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Lymphatic filariasis and onchocerciasis are two major neglected tropical diseases that are responsible for causing severe disability in 50 million people worldwide, whilst veterinary filariasis (heartworm) is a potentially lethal parasitic infection of companion animals. There is an urgent need for safe, short-course curative (macrofilaricidal) drugs to eliminate these debilitating parasite infections. We investigated combination treatments of the novel anti-Wolbachia azaquinazoline small molecule, AWZ1066S, with benzimidazole drugs (albendazole or oxfendazole) in up to four different rodent filariasis infection models: Brugia malayi—CB.17 SCID mice, B. malayi—Mongolian gerbils, B. pahangi—Mongolian gerbils, and Litomosoides sigmodontis—Mongolian gerbils. Combination treatments synergised to elicit threshold (>90%) Wolbachia depletion from female worms in 5 days of treatment, using 2-fold lower dose-exposures of AWZ1066S than monotherapy. Short-course lowered dose AWZ1066S-albendazole combination treatments also delivered partial adulticidal activities and/or long-lasting inhibition of embryogenesis, resulting in complete transmission blockade in B. pahangi and L. sigmodontis gerbil models. We determined that short-course AWZ1066S-albendazole co-treatment significantly augmented the depletion of Wolbachia populations within both germline and hypodermal tissues of B. malayi female worms and in hypodermal tissues in male worms, indicating that anti-Wolbachia synergy is not limited to targeting female embryonic tissues. Our data provides pre-clinical proof-of-concept that sub-seven-day combinations of rapid-acting novel anti-Wolbachia agents with benzimidazole anthelmintics are a promising curative and transmission-blocking drug treatment strategy for filarial diseases of medical and veterinary importance.

Abstract Background Fine needle aspiration cytology has been established as a minimally invasive, non-tumour seeding investigation of choice in the initial diagnostic pathway of parotid lesions. The purpose of this study was to compare the accuracy of fine needle aspiration cytology performed with and without ultrasound to determine whether one method should be preferred to the other. A retrospective review of all patients undergoing fine needle aspiration cytology with and without ultrasound for parotid masses in a large district general hospital between 2012 and 2016 was performed. Specificity, sensitivity, accuracy, positive and negative predictive value, percentage of inconclusive fine needle aspiration cytology and percentage of second fine needle aspiration cytology were determined for each group. Results A total of 397 fine needle aspiration cytology results were available for analysis. The numbers performed with ultrasound guidance and free-hand were roughly equal (208 (52.3%) versus 189 (47.7%)). The number of inconclusive fine needle aspiration cytology reports was significantly higher in the free-hand group (65/189 (34.4%)) than the ultrasound group (25/208 (12%)) (p < 0.0001). A significantly higher number of repeated fine needle aspiration cytology were undertaken in the free-hand group vs ultrasound group (43 vs 15, p < 0.0001); overall 7.2% of ultrasound-guided fine needle aspiration cytology required a second fine needle aspiration cytology, compared to 22.8% in the free-hand group. The sensitivity, specificity, positive and negative predictive values were all higher in the ultrasound group versus the free-hand group. Conclusions Ultrasound-guided fine needle aspiration cytology is superior to free-hand fine needle aspiration cytology in the investigation of parotid tumours. There is a significant benefit in reducing the number of inconclusive results and repeat fine needle aspiration cytology, and a potential benefit in improving the sensitivity and positive predictive value, when immediate cytology assessment of the sample quality is not performed.

Lymphatic filariasis and onchocerciasis are major neglected tropical diseases affecting over 90 million people worldwide with painful and profoundly disfiguring pathologies (such as lymphoedema or blindness). Type 2 inflammation is a hallmark of filarial nematode tissue infection and is implicated both in eosinophil dependent immunity and lymphatic or ocular immunopathologies. Type-2 innate lymphoid cells (ILC2) are known to play an important role in the initiation of type 2 inflammation in helminth infection. We therefore tracked comparative IL-12Rβ2+ ILC1, ST2+ ILC2 and NKp46+ natural killer (NK) innate lymphoid cell population expansions during Brugia malayi experimental peritoneal filarial infections using either immunocompetent or immunodeficient mice. In immunocompetent BALB/c animals, NKp46+ NK cells rapidly expanded representing over 90% of the ILC population in the first week of infection, whereas, surprisingly, ST2+ ILC2 failed to expand. NKp46+ NK cell expansions were confirmed in RAG2 deficient mice lacking adaptive immunity. Ablation of the NKp46+ NK cell compartment in RAG2 common gamma chain (gc) mice led to increased susceptibility to chronic adult B. malayi infection. This data was recapitulated using an Onchocerca ochengi male worm peritoneal implant model. When NKp46+ NK cells were depleted in RAG2 deficient mice using anti-NKp46 or asialo GM1 antibody injections over the first five weeks of B. malayi infection, susceptibility to adult B. malayi infection was significantly increased by 2-3 fold with concomitant impairment in eosinophil or neutrophil recruitments. Finally, we demonstrate that in RAG2 deficient mice, drug clearance of a primary adult B. malayi infection followed by challenge infection leads to resistance against early larval B. malayi establishment. This innate resistance is associated with bolstered NK and eosinophils whereby NKp46+ NK cells express markers of memory-like/enhanced activation (increased expression of interferon gamma and Ly6C). Our data promotes a novel functional role for NKp46+ NK cells in immunoprotection against experimental primary and secondary filarial infection which can proceed in the absence of adaptive immune regulation.

The development of new drugs targeting adult-stage lymphatic filarial nematodes is hindered by the lack of a robust long-term in vitro culture model. Testing potential direct-acting and anti-Wolbachia therapeutic candidates against adult lymphatic filariae in vitro requires their propagation via chronic infection of gerbils. We evaluated Brugia malayi parasite burden data from male Mongolian gerbils compared with two immune-deficient mouse strains highly susceptible to B. malayi: CB.17 Severe-Combined Immmuno-Deficient (SCID) and interleukin-4 receptor alpha, interleukin-5 double knockout (IL-4Rα-/-IL-5-/-) mice. Adult worms generated in IL-4Rα-/-IL-5-/- mice were tested with different feeder cells (human embryonic kidney cells, human adult dermal lymphatic endothelial cells and human THP-1 monocyte differentiated macrophages) and comparative cell-free conditions to optimise and validate a long-term in vitro culture system. Cultured parasites were compared against those isolated from mice using motility scoring, metabolic viability assay (MTT), ex vivo microfilariae release assay and Wolbachia content by qPCR. A selected culture system was validated as a drug screen using reference anti-Wolbachia (doxycycline, ABBV-4083 / flubentylosin) or direct-acting compounds (flubendazole, suramin). BALB/c IL-4Rα-/-IL-5-/- or CB.17 SCID mice were superior to Mongolian gerbils in generating adult worms and supporting in vivo persistence for periods of up to 52 weeks. Adult females retrieved from BALB/c IL-4Rα-/-IL-5-/- mice could be cultured for up to 21 days in the presence of a lymphatic endothelial cell co-culture system with comparable motility, metabolic activity and Wolbachia titres to those maintained in vivo. Drug studies confirmed significant Wolbachia depletions or direct macrofilaricidal activities could be discerned when female B. malayi were cultured for 14 days. We therefore demonstrate a novel methodology to generate adult B. malayi in vivo and accurately evaluate drug efficacy ex vivo which may be adopted for drug screening with the dual benefit of reducing overall animal use and improving anti-filarial drug development.

We conducted a narrative review of existing literature to identify the knowledge, skills, and abilities (KSAs) necessary for officers who police in democratic societies to successfully manage potentially volatile police–public interactions. This review revealed 10 such KSAs that are frequently discussed in the literature. These KSAs include: (1) knowledge of policies and laws; (2) an understanding of mental health-related issues; (3) an ability to interact effectively with, and show respect for, individuals from diverse community groups; (4) awareness and management of stress effects; (5) communication skills; (6) decision-making and problem-solving skills; (7) perceptual skills; (8) motor skills related to use-of-force; (9) emotion and behavior regulation; and (10) an ability to treat people in a procedurally just manner. Following our review, we conducted semi-structured interviews (N = 7) with researchers who specialize in police training and adult education, interactions with individuals in crisis, and racialized policing, as well as two police trainers with expertise in de-escalation and use-of-force training. These interviews confirmed the importance of the 10 KSAs and highlighted two additional KSAs that are likely to be critical: understanding the role of policing in a free and democratic society and tactical knowledge and skills. To ensure that police–public interactions are managed effectively, police trainers may want to focus on the development and evaluation of these KSAs—something that is not always done currently.

The quinazolines CBR417 and CBR490 were previously shown to be potent anti-wolbachials that deplete Wolbachia endosymbionts of filarial nematodes and present promising pre-clinical candidates for human filarial diseases such as onchocerciasis. In the present study we tested both candidates in two models of chronic filarial infection, namely the Litomosoides sigmodontis and Brugia pahangi jird model and assessed their long-term effect on Wolbachia depletion, microfilariae counts and filarial embryogenesis 16−18 weeks after treatment initiation (wpt). Once per day (QD) oral treatment with CBR417 (50 mg/kg) for 4 days or twice per day (BID) with CBR490 (25 mg/kg) for 7 days during patent L. sigmodontis infection reduced the Wolbachia load by >99% and completely cleared peripheral microfilaremia from 10–14 wpt. Similarly, 7 days of QD treatments (40 mg/kg) with CBR417 or CBR490 cleared >99% of Wolbachia from B. pahangi and reduced peritoneal microfilariae counts by 93% in the case of CBR417 treatment. Transmission electron microscopy analysis indicated intensive damage to the B. pahangi ovaries following CBR417 treatment and in accordance filarial embryogenesis was inhibited in both models after CBR417 or CBR490 treatment. Suboptimal treatment regimens of CBR417 or CBR490 did not lead to a maintained reduction of the microfilariae and Wolbachia load. In conclusion, CBR417 or CBR490 are pre-clinical candidates for filarial diseases, which achieve long-term clearance of Wolbachia endosymbionts of filarial nematodes, inhibit filarial embryogenesis and clear microfilaremia with treatments as short as 7 days. Keywords: Quinazoline, Litomosoides sigmodontis, Brugia pahangi, Filaria, Doxycycline, Wolbachia, Macrofilaricidal, Microfilariae

Introduction: Septic arthritis is a destructive form of acute arthritis that requires expeditious recognition. as delayed treatment yields significant morbidity and mortality. Case Report: A 40-year-old male presented to the emergency department with right elbow pain. Examination revealed tachycardia, swelling, redness, tenderness, and decreased range of motion of the right humeroulnar joint. Synovial fluid analysis was consistent with an inflammatory etiology, but blood and joint cultures ultimately revealed Haemophilus influenzae. Discussion: This case highlights the importance of trusting clinical findings over laboratory evidence in patients with suspected septic arthritis.

The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping in order to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (πX/πA = 0.2), which is lower than the expected (πX/πA = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication. Author summary Almost a billion people receive >7.7 billion doses of treatment aimed at eliminating lymphatic filariasis, which is caused by three filarial nematodes: Wuchereria bancrofti, Brugia malayi, and Brugia timori. Drug resistance and adaptation are both associated with pathogen success as well as higher levels of genetic diversity. In an examination of genetic diversity in Brugia malayi and Brugia pahangi, we observed a lack of genetic diversity over a third of the genome that is found on chromosome X. These species have neo-X chromosomes where a chromosome X fused with an autosome. Using publicly-available published data, the other filarial nematodes of greatest human significance are also found to have a similar lack of genetic diversity on their neo-X chromosomes. The two filarial nematodes with publicly-available data that lack neo-X chromosomes did not have this lack of genetic diversity. This lack of sequence diversity in B. malayi, W. bancrofti, and O. volvulus could have profound effects on all traits encoded on chromosome X.

As the demand for drought hardy tree seedlings rises alongside global temperatures, there is a need to optimize nursery drought preconditioning methods to improve field performance of planted seedlings. This perspective article advocates for a more holistic approach to drought preconditioning research that considers the moderating role of plant developmental stage on the effects of drought preconditioning. We identify discrepancies in past studies of root growth potential (RGP) responses to drought preconditioning and highlight studies that suggest such discrepancies may result from inconsistencies among studies in the timing of drought preconditioning implementation. We then illustrate our perspective by presenting original research from an aeroponic RGP trial of 1st-year western larch (Larix occidentalis Nutt.) seedlings exposed to three soil moisture contents for 6months. We evaluated whether drought preconditioning could be used to increase the ratio of root: foliar tissue mass or enhance seedling physiological vigor during a subsequent growth period. Drought preconditioning was found to increase the ratio of root: foliar tissue mass and enhance seedling physiological vigor. Specifically, soil moisture content related negatively with new root biomass, positively with new foliar biomass, and negatively with the length and number of new roots (p

Multidrug-resistant bacteria are of critical importance and a problem for human health and food preservation; the discovery of new antimicrobial substances to control their proliferation is part of the solution. This work reports on 57 antagonistic Aeromonas strains, of which 38 strains were antagonistic towards problematic human pathogens. The genome of the most antagonistic strain was sequenced and identified as Aeromonas allosaccharophila. Its genome was fully annotated and mined for genes that might explain that activity. Strain AE59-TE was antagonistic toward clinically relevant gram-negative and gram-positive multidrug-resistant bacteria, including Klebsiella pneumoniae KPC, Escherichia coli ESBL, Salmonella typhimurium, and Staphylococcus aureus MRSA. Strain AE59-TE2 was identified by multilocus sequence analysis. Genome mining identified four genes homologous to the bacteriocin, zoocin A from Streptococcus equi and a gene 98% similar to cvpA linked to colicin V production. A. allosaccharophila strain AE59-TE2 produced antimicrobial activity against a broad range of bacteria, including important gram-negative bacteria, not typically targeted by bacteriocins. Herewere described novel zoocin genes that are promising for industrial applications in the food and health sectors. Interesting and important antagonistic activity is described combined with the first detailed genomic analysis of the species Aeromonas allosaccharophila.

Parasitic nematodes causing onchocerciasis and lymphatic filariasis rely on a bacterial endosymbiont, Wolbachia, which is a validated therapeutic target. Here, Clare et al. perform a high-throughput screen of 1.3 million compounds and identify 5 chemotypes with faster kill rates than existing anti-Wolbachia drugs.

Basic and translational research on loiasis, a filarial nematode infection of medical importance, is impeded by a lack of suitable Loa loa infection models and techniques of obtaining and culturing life cycle stages. We describe the development of a new method for routine production of infective third-stage larvae (L3) of L. loa from the natural intermediate arthropod vector host, Chrysops silacea, following experimental infection with purified microfilariae. At 14-days post-infection of C. silacea, the fly survival rate was 43%. Survival was significantly higher in flies injected with 50 mf (55.2%) than those that received 100 mf (31.0%). However, yield per surviving fly and total yield of L3 was markedly higher in the group of flies inoculated with 100 mf (3474 vs 2462 L3 produced). The abdominal segment hosted the highest percentage recovery of L3 (47.7%) followed by head (34.5%) and thorax (17.9%). L. loa larval survival was higher than 90% after 30 days of in vitro culture. The in vitro moulting success rate to the L4 larval stage was 59.1%. After experimental infection of RAG2-/-IL-2γc-/-mice, the average L. loa juvenile adult worm recovery rate was 10.5% at 62 dpi. More than 87% of the worms were recovered from the muscles and subcutaneous tissues. Worms recovered measured an average 24.3 mm and 11.4 mm in length for females (n = 5) and males (n = 5), respectively. In conclusion, L. loa mf injected into C. silacea intrathoracically develop into infective larvae that remain viable and infective comparable to L3 obtained through natural feeding on the human host. This technique further advances the development of a full laboratory life cycle of L. loa where mf derived from experimentally-infected animals may be utilized to passage life cycle generations via intrathoracic injections of wild-caught vector hosts.

Abstract Filariasis is a global health problem targeted for elimination. Curative drugs (macrofilaricides) are required to accelerate elimination. Candidate macrofilaricides require testing in preclinical models of filariasis. The incidence of infection failures and high intra-group variation means that large group sizes are required for drug testing. Further, a lack of accurate, quantitative adult biomarkers results in protracted timeframes or multiple groups for endpoint analyses. Here we evaluate intra-vital ultrasonography (USG) to identify B. malayi in the peritonea of gerbils and CB.17 SCID mice and assess prognostic value in determining drug efficacy. USG operators, blinded to infection status, could detect intra-peritoneal filarial dance sign (ipFDS) with 100% specificity and sensitivity, when >5 B. malayi worms were present in SCID mice. USG ipFDS was predictive of macrofilaricidal activity in randomized, blinded studies comparing flubendazole, albendazole and vehicle-treated SCID mice. Semi-quantification of ipFDS could predict worm burden >10 with 87–100% accuracy in SCID mice or gerbils. We estimate that pre-assessment of worm burden by USG could reduce intra-group variation, obviate the need for surgical implantations in gerbils, and reduce total SCID mouse use by 40%. Thus, implementation of USG may reduce animal use, refine endpoints and negate invasive techniques for assessing anti-filarial drug efficacy.

Abstract Lymphatic filariasis (LF) and onchocerciasis are priority neglected tropical diseases targeted for elimination. The only safe drug treatment with substantial curative activity against the filarial nematodes responsible for LF (Brugia malayi, Wuchereria bancrofti) or onchocerciasis (Onchocerca volvulus) is doxycycline. The target of doxycycline is the essential endosymbiont, Wolbachia. Four to six weeks doxycycline therapy achieves >90% depletion of Wolbachia in worm tissues leading to blockade of embryogenesis, adult sterility and premature death 18–24 months post-treatment. Long treatment length and contraindications in children and pregnancy are obstacles to implementing doxycycline as a public health strategy. Here we determine, via preclinical infection models of Brugia malayi or Onchocerca ochengi that elevated exposures of orally-administered rifampicin can lead to Wolbachia depletions from filariae more rapidly than those achieved by doxycycline. Dose escalation of rifampicin achieves >90% Wolbachia depletion in time periods of 7 days in B. malayi and 14 days in O. ochengi. Using pharmacokinetic-pharmacodynamic modelling and mouse-human bridging analysis, we conclude that clinically relevant dose elevations of rifampicin, which have recently been determined as safe in humans, could be administered as short courses to filariasis target populations with potential to reduce anti-Wolbachia curative therapy times to between one and two weeks.

The Onchocerca ochengi adult implant and Brugia malayi microfilariemic Severe-Combined Immunodeficient (SCID) mouse models are validated screens to measure macrofilaricidal and microfilaricidal activities of candidate onchocerciasis drugs. The purpose of this study was to assess whether 5 daily sub-cutaneous (s.c.) injections of standard flubendazole (FBZ) suspension (10mg/kg), a single s.c. injection (10mg/kg) or 5 daily repeated oral doses of FBZ amorphous solid dispersion (ASD) formulation (0.2, 1.5 or 15mg/kg) mediated macrofilaricidal efficacy against O. ochengi male worms implanted into SCID mice. The direct microfilaricidal activity against circulating B. malayi microfilariae of single dose FBZ ASD formulation (2 or 40 mg/kg) was also evaluated and compared against the standard microfilaricide, ivermectin (IVM). Systemic exposures of FBZ/FBZ metabolites achieved following dosing were measured by pharmacokinetic (PK) bioanalysis. At necropsy, five weeks following start of FBZ SC injections, there were significant reductions in burdens of motile O. ochengi worms following multiple injections (93%) or single injection (82%). Further, significant proportions of mice dosed following multiple injections (5/6; 83%) or single injection (6/10; 60%) were infection negative (drug-cured). In comparison, no significant reduction in recovery of motile adult O. ochengi adult worms was obtained in any multiple-oral dosage group. Single oral-dosed FBZ did not mediate any significant microfilaricidal activity against circulating B. malayi mf at 2 or 7 days compared with >80% efficacy of single dose IVM. In conclusion, multiple oral FBZ formulation doses, whilst achieving substantial bioavailability, do not emulate the efficacy delivered by the parenteral route in vivo against adult O. ochengi. PK analysis determined FBZ efficacy was related to sustained systemic drug levels rather than achievable Cmax. PK modelling predicted that oral FBZ would have to be given at low dose for up to 5 weeks in the mouse model to achieve a matching efficacious exposure profile.

Eosinophils are effectors in immunity to tissue helminths but also induce allergic immunopathology. Mechanisms of eosinophilia in non-mucosal tissues during infection remain unresolved. Here we identify a pivotal function of tissue macrophages (Mϕ) in eosinophil anti-helminth immunity using a BALB/c mouse intra-peritoneal Brugia malayi filarial infection model. Eosinophilia, via C-C motif chemokine receptor (CCR)3, was necessary for immunity as CCR3 and eosinophil impairments rendered mice susceptible to chronic filarial infection. Post-infection, peritoneal Mϕ populations proliferated and became alternatively-activated (AAMϕ). Filarial AAMϕ development required adaptive immunity and interleukin-4 receptor-alpha. Depletion of Mϕ prior to infection suppressed eosinophilia and facilitated worm survival. Add back of filarial AAMϕ in Mϕ-depleted mice recapitulated a vigorous eosinophilia. Transfer of filarial AAMϕ into Severe-Combined Immune Deficient mice mediated immunological resistance in an eosinophil-dependent manner. Exogenous IL-4 delivery recapitulated tissue AAMϕ expansions, sustained eosinophilia and mediated immunological resistance in Mϕ-intact SCID mice. Co-culturing Brugia with filarial AAMϕ and/or filarial-recruited eosinophils confirmed eosinophils as the larvicidal cell type. Our data demonstrates that IL-4/IL-4Rα activated AAMϕ orchestrate eosinophil immunity to filarial tissue helminth infection.

Abstract Background New drugs effective against adult filariae (macrofilaricides) would accelerate the elimination of lymphatic filariasis and onchocerciasis. Anti-Onchocerca drug development is hampered by the lack of a facile model. We postulated that SCID mice could be developed as a fmacrofilaricide screening model. Methods The filaricides: albendazole (ABZ), diethylcarbamazine (DEC), flubendazole (FBZ), ivermectin (IVM) and the anti-Wolbachia macrofilaricide, minocycline (MIN) were tested in Brugia malayi (Bm)-parasitized BALB/c SCID mice vs vehicle control (VC). Responses were compared to BALB/c wild type (WT). Onchocerca ochengi male worms or onchocercomata were surgically implanted into BALB/c SCID, CB.17 SCID, BALB/c WT mice or Meriones gerbils. Survival was evaluated at 7–15 days. BALB/c SCID were tested to evaluate the responsiveness of pre-clinical macrofilaricides FBZ and rifapentine (RIFAP) against male Onchocerca. Results WT and SCID responded with >95% efficacy following ABZ or DEC treatments against Bm larvae (P < 0.0001). IVM was partially filaricidal against Bm larvae in WT and SCID (WT; 39.8%, P = 0.0356 and SCID; 56.7%, P = 0.026). SCID responded similarly to WT following IVM treatment of microfilaraemias (WT; 79%, P = 0.0194. SCID; 76%, P = 0.0473). FBZ induced a total macrofilaricidal response against adult Bm in WT and SCID (WT; P = 0.0067, SCID; P = 0.0071). MIN induced a >90% reduction in Bm Wolbachia burdens (P < 0.0001) and a blockade of microfilarial release (P = 0.0215) in SCID. Male Onchocerca survival was significantly higher in SCID vs WT mice, but not gerbils, after +15 days (60% vs 22% vs 39% P = 0.0475). Onchocercoma implants had engrafted into host tissues, with evidence of neovascularisation, after +7 days and yielded viable macro/microfilariae ex vivo. FBZ induced a macrofilaricidal effect in Onchocerca male implanted SCID at +5 weeks (FBZ; 1.67% vs VC; 43.81%, P = 0.0089). Wolbachia loads within male Onchocerca were reduced by 99% in implanted SCID receiving RIFAP for +2 weeks. Conclusions We have developed a `pan-filarial’ small animal research model that is sufficiently robust, with adequate capacity and throughput, to screen existing and future pre-clinical candidate macrofilaricides. Pilot data suggests a murine onchocercoma xenograft model is achievable.

The devil facial tumor disease (DFTD) is caused by clonal transmissible cancers that have led to a catastrophic decline in the wild Tasmanian devil (Sarcophilus harrisii) population. The first transmissible tumor, now termed DFT1, was first discovered in 1996 and has been continually transmitted to new hosts for at least 20 years. In 2015 a second transmissible cancer (DFT2) was discovered in wild devils, and the DFT2 is genetically distinct and independent from the DFT1. Despite the estimated 136,559 base pair substitutions and 14,647 insertions/deletions in the DFT1 genome as compared to two normal devil reference genomes, the allograft tumors are not rejected by the host immune system. Additionally, genome sequencing of two sub-strains of DFT1 detected greater than 15,000 single-base substitutions that were found in only one of the DFT1 sub-strains, demonstrating the transmissible tumors are evolving and that generation of neoantigens is likely ongoing. Recent evidence in human clinical trials suggests that blocking PD-1:PD-L1 interactions promote anti-tumor immune responses and is most effective in cancers with a high number of mutations. We hypothesized that DFTD cells could exploit the PD-1:PD-L1 inhibitory pathway to evade anti-tumor immune responses. We developed recombinant proteins and monoclonal antibodies to provide the first demonstration that PD-1 binds to both PD-L1 and PD-L2 in a non-placental mammal and show that PD-L1 is upregulated in DFTD cells in response to IFN-γ. Immunohistochemistry showed that PD-L1 is rarely expressed in primary tumor masses, but low numbers of PD-L1+ non-tumor cells were detected in the microenvironment of several metastatic tumors. Importantly, in vitro testing suggests that PD-1 binding to PD-L1 and PD-L2 can be blocked by monoclonal antibodies, which could be critical to understanding how the DFT allografts evade the immune system.

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The effect of different cropping systems on CO2 fixation by soil microorganisms was studied by comparing soils from three exemplary cropping systems: continuous cropping of paddy rice (rice-rice), rotation of paddy rice and rapeseed (rice-rapeseed), and rotated cropping of rapeseed and corn (rapeseed-corn). Soils from different cropping systems were continuously labeling with 14C-CO2 for 110 days. The CO2-fixing bacterial communities were investigated by analyzing the cbbL gene encoding ribulose-1,5-bisphosphate carboxylase oxygenase (RubisCO). Abundance, diversity and activity of cbbL-carrying bacteria were analyzed by quantitative PCR, cbbL clone libraries and enzyme assays. After 110 days incubation, substantial amounts of 14C-CO2 were incorporated into soil organic carbon (14C-SOC) and microbial organic carbon (14C-MBC). Rice-rice rotated soil showed stronger incorporation rates when looking at 14C-SOC and 14C-MBC contents. These differences in incorporation rates were also reflected by RubisCO activities. 14C-MBC, cbbL gene abundances and RubisCO activity were found to correlate significantly with 14C-SOC, indicating cbbL-carrying bacteria to be key players for CO2 fixation in these soils. The analysis of clone libraries revealed distinct cbbL-carrying bacterial communities for the individual soils analyzed. Most of the identified operational taxonomic units (OTU) were related to Nitrobacter hamburgensis, Methylibium petroleiphilum, Rhodoblastus acidophilus, Bradyrhizobium, Cupriavidus metallidurans, Rubrivivax, Burkholderia, stappia and Thiobacillus thiophilus. OTUs related to Rubrivivax gelatinosus were specific for rice-rice soil. OTUs linked to Methylibium petroleiphilum were exclusively found in rice-rapeseed soil. Observed differences could be linked to differences in soil parameters such as SOC. We conclude that the long-term application of cropping systems alters underlying soil parameters, which in turn selects for distinct autotrophic communities.

The anomaly that Anopheles culicifacies (Diptera: Culicidae) species B is a major vector of malaria in Sri Lanka, but a non-vector in India, has been noted for several years. In 1999, a Y chromosome dimorphism associated with Plasmodium vivax infectivity within the Indian A. culicifacies species B suggested that this was itself a complex of two sibling species, B and E. A recent cytogenetic analysis shows the sympatric presence of these sibling species in Sri Lanka, a situation similar to that reported from nearby Rameshwaram Island, India. Species E, with a submetacentric Y chromosome, is a more effective vector of P. vivax than species B with an acrocentric Y chromosome. Larval karyotyping, however, is time-consuming and labour-intensive. Recently, the development of a PCR-RFLP assay distinguishing species B and E of A. culicifacies from India, based on differences in one region of the cytochrome oxidase subunit II (COII) gene, was reported. Here we show that whilst this diagnostic approach reveals polymorphism in Sri Lankan A. culicifacies, this variation is not correlated with Y chromosome karyotype. Hence this assay will not be useful for distinguishing species B and E in Sri Lanka. Further, we found no difference between the sequences of Sri Lankan specimens in any of three other regions (ITS2, D3 region of 28S rDNA, and guanylate cyclase intron) often used for species discrimination.

The susceptibility status of Anopheles funestus to insecticides remains largely unknown in most parts of Africa because of the difficulty in rearing field-caught mosquitoes of this malaria vector. Here we report the susceptibility status of the An. funestus population from Tororo district in Uganda and a preliminary characterisation of the putative resistance mechanisms involved.A new forced egg laying technique used in this study significantly increased the numbers of field-caught females laying eggs and generated more than 4000 F1 adults. WHO bioassays indicated that An. funestus in Tororo is resistant to pyrethroids (62% mortality after 1 h exposure to 0.75% permethrin and 28% mortality to 0.05% deltamethrin). Suspected DDT resistance was also observed with 82% mortality. However this population is fully susceptible to bendiocarb (carbamate), malathion (organophosphate) and dieldrin with 100% mortality observed after exposure to each of these insecticides. Sequencing of a fragment of the sodium channel gene containing the 1014 codon conferring pyrethroid/DDT resistance in An. gambiae did not detect the L1014F kdr mutation but a correlation between haplotypes and resistance phenotype was observed indicating that mutations in other exons may be conferring the knockdown resistance in this species. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA compared to a susceptible strain of Anopheles gambiae. RT-PCR further confirmed the involvement of P450s with a 12-fold over-expression of CYP6P9b in the Tororo population compared to the fully susceptible laboratory colony FANG.This study represents the first report of pyrethroid/DDT resistance in An. funestus from East Africa. With resistance already reported in southern and West Africa, this indicates that resistance in An. funestus may be more widespread than previously assumed and therefore this should be taken into account for the implementation and management of vector control programs in Africa.

Although Anopheles funestus is difficult to rear, it is crucial to analyse field populations of this malaria vector in order to successfully characterise mechanisms of insecticide resistance observed in this species in Africa. In this study we carried out a large-scale field collection and rearing of An. funestus from Mozambique in order to analyse its susceptibility status to insecticides and to broadly characterise the main resistance mechanisms involved in natural populations.3,000 F(1) adults were obtained through larval rearing. WHO susceptibility assays indicated a very high resistance to pyrethroids with no mortality recorded after 1 h 30 min exposure and less than 50% mortality at 3 h 30 min. Resistance to the carbamate, bendiocarb was also noted, with 70% mortality after 1h exposure. In contrast, no DDT resistance was observed, indicating that no kdr-type resistance was involved. The sequencing of the acetylcholinesterase gene indicated the absence of the G119S and F455W mutations associated with carbamate and organophosphate resistance. This could explain the absence of malathion resistance in this population. Both biochemical assays and quantitative PCR implicated up-regulated P450 genes in pyrethroid resistance, with GSTs playing a secondary role. The carbamate resistance observed in this population is probably conferred by the observed altered AChE with esterases also involved.The high level of pyrethroid resistance in this population despite the cessation of pyrethroid use for IRS in 1999 is a serious concern for resistance management strategies such as rotational use of insecticides. As DDT has now been re-introduced for IRS, susceptibility to DDT needs to be closely monitored to prevent the appearance and spread of resistance to this insecticide.

BACKGROUND: Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. METHODOLOGY/PRINCIPAL FINDINGS: We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. CONCLUSIONS/SIGNIFICANCE: The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control.

Insects exposed to pesticides undergo strong natural selection and have developed various adaptive mechanisms to survive. Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is receiving increasing attention because it threatens the sustainability of malaria vector control programs in sub-Saharan Africa. An understanding of the molecular mechanisms conferring pyrethroid resistance gives insight into the processes of evolution of adaptive traits and facilitates the development of simple monitoring tools and novel strategies to restore the efficacy of insecticides. For this purpose, it is essential to understand which mechanisms are important in wild mosquitoes. Here, our aim was to identify enzymes that may be important in metabolic resistance to pyrethroids by measuring gene expression for over 250 genes potentially involved in metabolic resistance in phenotyped individuals from a highly resistant, wild A. gambiae population from Ghana. A cytochrome P450, CYP6P3, was significantly overexpressed in the survivors, and we show that the translated enzyme metabolises both alpha-cyano and non-alpha-cyano pyrethroids. This is the first study to demonstrate the capacity of a P450 identified in wild A. gambiae to metabolise insecticides. The findings add to the understanding of the genetic basis of insecticide resistance in wild mosquito populations.

PurposeTo compare standard reliability metrics and gaze tracking (GT) metrics on the Humphrey field analyzer (HFA).DesignRetrospective cross-sectional study.MethodsThe study was performed at the VA Medical Center, San Diego, and included 494 glaucoma and glaucoma suspect patients who had an HFA 24-2 SITA Fast visual field (VF) performed in both eyes. Standard reliability metrics (fixation loss [FL], false-positive [FP], and false-negative [FN]) were compared to GT metrics (deviations of 1°-2° [M1], deviations of 3°-5° [M3], deviations >6° [M6], and tracking failure frequency [TFF]). The main outcome measures were Spearman rank-based correlation coefficient and area under the receiver operating characteristic (AUROC) curves between standard and GT reliability metrics.ResultsThe 95th percentile limits for GT metrics were 66.7% for M1, 67.5% for M3, 49.5% for M6, and 79.8% for TFF. There were statistically significant correlations between standard and GT reliability metrics using the 95th percentile as a binary cutoff for GT metrics. However, low Spearman correlation values and AUROC calculations suggest little clinical significance of the associations. FN increased as VF severity worsened (P < .001). M6 was lower in eyes with mild compared to moderate and advanced VF loss (P = .012).ConclusionsGT metrics do not have a clinically significant association with standard reliability metrics. Both FN and M6 are influenced by VF severity. Aggregate GT metrics do not aid in reliability assessment. These findings suggest that GT metrics may provide an alternative or complementary measure of VF reliability.

Introduction: Septic arthritis is a destructive form of acute arthritis that requires expeditious recognition. as delayed treatment yields significant morbidity and mortality.Case Report: A 40-year-old male presented to the emergency department with right elbow pain. Examination revealed tachycardia, swelling, redness, tenderness, and decreased range of motion of the right humeroulnar joint. Synovial fluid analysis was consistent with an inflammatory etiology, but blood and joint cultures ultimately revealed Haemophilus influenzae.Discussion: This case highlights the importance of trusting clinical findings over laboratory evidence in patients with suspected septic arthritis.