Programmed Death-1 Ligand 2-Mediated Regulation of the PD-L1 to PD-1 Axis Is Essential for Establishing CD4(+) T Cell Immunity.

Many pathogens, including Plasmodium spp., exploit the interaction of programmed death-1 (PD-1) with PD-1-ligand-1 (PD-L1) to "deactivate" T cell functions, but the role of PD-L2 remains unclear. We studied malarial infections to understand the contribution of PD-L2 to immunity. Here we have shown that higher PD-L2 expression on blood dendritic cells, from Plasmodium falciparum-infected individuals, correlated with lower parasitemia. Mechanistic studies in mice showed that PD-L2 was indispensable for establishing effective CD4(+) T cell immunity against malaria, because it not only inhibited PD-L1 to PD-1 activity but also increased CD3 and inducible co-stimulator (ICOS) expression on T cells. Importantly, administration of soluble multimeric PD-L2 to mice with lethal malaria was sufficient to dramatically improve immunity and survival. These studies show immuno-regulation by PD-L2, which has the potential to be translated into an effective treatment for malaria and other diseases where T cell immunity is ineffective or short-lived due to PD-1-mediated signaling.

Beacon Success Rate versus Gateway Density in Sub-GHz Sensor Networks.

Multiple Gateways (GWs) provide network connectivity to Internet of Things (IoT) sensors in a Wide Area Network (WAN). The End Nodes (ENs) can connect to any GW by discovering and acquiring its periodic beacons. This provides GW diversity, improving coverage area. However, simultaneous periodic beacon transmissions among nearby GWs lead to interference and collisions. In this study, the impact of such intra-network interference is analyzed to determine the maximum number of GWs that can coexist. The paper presents a new collision model that considers the combined effects of the Medium Access Control (MAC) and Physical (PHY) layers. The model takes into account the partial overlap durations and relative power of all colliding events. It also illustrates the relationship between the collisions and the resulting packet loss rates. A performance evaluation is presented using a combination of analytical and simulation methods, with the former validating the simulation results. The system models are developed from experimental data obtained from field measurements. Numerical results are provided with Gaussian Frequency Shift Keying (GFSK) modulation. This paper provides guidance on selecting GFSK modulation parameters for low bit-rate and narrow-bandwidth IoT applications. The analysis and simulation results show that larger beacon intervals and frequency hopping help in reducing beacon loss rates, at the cost of larger beacon acquisition latency. On the flip side, the gateway discovery latency reduces with increasing GW density, thanks to an abundance of beacons.

A comparison study of superovulation strategies for C57BL/6J and B6D2F1 mice in CRISPR-Cas9 mediated genome editing.

Reproductive techniques such as superovulation and in vitro fertilisation (IVF) have been widely used in generating genetically modified animals. The current gold standard for superovulation in mice is using coherent treatments of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). An alternative method using inhibin antiserum (IAS) instead of eCG has been recently reported. Here, we evaluate different superovulation strategies in C57BL/6J and B6D2F1 mice. Firstly, we found that using 5-week-old C57BL/6J and 4-week-old B6D2F1 donors could achieve better superovulation outcomes. Then, we compared eCG-hCG, IAS-hCG and eCG-IAS-hCG with different dosages in both mouse strains. Significantly increased numbers of oocytes were obtained by using IAS-hCG and eCG-IAS-hCG methods. However, low fertilisation rates (36.3-38.8%) were observed when natural mating was applied. We then confirmed that IVF could dramatically ameliorate the fertilisation rates up to 89.1%. Finally, we performed CRISPR-Cas9 mediated genome editing targeting Scn11a and Kcnh1 loci, and successfully obtained mutant pups using eCG-hCG and IAS-hCG induced zygotes, which were fertilised by either natural mating or IVF. Our results showed that IAS is a promising superovulation reagent, and the efficiency of genome editing is unlikely to be affected by using IAS-induced zygotes.

A novel murine antibody and an open sandwich immunoassay for the detection of clenbuterol.

Clenbuterol (CLEN) is a sympathomimetic amine used as a decongestant and bronchodilator while treating breathing disorders. It is also used in food-producing animals as it improves the rate of red meat production. However, it is prohibited in many countries nowadays due to human health and safety concerns. Unfortunately, the illegal use of CLEN is still rampant. Thus, monitoring it in food and livestock is important. Here, we report a novel murine antibody and an open sandwich enzyme linked immunosorbent assay (OS-ELISA) to detect CLEN based on antigen-antibody reactions. The genes of antibody variable regions in mice immunized with CLEN conjugated with bovine serum albumin were cloned into a phagemid (pDong1/Fab) to construct a phage-display antibody library, from which a novel antibody, A12, was selected. Then, an OS-ELISA was developed to detect CLEN using separated variable regions of the A12 antibody. The limit of detection of the assay was found to be 8 ng/mL, which was useful for monitoring CLEN usage.

Structure-Function Studies of Polymyxin B Lipononapeptides.

The emerging threat of infections caused by highly drug-resistant bacteria has prompted a resurgence in the use of the lipodecapeptide antibiotics polymyxin B and colistin as last resort therapies. Given the emergence of resistance to these drugs, there has also been a renewed interest in the development of next generation polymyxins with improved therapeutic indices and spectra of action. We report structure-activity studies of 36 polymyxin lipononapeptides structurally characterised by an exocyclic FA-Thr²-Dab³ lipodipeptide motif instead of the native FA-Dab¹-Thr²-Dab³ tripeptide motif found in polymyxin B, removing one of the positively charged residues believed to contribute to nephrotoxicity. The compounds were prepared by solid phase synthesis using an on-resin cyclisation approach, varying the fatty acid and the residues at position 2 (P2), P3 and P4, then assessing antimicrobial potency against a panel of Gram-negative bacteria, including polymyxin-resistant strains. Pairwise comparison of N-acyl nonapeptide and decapeptide analogues possessing different fatty acids demonstrated that antimicrobial potency is strongly influenced by the N-terminal L-Dab-1 residue, contingent upon the fatty acid. This study highlights that antimicrobial potency may be retained upon truncation of the N-terminal L-Dab-1 residue of the native exocyclic lipotripeptide motif found in polymyxin B. The strategy may aid in the design of next generation polymyxins.

Synthesis of octapeptin C4 and biological profiling against NDM-1 and polymyxin-resistant bacteria.

The first synthesis of octapeptin C4 was achieved using a combination of solid phase synthesis and off-resin cyclisation. Octapeptin C4 displayed antibiotic activity against multi-drug resistant, NDM-1 and polymyxin-resistant Gram-negative bacteria, with moderate activity against Staphylococcus aureus. The linear analogue of octapeptin C4 was also prepared, which showed reduced activity.

Hypomagnesemia and the Risk of New-Onset Diabetes Mellitus after Kidney Transplantation.

Several studies suggest a link between post-transplant hypomagnesemia and new-onset diabetes after transplantation (NODAT), but this relationship remains controversial. We conducted a retrospective cohort study of 948 nondiabetic kidney transplant recipients from January 1, 2000, to December 31, 2011, to examine the association between serum magnesium level and NODAT. Multivariable Cox proportional hazards models were fitted to evaluate the risk of NODAT as a function of baseline (at 1 month), time-varying (every 3 months), and rolling-average (i.e., mean for 3 months moving at 3-month intervals) serum magnesium levels while adjusting for potential confounders. A total of 182 NODAT events were observed over 2951.2 person-years of follow-up. Multivariable models showed an inverse relationship between baseline serum magnesium level and NODAT (hazard ratio [HR], 1.24 per 0.1 mmol/L decrease; 95% confidence interval [95% CI], 1.05 to 1.46; P=0.01). The association with the risk of NODAT persisted in conventional time-varying (HR, 1.32; 95% CI, 1.14 to 1.52; P<0.001) and rolling-average models (HR, 1.34; 95% CI, 1.13 to 1.57; P=0.001). Hypomagnesemia (serum magnesium <0.74 mmol/L) also significantly associated with increased risk of NODAT in baseline (HR, 1.58; 95% CI, 1.07 to 2.34; P=0.02), time-varying (HR, 1.78; 95% CI, 1.29 to 2.45; P<0.001), and rolling-average models (HR, 1.83; 95% CI, 1.30 to 2.57; P=0.001). Our results suggest that lower post-transplant serum magnesium level is an independent risk factor for NODAT in kidney transplant recipients. Interventions targeting serum magnesium to reduce the risk of NODAT should be evaluated.

α-Conotoxin dendrimers have enhanced potency and selectivity for homomeric nicotinic acetylcholine receptors.

Covalently attached peptide dendrimers can enhance binding affinity and functional activity. Homogenous di- and tetravalent dendrimers incorporating the α7-nicotinic receptor blocker α-conotoxin ImI (α-ImI) with polyethylene glycol spacers were designed and synthesized via a copper-catalyzed azide-alkyne cycloaddition of azide-modified α-ImI to an alkyne-modified polylysine dendron. NMR and CD structural analysis confirmed that each α-ImI moiety in the dendrimers had the same 3D structure as native α-ImI. The binding of the α-ImI dendrimers to binding protein Ac-AChBP was measured by surface plasmon resonance and revealed enhanced affinity. Quantitative electrophysiology showed that α-ImI dendrimers had ∼100-fold enhanced potency at hα7 nAChRs (IC50 = 4 nM) compared to native α-ImI (IC50 = 440 nM). In contrast, no significant potency enhancement was observed at heteromeric hα3ß2 and hα9α10 nAChRs. These findings indicate that multimeric ligands can significantly enhance conotoxin potency and selectivity at homomeric nicotinic ion channels.

Mucin Binding Reduces Colistin Antimicrobial Activity.

Colistin has found increasing use in treating drug-resistant bacterial lung infections, but potential interactions with pulmonary biomolecules have not been investigated. We postulated that colistin, like aminoglycoside antibiotics, may bind to secretory mucin in sputum or epithelial mucin that lines airways, reducing free drug levels. To test this hypothesis, we measured binding of colistin and other antibiotics to porcine mucin, a family of densely glycosylated proteins used as a surrogate for human sputum and airway mucin. Antibiotics were incubated in dialysis tubing with or without mucin, and concentrations of unbound antibiotics able to penetrate the dialysis tubing were measured over time using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The percentage of antibiotic measured in the dialysate after 4 h in the presence of mucin, relative to the amount without mucin, was 15% for colistin, 16% for polymyxin B, 19% for tobramycin, 52% for ciprofloxacin, and 78% for daptomycin. Antibiotics with the strongest mucin binding had an overall polybasic positive charge, whereas those with comparatively little binding were less basic. When comparing MICs measured with or without added mucin, colistin and polymyxin B showed >100-fold increases in MICs for multiple Gram-negative bacteria. Preclinical evaluation of mucin binding should become a standard procedure when considering the potential pulmonary use of new or existing antibiotics, particularly those with a polybasic overall charge. In the airways, mucin binding may reduce the antibacterial efficacy of inhaled or intravenously administered colistin, and the presence of sub-MIC effective antibiotic concentrations could result in the development of antibiotic resistance.

Drug-drug plasma protein binding interactions of ivacaftor.

Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR-protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co-administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co-administered CF drugs for human serum albumin (HSA) and α1 -acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site-selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug-drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug-drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug-drug interactions of ivacaftor with co-administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor.

Synthesis and evaluation of cationic norbornanes as peptidomimetic antibacterial agents.

A series of structurally amphiphilic biscationic norbornanes have been synthesised as rigidified, low molecular weight peptidomimetics of cationic antimicrobial peptides. A variety of charged hydrophilic functionalities were attached to the norbornane scaffold including aminium, guanidinium, imidazolium and pyridinium moieties. Additionally, a range of hydrophobic groups of differing sizes were incorporated through an acetal linkage. The compounds were evaluated for antibacterial activity against both Gram-negative and Gram-positive bacteria. Activity was observed across the series; the most potent of which exhibited an MIC's ≤ 1 µg mL(-1) against Streptococcus pneumoniae, Enterococcus faecalis and several strains of Staphylococcus aureus, including multi-resistant methicillin resistant (mMRSA), glycopeptide-intermediate (GISA) and vancomycin-intermediate (VISA) S. aureus.

Flemingin-Type Prenylated Chalcones from the Sarawak Rainforest Plant Desmodium congestum.

In an ongoing program to identify new anti-infective leads, an extract derived from whole plant material of Desmodium congestum collected in the Sarawak rainforest was found to have anti-MRSA activity. Bioassay-guided isolation led to the isolation of two new prenylated chalcones, 5'-O-methyl-3-hydroxyflemingin A (1) and 5'-O-methylflemingin C (2), which were closely related to the flemingins previously isolated from various Flemingia species. Chalcones 1 and 2, which were determined to be 4:6 enantiomeric mixtures by chiral HPLC, exhibited moderate activity against a panel of Gram-positive bacteria and were also cytotoxic to the HEK293 human embryonic kidney cell line.

Expansion, folding, and abnormal lamination of the chick optic tectum after intraventricular injections of FGF2.

Comparative research has shown that evolutionary increases in brain region volumes often involve delays in neurogenesis. However, little is known about the influence of such changes on subsequent development. To get at this question, we injected FGF2--which delays cell cycle exit in mammalian neocortex--into the cerebral ventricles of chicks at embryonic day (ED) 4. This manipulation alters the development of the optic tectum dramatically. By ED7, the tectum of FGF2-treated birds is abnormally thin and has a reduced postmitotic layer, consistent with a delay in neurogenesis. FGF2 treatment also increases tectal volume and ventricular surface area, disturbs tectal lamination, and creates small discontinuities in the pia mater overlying the tectum. On ED12, the tectum is still larger in FGF2-treated embryos than in controls. However, lateral portions of the FGF2-treated tectum now exhibit volcano-like laminar disturbances that coincide with holes in the pia, and the caudomedial tectum exhibits prominent folds. To explain these observations, we propose that the tangential expansion of the ventricular surface in FGF2-treated tecta outpaces the expansion of the pial surface, creating abnormal mechanical stresses. Two alternative means of alleviating these stresses are tectal foliation and the formation of pial holes. The latter probably alter signaling gradients required for normal cell migration and may generate abnormal patterns of cerebrospinal fluid flow; both abnormalities would generate disturbances in tectal lamination. Overall, our findings suggest that evolutionary expansion of sheet-like, laminated brain regions requires a concomitant expansion of the pia mater.

Molecular Characterisation of the Haemagglutinin Glycan-Binding Specificity of Egg-Adapted Vaccine Strains of the Pandemic 2009 H1N1 Swine Influenza A Virus.

The haemagglutinin (HA) glycan binding selectivity of H1N1 influenza viruses is an important determinant for the host range of the virus and egg-adaption during vaccine production. This study integrates glycan binding data with structure-recognition models to examine the impact of the K123N, D225G and Q226R mutations (as seen in the HA of vaccine strains of the pandemic 2009 H1N1 swine influenza A virus). The glycan-binding selectivity of three A/California/07/09 vaccine production strains, and purified recombinant A/California/07/09 HAs harboring these mutations was examined via a solid-phase ELISA assay. Wild-type A/California/07/09 recombinant HA bound specifically to α2,6-linked sialyl-glycans, with no affinity for the α2,3-linked sialyl-glycans in the array. In contrast, the vaccine virus strains and recombinant HA harboring the Q226R HA mutation displayed a comparable pattern of highly specific binding to α2,3-linked sialyl-glycans, with a negligible affinity for α2,6-linked sialyl-glycans. The D225G A/California/07/09 recombinant HA displayed an enhanced binding affinity for both α2,6- and α2,3-linked sialyl-glycans in the array. Notably its α2,6-glycan affinity was generally higher compared to its α2,3-glycan affinity, which may explain why the double mutant was not naturally selected during egg-adaption of the virus. The K123N mutation which introduces a glycosylation site proximal to the receptor binding site, did not impact the α2,3/α2,6 glycan selectivity, however, it lowered the overall glycan binding affinity of the HA; suggesting glycosylation may interfere with receptor binding. Docking models and 'per residues' scoring were employed to provide a structure-recognition rational for the experimental glycan binding data. Collectively, the glycan binding data inform future vaccine design strategies to introduce the D225G or Q226R amino acid substitutions into recombinant H1N1 viruses.

Ramoplanin at bactericidal concentrations induces bacterial membrane depolarization in Staphylococcus aureus.

Ramoplanin is an actinomycetes-derived antibiotic with broad-spectrum activity against Gram-positive bacteria that has been evaluated in clinical trials for the treatment of gastrointestinal vancomycin-resistant enterococci (VRE) and Clostridium difficile infections. Recent studies have proposed that ramoplanin binds to bacterial membranes as a C2 symmetrical dimer that can sequester Lipid II, which causes inhibition of cell wall peptidoglycan biosynthesis and cell death. In this study, ramoplanin was shown to bind to anionic and zwitterionic membrane mimetics with a higher affinity for anionic membranes and to induce membrane depolarization of methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 25923 at concentrations at or above the minimal bactericidal concentration (MBC). The ultrastructural effects of ramoplanin on S. aureus were also examined by transmission electron microscopy (TEM), and this showed dramatic changes to bacterial cell morphology. The correlation observed between membrane depolarization and bacterial cell viability suggests that this mechanism may contribute to the bactericidal activity of ramoplanin.

An azido-oxazolidinone antibiotic for live bacterial cell imaging and generation of antibiotic variants.

An azide-functionalised analogue of the oxazolidinone antibiotic linezolid was synthesised and shown to retain antimicrobial activity. Using facile 'click' chemistry, this versatile intermediate can be further functionalised to explore antimicrobial structure-activity relationships or conjugated to fluorophores to generate fluorescent probes. Such probes can report bacteria and their location in a sample in real time. Modelling of the structures bound to the cognate 50S ribosome target demonstrates binding to the same site as linezolid is possible. The fluorescent probes were successfully used to image Gram-positive bacteria using confocal microscopy.

Efficient synthesis of anacardic acid analogues and their antibacterial activities.

Anacardic acid derivatives exhibit a broad range of biological activities. In this report, an efficient method for the synthesis of anacardic acid derivatives was explored, and a small set of salicylic acid variants synthesised retaining a constant hydrophobic element (a naphthyl tail). The naphthyl side chain was introduced via Wittig reaction and the aldehyde installed using directed ortho-metalation reaction of the substituted o-anisic acids. The failure of ortho-metalation using unprotected carboxylic acid group compelled us to use directed ortho-metalation in which a tertiary amide was used as a strong ortho-directing group. In the initial route, tertiary amide cleavage during final step was challenging, but cleaving the tertiary amide before Wittig reaction was beneficial. The Wittig reaction with protected carboxylic group (methyl ester) resulted in side-products whereas using sodium salt resulted in higher yields. The novel compounds were screened for antibacterial activity and cytotoxicity. Although substitution on the salicylic head group enhanced antibacterial activities they also enhanced cytotoxicity.

Advance care and resuscitation plans in a tertiary hospital: a multimodal approach.

OBJECTIVES: Advance care planning (ACP) is the process of documenting a person's preference for medical treatment in the event of future deterioration. This audit aimed to improve discussion and documentation of ACP in patients who die during a hospital admission. METHODS: We performed a clinical audit in 2021 of inpatients at a tertiary hospital in Sydney, Australia to evaluate the benefit of multimodal interventions to improve ACP compared with previous audits from 2016 and 2011. RESULTS: In 2021, 97% of audited patients had a documented ACP prior to death compared with 80% in the 2016 audit. The completion of NFR documentation on admission in 2016 was 33%, while in 2021 65% of ACPs were completed within 24 hours of admission.In 2021, 94% of patients had a paper resuscitation form filled; however, identification stickers, which are associated with risk of error, were used in 64%; and 25% of forms were only partially completed. Palliative care was consulted for 44% of patients prior to death; 33% on the day of or prior to death. CONCLUSIONS: Improvement in prevalence and timing of ACP prior to death is seen in the postintervention audit. A repeat audit in 5 years will be conducted, with interventions focused on improving documentation of ACP.