDC activation and IFN-α production using murine bone marrow (BM)-derived Flt-3L induced dendritic cell culture. Microbial strains with such activity on pDC were absent in a diversity of bacillary strains, but were observed in certain spherical species (Lactococcus, Leuconostoc, Streptococcus and Pediococcus), which was correlated with their capacity for uptake by pDC. Detailed study of Lactococcus lactis subsp. lactis JCM5805 and JCM20101 revealed that the major type I and type III interferons were induced (IFN-α, -β, and λ). IFN-α induction was TLR9 and MyD88-dependent; a slight impairment was also observed in TLR4-/- cells. While these responses occurred with purified pDC, IFN-α production was synergistic upon co-culture with myeloid dendritic cells (mDC), an interaction that required direct mDC-pDC contact. L. lactis strains also stimulated expression of immunoregulatory receptors on pDC (ICOS-L and PD-L1), and accordingly augmented pDC induction of CD4+CD25+FoxP3+ Treg compared to the Lactobacillus strain. Oral administration of L. lactis JCM5805 induced significant activation of pDC resident in the intestinal draining mesenteric lymph nodes, but not in a remote lymphoid site (spleen). Taken together, certain non-pathogenic spherical LAB in wide dietary use has potent and diverse immunomodulatory effects on pDC potentially relevant to anti-viral immunity and chronic inflammatory disease

Raltitrexed plus oxaliplatin (TOMOX) as first-line chemotherapy for metastatic colorectal cancer. A phase ii study of the italian group for the study of gastrointestinal tract carcinomas (GISCAD)

Background: To evaluate the safety and efficacy of the novel raltitrexed/oxaliplatin combination (TOMOX) as first-line chemotherapy for patients with advanced colorectal cancer. Materials and methods: Previously untreated patients with metastatic colorectal cancer received raltitrexed 3 mg/m2 plus oxaliplatin 100 mg/m2, both intravenously, on day 1 every 3 weeks. Patients were re-evaluated after every third cycle and chemotherapy was continued up to tolerance or disease progression. Results: Fifty-eight patients from 13 Italian Group for the Study of Gastrointestinal Tract Carcinomas (GISCAD) centers were accrued from September 1999 to November 2000. According to the intention-to-treat analysis from 58 patients, the overall response rate was 50% [95% confidence interval (CI) 38% to 62%], with three complete responses and 26 partial responses. The median overall survival (44 patients currently alive) was >9 months and the median time to disease progression was 6.5 months (range 1-15 months). The main hematological toxicity was grade III/IV neutropenia, which occurred in 17% of patients, while anemia and thrombocytopenia were uncommon. Grade III/IV non-hematological toxicities were transient transaminitis (17% of patients); asthenia (16% of patients); neurotoxicity (10% of patients) and diarrhea (7% of patients). No toxic death was observed, one patient with grade IV asthenia after the first cycle refused chemotherapy. Conclusions: The results of this study suggest that the TOMOX combination is an effective and well tolerated regimen for the treatment of advanced colorectal cancer. Its ease of administration and patient tolerance warrant further investigation as an alternative to fluoropyrimidine-based regimens with repeated and prolonged fluorouracil infusions

RsmW, Pseudomonas aeruginosa small non-coding RsmA-binding RNA upregulated in biofilm versus planktonic growth conditions

BACKGROUND: Biofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state. RESULTS: A computational algorithm was devised to detect and categorize sRNAs into 5 types: intergenic, intragenic, 5′-UTR, 3′-UTR, and antisense. Here we report a novel RsmY/RsmZ-type sRNA, termed RsmW, in P. aeruginosa up-transcribed in biofilm versus planktonic growth. RNA-Seq, 5’-RACE and Mfold predictions suggest RsmW has a secondary structure with 3 of 7 GGA motifs located on outer stem loops. Northern blot revealed two RsmW binding bands of 400 and 120 bases, suggesting RsmW is derived from the 3’-UTR of the upstream hypothetical gene, PA4570. RsmW expression is elevated in late stationary versus logarithmic growth phase in PB minimal media, at higher temperatures (37°C versus 28°C), and in both gacA and rhlR transposon mutants versus wild-type. RsmW specifically binds to RsmA protein in vitro and restores biofilm production and reduces swarming in an rsmY/rsmZ double mutant. PA4570 weakly resembles an RsmA/RsmN homolog having 49% and 51% similarity, and 16% and 17% identity to RsmA and RsmN amino acid sequences, respectively. PA4570 was unable to restore biofilm and swarming phenotypes in ΔrsmA deficient strains. CONCLUSION: Collectively, our study reveals an interesting theme regarding another sRNA regulator of the Rsm system and further unravels the complexities regulating adaptive responses for Pseudomonas species

Inhibition of Nipah Virus Infection In Vivo: Targeting an Early Stage of Paramyxovirus Fusion Activation during Viral Entry

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses

SN 2021gno: a Calcium-rich transient with double-peaked light curves

We present extensive ultraviolet (UV) and optical photometric and optical spectroscopic follow-up of supernova (SN)~2021gno by the "Precision Observations of Infant Supernova Explosions" (POISE) project, starting less than two days after the explosion. Given its intermediate luminosity, fast photometric evolution, and quick transition to the nebular phase with spectra dominated by [Ca~II] lines, SN~2021gno belongs to the small family of Calcium-rich transients. Moreover, it shows double-peaked light curves, a phenomenon shared with only four other Calcium-rich events. The projected distance from the center of the host galaxy is not as large as other objects in this family. The initial optical light-curve peaks coincide with a very quick decline of the UV flux, indicating a fast initial cooling phase. Through hydrodynamical modelling of the bolometric light curve and line velocity evolution, we found that the observations are compatible with the explosion of a highly-stripped massive star with an ejecta mass of $0.8\,M_\odot$ and a $^{56}$Ni mass of $0.024~M_{\odot}$. The initial cooling phase (first light curve peak) is explained by the presence of an extended circumstellar material comprising $\sim$$10^{-2}\,M_{\odot}$ with an extension of $1100\,R_{\odot}$. We discuss if hydrogen features are present in both maximum-light and nebular spectra, and its implications in terms of the proposed progenitor scenarios for Calcium-rich transients.Comment: 21 pages, 13 figures, accepted for publication in MNRA

Magnesium Limitation Is an Environmental Trigger of the Pseudomonas aeruginosa Biofilm Lifestyle

Biofilm formation is a conserved strategy for long-term bacterial survival in nature and during infections. Biofilms are multicellular aggregates of cells enmeshed in an extracellular matrix. The RetS, GacS and LadS sensors control the switch from a planktonic to a biofilm mode of growth in Pseudomonas aeruginosa. Here we detail our approach to identify environmental triggers of biofilm formation by investigating environmental conditions that repress expression of the biofilm repressor RetS. Mg2+ limitation repressed the expression of retS leading to increased aggregation, exopolysaccharide (EPS) production and biofilm formation. Repression of retS expression under Mg2+ limitation corresponded with induced expression of the GacA-controlled small regulatory RNAs rsmZ and rsmY and the EPS biosynthesis operons pel and psl. We recently demonstrated that extracellular DNA sequesters Mg2+ cations and activates the cation-sensing PhoPQ two-component system, which leads to increased antimicrobial peptide resistance in biofilms. Here we show that exogenous DNA and EDTA, through their ability to chelate Mg2+, promoted biofilm formation. The repression of retS in low Mg2+ was directly controlled by PhoPQ. PhoP also directly controlled expression of rsmZ but not rsmY suggesting that PhoPQ controls the equilibrium of the small regulatory RNAs and thus fine-tunes the expression of genes in the RetS pathway. In summary, Mg2+ limitation is a biologically relevant environmental condition and the first bonafide environmental signal identified that results in transcriptional repression of retS and promotes P. aeruginosa biofilm formation

The Gac-Rsm and SadB Signal Transduction Pathways Converge on AlgU to Downregulate Motility in Pseudomonas fluorescens

Flagella mediated motility in Pseudomonas fluorescens F113 is tightly regulated. We have previously shown that motility is repressed by the GacA/GacS system and by SadB through downregulation of the fleQ gene, encoding the master regulator of the synthesis of flagellar components, including the flagellin FliC. Here we show that both regulatory pathways converge in the regulation of transcription and possibly translation of the algU gene, which encodes a sigma factor. AlgU is required for multiple functions, including the expression of the amrZ gene which encodes a transcriptional repressor of fleQ. Gac regulation of algU occurs during exponential growth and is exerted through the RNA binding proteins RsmA and RsmE but not RsmI. RNA immunoprecipitation assays have shown that the RsmA protein binds to a polycistronic mRNA encoding algU, mucA, mucB and mucD, resulting in lower levels of algU. We propose a model for repression of the synthesis of the flagellar apparatus linking extracellular and intracellular signalling with the levels of AlgU and a new physiological role for the Gac system in the downregulation of flagella biosynthesis during exponential growth