Long-Term Epidemiology and Evolution of Swine Influenza Viruses, Vietnam.

Influenza A viruses are a One Health threat because they can spill over between host populations, including among humans, swine, and birds. Surveillance of swine influenza virus in Hanoi, Vietnam, during 2013-2019 revealed gene pool enrichment from imported swine from Asia and North America and showed long-term maintenance, persistence, and reassortment of virus lineages. Genome sequencing showed continuous enrichment of H1 and H3 diversity through repeat introduction of human virus variants and swine influenza viruses endemic in other countries. In particular, the North American H1-δ1a strain, which has a triple-reassortant backbone that potentially results in increased human adaptation, emerged as a virus that could pose a zoonotic threat. Co-circulation of H1-δ1a viruses with other swine influenza virus genotypes raises concerns for both human and animal health.

Independent representations of self-motion and object location in barrel cortex output.

During active tactile exploration, the dynamic patterns of touch are transduced to electrical signals and transformed by the brain into a mental representation of the object under investigation. This transformation from sensation to perception is thought to be a major function of the mammalian cortex. In primary somatosensory cortex (S1) of mice, layer 5 (L5) pyramidal neurons are major outputs to downstream areas that influence perception, decision-making, and motor control. We investigated self-motion and touch representations in L5 of S1 with juxtacellular loose-seal patch recordings of optogenetically identified excitatory neurons. We found that during rhythmic whisker movement, 54 of 115 active neurons (47%) represented self-motion. This population was significantly more modulated by whisker angle than by phase. Upon active touch, a distinct pattern of activity was evoked across L5, which represented the whisker angle at the time of touch. Object location was decodable with submillimeter precision from the touch-evoked spike counts of a randomly sampled handful of these neurons. These representations of whisker angle during self-motion and touch were independent, both in the selection of which neurons were active and in the angle-tuning preference of coactive neurons. Thus, the output of S1 transiently shifts from a representation of self-motion to an independent representation of explored object location during active touch.

Determining Existing Human Population Immunity as Part of Assessing Influenza Pandemic Risk.

Zoonotic influenza infections continue to threaten human health. Ongoing surveillance and risk assessment of animal viruses are needed for pandemic preparedness, and population immunity is an important component of risk assessment. We determined age-stratified hemagglutinin inhibition seroprevalence against 5 swine influenza viruses circulating in Hong Kong and Guangzhou in China. Using hemagglutinin inhibition seroprevalence and titers, we modeled the effect of population immunity on the basic reproduction number (R0) if each virus were to become transmissible among humans. Among 353 individual serum samples, we reported low seroprevalence for triple-reassortant H1N2 and Eurasian avian-like H1N1 influenza viruses, which would reduce R0 by only 18%-20%. The smallest R0 needed to cause a pandemic was 1.22-1.24, meaning existing population immunity would be insufficient to block the spread of these H1N1 or H1N2 variants. For human-origin H3N2, existing population immunity could suppress R0 by 47%, thus reducing pandemic risk.

Novel Zoonotic Avian Influenza A(H3N8) Virus in Chicken, Hong Kong, China.

Zoonotic and pandemic influenza continue to pose threats to global public health. Pandemics arise when novel influenza A viruses, derived in whole or in part from animal or avian influenza viruses, adapt to transmit efficiently in a human population that has little population immunity to contain its onward transmission. Viruses of previous pandemic concern, such as influenza A(H7N9), arose from influenza A(H9N2) viruses established in domestic poultry acquiring a hemagglutinin and neuraminidase from influenza A viruses of aquatic waterfowl. We report a novel influenza A(H3N8) virus in chicken that has emerged in a similar manner and that has been recently reported to cause zoonotic disease. Although they are H3 subtype, these avian viruses are antigenically distant from contemporary human influenza A(H3N2) viruses, and there is little cross-reactive immunity in the human population. It is essential to heighten surveillance for these avian A(H3N8) viruses in poultry and in humans.

Congenital myasthenic syndrome due to mutations in MUSK suggests that the level of MuSK phosphorylation is crucial for governing synaptic structure.

MUSK encodes the muscle-specific receptor tyrosine kinase (MuSK), a key component of the agrin-LRP4-MuSK-DOK7 signaling pathway, which is essential for the formation and maintenance of highly specialized synapses between motor neurons and muscle fibers. We report a patient with severe early-onset congenital myasthenic syndrome and two novel missense mutations in MUSK (p.C317R and p.A617V). Functional studies show that MUSK p.C317R, located at the frizzled-like cysteine-rich domain of MuSK, disrupts an integral part of MuSK architecture resulting in ablated MuSK phosphorylation and acetylcholine receptor (AChR) cluster formation. MUSK p.A617V, located at the kinase domain of MuSK, enhances MuSK phosphorylation resulting in anomalous AChR cluster formation. The identification and evidence for pathogenicity of MUSK mutations supported the initiation of treatment with ß2-adrenergic agonists with a dramatic improvement of muscle strength in the patient. This work suggests uncharacterized mechanisms in which control of the precise level of MuSK phosphorylation is crucial in governing synaptic structure.

ß2-Adrenergic receptor agonists ameliorate the adverse effect of long-term pyridostigmine on neuromuscular junction structure.

Acetylcholine receptor deficiency is the most common form of the congenital myasthenic syndromes, a heterogeneous collection of genetic disorders of neuromuscular transmission characterized by fatiguable muscle weakness. Most patients with acetylcholine receptor deficiency respond well to acetylcholinesterase inhibitors; however, in some cases the efficacy of acetylcholinesterase inhibitors diminishes over time. Patients with acetylcholine receptor deficiency can also benefit from the addition of a ß2-adrenergic receptor agonist to their medication. The working mechanism of ß2-adrenergic agonists in myasthenic patients is not fully understood. Here, we report the long-term follow-up for the addition of ß2-adrenergic agonists for a cohort of patients with acetylcholine receptor deficiency on anticholinesterase medication that demonstrates a sustained quantitative improvement. Coincidently we used a disease model to mirror the treatment of acetylcholine receptor deficiency, and demonstrate improved muscle fatigue, improved neuromuscular transmission and improved synaptic structure resulting from the addition of the ß2-adrenergic agonist salbutamol to the anticholinesterase medication pyridostigmine. Following an initial improvement in muscle fatiguability, a gradual decline in the effect of pyridostigmine was observed in mice treated with pyridostigmine alone (P < 0.001). Combination therapy with pyridostigmine and salbutamol counteracted this decline (P < 0.001). Studies of compound muscle action potential decrement at high nerve stimulation frequencies (P < 0.05) and miniature end-plate potential amplitude analysis (P < 0.01) showed an improvement in mice following combination therapy, compared to pyridostigmine monotherapy. Pyridostigmine alone reduced postsynaptic areas (P < 0.001) and postsynaptic folding (P < 0.01). Combination therapy increased postsynaptic area (P < 0.001) and promoted the formation of postsynaptic junctional folds (P < 0.001), in particular in fast-twitch muscles. In conclusion, we demonstrate for the first time how the improvement seen in patients from adding salbutamol to their medication can be explained in an experimental model of acetylcholine receptor deficiency, the most common form of congenital myasthenic syndrome. Salbutamol enhances neuromuscular junction synaptic structure by counteracting the detrimental effects of long-term acetylcholinesterase inhibitors on the postsynaptic neuromuscular junction. The results have implications for both autoimmune and genetic myasthenias where anticholinesterase medication is a standard treatment.

A causal role for TRESK loss of function in migraine mechanisms.

The two-pore potassium channel, TRESK has been implicated in nociception and pain disorders. We have for the first time investigated TRESK function in human nociceptive neurons using induced pluripotent stem cell-based models. Nociceptors from migraine patients with the F139WfsX2 mutation show loss of functional TRESK at the membrane, with a corresponding significant increase in neuronal excitability. Furthermore, using CRISPR-Cas9 engineering to correct the F139WfsX2 mutation, we show a reversal of the heightened neuronal excitability, linking the phenotype to the mutation. In contrast we find no change in excitability in induced pluripotent stem cell derived nociceptors with the C110R mutation and preserved TRESK current; thereby confirming that only the frameshift mutation is associated with loss of function and a migraine relevant cellular phenotype. We then demonstrate the importance of TRESK to pain states by showing that the TRESK activator, cloxyquin, can reduce the spontaneous firing of nociceptors in an in vitro human pain model. Using the chronic nitroglycerine rodent migraine model, we demonstrate that mice lacking TRESK develop exaggerated nitroglycerine-induced mechanical and thermal hyperalgesia, and furthermore, show that cloxyquin conversely is able to prevent sensitization. Collectively, our findings provide evidence for a role of TRESK in migraine pathogenesis and its suitability as a therapeutic target.

Rapsyn facilitates recovery from desensitization in fetal and adult acetylcholine receptors expressed in a muscle cell line.

KEY POINTS: The physiological significance of the developmental switch from fetal to adult acetylcholine receptors in muscle (AChRs) and the functional impact of AChR clustering by rapsyn are not well studied. Using patch clamp experiments, we show that recovery from desensitization is faster in the adult AChR isoform. Recovery from desensitization is determined by the AChR isoform-specific cytoplasmic M3-M4 domain. The co-expression of rapsyn in muscle cells induced AChR clustering and facilitated recovery from desensitization in both fetal and adult AChRs. In fetal AChRs, facilitation of recovery kinetics by rapsyn was independent of AChR clustering. These effects could be crucial adaptations to motor neuron firing rates, which, in rodents, have been shown to increase around the time of birth when AChRs cluster at the developing neuromuscular junctions. ABSTRACT: The neuromuscular junction (NMJ) is the site of a number of autoimmune and genetic disorders, many involving the muscle-type nicotinic acetylcholine receptor (AChR), although there are aspects of normal NMJ development and function that need to be better understood. In particular, there are still questions regarding the implications of the developmental switch from fetal to adult AChRs, as well as how their functions might be modified by rapsyn that clusters the AChRs. Desensitization of human muscle AChRs was investigated using the patch clamp technique to measure whole-cell currents in muscle-type (TE671/CN21) and non-muscle (HEK293) cell lines expressing either fetal or adult AChRs. Desensitization time constants were similar with both AChR isoforms but recovery time constants were shorter in cells expressing adult compared to fetal AChRs (P < 0.0001). Chimeric experiments showed that recovery from desensitization was determined by the M3-M4 cytoplasmic loops of the γ- and ε-subunits. Expression of rapsyn in TE671/CN21 cells induced AChR aggregation and also, surprisingly, shortened recovery time constants in both fetal and adult AChRs. However, this was not dependent on clustering because rapsyn also facilitated recovery from desensitization in HEK293 cells expressing a δ-R375H AChR mutant that did not form clusters in C2C12 myotubes. Thus, rapsyn interactions with AChRs lead not only to clustering, but also to a clustering independent faster recovery from desensitization. Both effects of rapsyn could be a necessary adjustment to the motor neuron firing rates that increase around the time of birth.

Novel Bacterial Topoisomerase Inhibitors with Potent Broad-Spectrum Activity against Drug-Resistant Bacteria.

The novel bacterial topoisomerase inhibitor class is an investigational type of antibacterial inhibitor of DNA gyrase and topoisomerase IV that does not have cross-resistance with the quinolones. Here, we report the evaluation of the in vitro properties of a new series of this type of small molecule. Exemplar compounds selectively and potently inhibited the catalytic activities of Escherichia coli DNA gyrase and topoisomerase IV but did not block the DNA breakage-reunion step. Compounds showed broad-spectrum inhibitory activity against a wide range of Gram-positive and Gram-negative pathogens, including biodefence microorganisms and Mycobacterium tuberculosis No cross-resistance with fluoroquinolone-resistant Staphylococcus aureus and E. coli isolates was observed. Measured MIC90 values were 4 and 8 µg/ml against a panel of contemporary multidrug-resistant isolates of Acinetobacter baumannii and E. coli, respectively. In addition, representative compounds exhibited greater antibacterial potency than the quinolones against obligate anaerobic species. Spontaneous mutation rates were low, with frequencies of resistance typically <10-8 against E. coli and A. baumannii at concentrations equivalent to 4-fold the MIC. Compound-resistant E. coli mutants that were isolated following serial passage were characterized by whole-genome sequencing and carried a single Arg38Leu amino acid substitution in the GyrA subunit of DNA gyrase. Preliminary in vitro safety data indicate that the series shows a promising therapeutic index and potential for low human ether-a-go-go-related gene (hERG) inhibition (50% inhibitory concentration [IC50], >100 µM). In summary, the compounds' distinct mechanism of action relative to the fluoroquinolones, whole-cell potency, low potential for resistance development, and favorable in vitro safety profile warrant their continued investigation as potential broad-spectrum antibacterial agents.

In vitro biological evaluation of novel broad-spectrum isothiazolone inhibitors of bacterial type II topoisomerases.

OBJECTIVES: To evaluate the in vitro biological properties of a novel class of isothiazolone inhibitors of the bacterial type II topoisomerases. METHODS: Inhibition of DNA gyrase and topoisomerase IV activity was assessed using DNA supercoiling and decatenation assays. MIC and MBC were determined according to CLSI guidelines. Antibacterial combinations were assessed using a two-dimensional chequerboard MIC method. Spontaneous frequency of resistance was measured at various multiples of the MIC. Resistant mutants were generated by serial passage at subinhibitory concentrations of antibacterials and genetic mutations were determined through whole genome sequencing. Mammalian cytotoxicity was evaluated using the HepG2 cell line. RESULTS: Representative isothiazolone compound REDX04957 and its enantiomers (REDX05967 and REDX05990) showed broad-spectrum bactericidal activity against the ESKAPE organisms, with the exception of Enterococcus spp., as well as against a variety of other human bacterial pathogens. Compounds retained activity against quinolone-resistant strains harbouring GyrA S83L and D87G mutations (MIC ≤4 mg/L). Compounds inhibited the supercoiling activity of wild-type DNA gyrase and the decatenation function of topoisomerase IV. Frequency of resistance of REDX04957 at 4× MIC was <9.1 × 10(-9). Against a panel of recent MDR isolates, REDX05967 demonstrated activity against Acinetobacter baumannii with MIC50 and MIC90 of 16 and 64 mg/L, respectively. Compounds showed a lack of cytotoxicity against HepG2 cells at 128 mg/L. CONCLUSIONS: Isothiazolone compounds show potent activity against Gram-positive and -negative pathogens with a dual targeting mechanism-of-action and a low potential for resistance development, meriting their continued investigation as broad-spectrum antibacterial agents.

Late presentations of congenital myasthenic syndromes: How many do we miss?

INTRODUCTION: Congenital myasthenic syndromes (CMS) usually present neonatally or in early childhood. When they present later, they may be mistaken for seronegative autoimmune myasthenia, and unnecessary immunosuppressive treatment may be administered. METHODS: Patients who met criteria for seronegative generalized myasthenia without congenital or early childhood onset, but with an affected sibling were tested for CMS associated genes using exome and Sanger sequencing. RESULTS: Four sibling pairs from nonconsanguineous families were identified. Three had mutations in the RAPSN gene, and 1 had a mutation in CHRNA1. One sibling of a pair with symptoms of fatigue but no convincing features of neuromuscular dysfunction tested negative on genetic studies. The definite CMS cases comprised 7 of 25 seronegative patients with definite generalized myasthenia in the clinic, and over half had been treated for autoimmune myasthenia. CONCLUSIONS: CMS is probably underdiagnosed in seronegative myasthenic disorders and should be considered in the differential diagnosis. Muscle Nerve 54: 721-727, 2016.

Discovery and structure-activity relationships of a novel isothiazolone class of bacterial type II topoisomerase inhibitors.

There is an urgent and unmet medical need for new antibacterial drugs that tackle infections caused by multidrug-resistant (MDR) pathogens. During the course of our wider efforts to discover and exploit novel mechanism of action antibacterials, we have identified a novel series of isothiazolone based inhibitors of bacterial type II topoisomerase. Compounds from the class displayed excellent activity against both Gram-positive and Gram-negative bacteria with encouraging activity against a panel of MDR clinical Escherichia coli isolates when compared to ciprofloxacin. Representative compounds also displayed a promising in vitro safety profile.

Toll-like receptor signaling adapter proteins govern spread of neuropathic pain and recovery following nerve injury in male mice.

BACKGROUND: Spinal Toll-like receptors (TLRs) and signaling intermediaries have been implicated in persistent pain states. We examined the roles of two major TLR signaling pathways and selected TLRs in a mononeuropathic allodynia. METHODS: L5 spinal nerve ligation (SNL) was performed in wild type (WT, C57BL/6) male and female mice and in male Tlr2-/-Tlr3-/-, Tlr4-/-, Tlr5-/-, Myd88-/-, Triflps2, Myd88/Triflps2, Tnf-/-, and Ifnar1-/- mice. We also examined L5 ligation in Tlr4-/- female mice. We examined tactile allodynia using von Frey hairs. Iba-1 (microglia) and GFAP (astrocytes) were assessed in spinal cords by immunostaining. Tactile thresholds were analyzed by 1- and 2-way ANOVA and the Bonferroni post hoc test was used. RESULTS: In WT male and female mice, SNL lesions resulted in a persistent and robust ipsilateral, tactile allodynia. In males with TLR2, 3, 4, or 5 deficiencies, tactile allodynia was significantly, but incompletely, reversed (approximately 50%) as compared to WT. This effect was not seen in female Tlr4-/- mice. Increases in ipsilateral lumbar Iba-1 and GFAP were seen in mutant and WT mice. Mice deficient in MyD88, or MyD88 and TRIF, showed an approximately 50% reduction in withdrawal thresholds and reduced ipsilateral Iba-1. In contrast, TRIF and interferon receptor null mice developed a profound ipsilateral and contralateral tactile allodynia. In lumbar sections of the spinal cords, we observed a greater increase in Iba-1 immunoreactivity in the TRIF-signaling deficient mice as compared to WT, but no significant increase in GFAP. Removing MyD88 abrogated the contralateral allodynia in the TRIF signaling-deficient mice. Conversely, IFNß, released downstream to TRIF signaling, administered intrathecally, temporarily reversed the tactile allodynia. CONCLUSIONS: These observations suggest a critical role for the MyD88 pathway in initiating neuropathic pain, but a distinct role for the TRIF pathway and interferon in regulating neuropathic pain phenotypes in male mice.

Influenza A virus transmission in swine farms and during transport in the swine supply chain.

The last influenza pandemic in 2009 emerged from swine and surveillance of swine influenza is important for pandemic preparedness. Movement of swine during husbandry, trade or marketing for slaughter provide opportunities for transfer and genetic reassortment of swine influenza viruses. Over 90% of the swine slaughtered at the central swine abattoir in Hong Kong are imported from farms located in multiple provinces in mainland China. There is opportunity for virus cross-infection during this transport and slaughter process. Of the 26,980 swabs collected in the slaughterhouse in Hong Kong from 5 January 2012 to 15 December 2016, we analysed sequence data on influenza A (H3N2) virus isolates (n = 174) in conjunction with date of sampling and originating farm. Molecular epidemiology provided evidence of virus cross-infection between swine originating from different farms during transport. The findings are also suggestive of a virus lineage persisting in a swine farm for over 2 years, although the lack of information on management practices at farm-level means that alternative explanations cannot be excluded. We used virus serology and isolation data from 4226 pairs of linked serum and swabs collected from the same pig at slaughter from swine originating from Guangdong Province to compare the force of infection (FOI) during transport and within farms. The mean weekly FOI during transport was λt  = 0.0286 (95% CI = 0.0211-0.0391) while the weekly FOI in farms was λf = 0.0089 (95% CI = 0.0084-0.0095), assuming a possible exposure duration in farm of 28 weeks, suggesting increased FOI during the transport process. Pigs sourced from farms with high seroprevalence were found to be a significant risk factor (adjusted OR = 2.24, p value = .015) for infection of imported pigs during transport by multivariable logistic regression analysis, whereas pigs with HAI titre of ≥1:40 were associated with a substantial reduction in infection risk by 67% (p value = 0.012). Transport may increase virus cross-infection rates and provide opportunities for virus reassortment potentially increasing zoonotic risk to those involved in the transportation and slaughtering processes.