Highly sensitive wastewater surveillance of SARS-CoV-2 variants by targeted next-generation amplicon sequencing provides early warning of incursion in Victoria, Australia.

The future of the COVID pandemic and its public health and societal impact will be determined by the profile and spread of emerging variants and the timely identification and response to them. Wastewater surveillance of SARS-CoV-2 has been widely adopted in many countries across the globe and has played an important role in tracking infection levels and providing useful epidemiological information that cannot be adequately captured by clinical testing alone. However, novel variants can emerge rapidly, spread globally, and markedly alter the trajectory of the pandemic, as exemplified by the Delta and Omicron variants. Most mutations linked to the emergence of new SARS-CoV-2 variants are found within variable regions of the SARS-CoV-2 Spike protein. We have developed a duplex hemi-nested PCR method that, coupled with short amplicon sequencing, allows simultaneous typing of two of the most highly variable and informative regions of the Spike gene: the N-terminal domain and the receptor binding motif. Using this method in an operationalized public health program, we identified the first known incursion of Omicron BA.1 into Victoria, Australia and demonstrated how sensitive amplicon sequencing methods can be combined with wastewater surveillance as a relatively low-cost solution for early warning of variant incursion and spread.IMPORTANCEThis study offers a rapid, cost-effective, and sensitive approach for monitoring SARS-CoV-2 variants in wastewater. The method's flexibility permits timely modifications, enabling the integration of emerging variants and adaptations to evolving SARS-CoV-2 genetics. Of particular significance for low- and middle-income regions with limited surveillance capabilities, this technique can potentially be utilized to study a range of pathogens or viruses that possess diverse genetic sequences, similar to influenza.

Hookworms dynamically respond to loss of Type 2 immune pressure.

The impact of the host immune environment on parasite transcription and fitness is currently unknown. It is widely held that hookworm infections have an immunomodulatory impact on the host, but whether the converse is true remains unclear. Immunity against adult-stage hookworms is largely mediated by Type 2 immune responses driven by the transcription factor Signal Transducer and Activator of Transcription 6 (STAT6). This study investigated whether serial passage of the rodent hookworm Nippostrongylus brasiliensis in STAT6-deficient mice (STAT6 KO) caused changes in parasites over time. After adaptation to STAT6 KO hosts, N. brasiliensis increased their reproductive output, feeding capacity, energy content, and body size. Using an improved N. brasiliensis genome, we found that these physiological changes corresponded with a dramatic shift in the transcriptional landscape, including increased expression of gene pathways associated with egg production, but a decrease in genes encoding neuropeptides, proteases, SCP/TAPS proteins, and transthyretin-like proteins; the latter three categories have been repeatedly observed in hookworm excreted/secreted proteins (ESPs) implicated in immunosuppression. Although transcriptional changes started to appear in the first generation of passage in STAT6 KO hosts for both immature and mature adult stages, downregulation of the genes putatively involved in immunosuppression was only observed after multiple generations in this immunodeficient environment. When STAT6 KO-adapted N. brasiliensis were reintroduced to a naive WT host after up to 26 generations, this progressive change in host-adaptation corresponded to increased production of inflammatory cytokines by the WT host. Surprisingly, however, this single exposure of STAT6 KO-adapted N. brasiliensis to WT hosts resulted in worms that were morphologically and transcriptionally indistinguishable from WT-adapted parasites. This work uncovers remarkable plasticity in the ability of hookworms to adapt to their hosts, which may present a general feature of parasitic nematodes.

Use of adrenaline to manage suspected anaphylaxis following COVID-19 vaccination: An Australian retrospective cohort study.

BACKGROUND: The rate of anaphylaxis following COVID-19 vaccinations is estimated to be 2-11 cases per million doses administered. However, adrenaline is occasionally used in individuals who are later diagnosed with immunisation stress-related responses, as their initial presenting signs and symptoms can appear similar to that of anaphylaxis. This study aims to describe the clinical profile of individuals who had received adrenaline following a COVID-19 vaccine and their subsequent revaccination outcomes. METHODS: We examined notifications of cases who had received adrenaline following a COVID-19 vaccine in New South Wales, Australia. The cases were classified into Brighton Collaboration Case Definition (BCCD) for anaphylaxis, their clinical presentation, management and subsequent revaccination outcomes were compared. RESULTS: From 22 February 2021 to 30 September 2021, there were 222 cases where adrenaline was administered. Of these, 32 (14 %) fulfilled Level 1 BCCD, 59 (27%) Level 2, 2 (1%) Level 3, 97 (44%) Level 4 and 32 (14 %) Level 5. The most commonly reported symptoms were sensation of throat closure (n = 116, 52%), difficulty breathing (n = 82, 37%) and nausea (n = 55, 25 %). Of the 176 (79%) individuals who proceeded to further vaccination, 89 (51%) received the same vaccine formulation and only 14 (8%) experienced another allergic adverse event with 9 (5%) receiving adrenaline. CONCLUSION: Less than one in five individuals who received adrenaline met Level 1 BCCD criteria for anaphylaxis. Many reactions that were treated with adrenaline had little to no diagnostic certainty of anaphylaxis and in such cases repeat vaccination had a high likelihood of being tolerated. Increased awareness and education on objective signs and symptoms of anaphylaxis is required to ensure appropriate use of adrenaline.

Transcriptomic analysis of albendazole resistance in human diarrheal parasite Giardia duodenalis.

Benzimidazole-2-carbamates (BZ, e.g., albendazole; ALB), which bind ß-tubulin to disrupt microtubule polymerization, are one of two primary compound classes used to treat giardiasis. In most parasitic nematodes and fungi, BZ-resistance is caused by ß-tubulin mutations and its molecular mode of action (MOA) is well studied. In contrast, in Giardia duodenalis BZ MOA or resistance is less well understood, may involve target-specific and broader impacts including cellular damage and oxidative stress, and its underlying cause is not clearly determined. Previously, we identified acquisition of a single nucleotide polymorphism, E198K, in ß-tubulin in ALB-resistant (ALB-R) G. duodenalis WB-1B relative to ALB-sensitive (ALB-S) parental controls. E198K is linked to BZ-resistance in fungi and its allelic frequency correlated with the magnitude of BZ-resistance in G. duodenalis WB-1B. Here, we undertook detailed transcriptomic comparisons of these ALB-S and ALB-R G. duodenalis WB-1B cultures. The primary transcriptional changes with ALB-R in G. duodenalis WB-1B indicated increased protein degradation and turnover, and up-regulation of tubulin, and related genes, associated with the adhesive disc and basal bodies. These findings are consistent with previous observations noting focused disintegration of the disc and associated structures in Giardia duodenalis upon ALB exposure. We also saw transcriptional changes with ALB-R in G. duodenalis WB-1B consistent with prior observations of a shift from glycolysis to arginine metabolism for ATP production and possible changes to aspects of the vesicular trafficking system that require further investigation. Finally, we saw mixed transcriptional changes associated with DNA repair and oxidative stress responses in the G. duodenalis WB-1B line. These changes may be indicative of a role for H2O2 degradation in ALB-R, as has been observed in other G. duodenalis cell cultures. However, they were below the transcriptional fold-change threshold (log2FC > 1) typically employed in transcriptomic analyses and appear to be contradicted in ALB-R G. duodenalis WB-1B by down-regulation of the NAD scavenging and conversion pathways required to support these stress pathways and up-regulation of many highly oxidation sensitive iron-sulphur (FeS) cluster based metabolic enzymes.

Multi-Organ Dysfunction in Cerebral Palsy.

Cerebral Palsy (CP) describes a heterogenous group of non-progressive disorders of posture or movement, causing activity limitation, due to a lesion in the developing brain. CP is an umbrella term for a heterogenous condition and is, therefore, descriptive rather than a diagnosis. Each case requires detailed consideration of etiology. Our understanding of the underlying cause of CP has developed significantly, with areas such as inflammation, epigenetics and genetic susceptibility to subsequent insults providing new insights. Alongside this, there has been increasing recognition of the multi-organ dysfunction (MOD) associated with CP, in particular in children with higher levels of motor impairment. Therefore, CP should not be seen as an unchanging disorder caused by a solitary insult but rather, as a condition which evolves over time. Assessment of multi-organ function may help to prevent complications in later childhood or adulthood. It may also contribute to an improved understanding of the etiology and thus may have an implication in prevention, interventional methods and therapies. MOD in CP has not yet been quantified and a scoring system may prove useful in allowing advanced clinical planning and follow-up of children with CP. Additionally, several biomarkers hold promise in assisting with long-term monitoring. Clinicians should be aware of the multi-system complications that are associated with CP and which may present significant diagnostic challenges given that many children with CP communicate non-verbally. A step-wise, logical, multi-system approach is required to ensure that the best care is provided to these children. This review summarizes multi-organ dysfunction in children with CP whilst highlighting emerging research and gaps in our knowledge. We identify some potential organ-specific biomarkers which may prove useful in developing guidelines for follow-up and management of these children throughout their lifespan.

In vitro selection of Giardia duodenalis for Albendazole resistance identifies a ß-tubulin mutation at amino acid E198K.

Benzimidazole-2-carbamate (BZ) compounds, including Albendazole (Alb), are one of just two drug classes approved to treat the gastrointestinal protist Giardia duodenalis. Benzimidazoles bind to the tubulin dimer interface overlapping the colchicine binding site (CBS) of ß-tubulin, thereby inhibiting microtubule polymerisation and disrupting microtubule networks. These BZ compounds are widely used as anthelmintic, anti-fungal and anti-giardial drugs. However, in helminths and fungi, BZ-resistance is widespread and caused by specific point mutations primarily occurring at F167, E198 and F200 in ß-tubulin isoform 1. BZ-resistance in Giardia is reported clinically and readily generated in vitro, with significant implications for Giardia control. In Giardia, BZ mode of action (MOA) and resistance mechanisms are presumed but not proven, and no mutations in ß-tubulin have been reported in association with Alb resistance (AlbR). Herein, we undertook detailed in vitro drug-susceptibility screens of 13 BZ compounds and 7 Alb structural analogues in isogenic G. duodenalis isolates selected for AlbR and podophyllotoxin, another ß-tubulin inhibitor, as well as explored cross-resistance to structurally unrelated, metronidazole (Mtz). AlbR lines exhibited co-resistance to many structural variants in the BZ-pharmacophore, and cross-resistance to podophyllotoxin. AlbR lines were not cross-resistant to Mtz, but MtzR lines had enhanced survival in Alb. Lastly, Alb analogues with longer thioether substituents had decreased potency against our AlbR lines. In silico modelling indicated the Alb-ß-tubulin interaction in Giardia partially overlaps the CBS and corresponds to residues associated with BZ-resistance in helminths and fungi (F167, E198, F200). Sequencing of Giardia ß-tubulin identified a single nucleotide polymorphism resulting in a mutation from glutamic acid to lysine at amino acid 198 (E198K). To our knowledge, this is the first ß-tubulin mutation reported for protistan BZ-resistance. This study provides insight into BZ mode of action and resistance in Giardia, and presents a potential avenue for a genetic test for clinically resistance isolates.

Passive Sampling of SARS-CoV-2 for Wastewater Surveillance.

The shedding of pathogens by infected humans enables the use of sewage monitoring to conduct wastewater-based epidemiology (WBE). Although most WBE studies use data from large sewage treatment plants, timely data from smaller catchments are needed for targeted public health action. Traditional sampling methods, like autosamplers or grab sampling, are not conducive to quick ad hoc deployments and high-resolution monitoring at these smaller scales. This study develops and validates a cheap and easily deployable passive sampler unit, made from readily available consumables, with relevance to the COVID-19 pandemic but with broader use for WBE. We provide the first evidence that passive samplers can be used to detect SARS-CoV-2 in wastewater from populations with low prevalence of active COVID-19 infections (0.034 to 0.34 per 10,000), demonstrating their ability for early detection of infections at three different scales (lot, suburb, and city). A side by side evaluation of passive samplers (n = 245) and traditionally collected wastewater samples (n = 183) verified that the passive samplers were sensitive at detecting SARS-CoV-2 in wastewater. On all 33 days where we directly compared traditional and passive sampling techniques, at least one passive sampler was positive when the average SARS-CoV-2 concentration in the wastewater equaled or exceeded the quantification limit of 1.8 gene copies per mL (n = 7). Moreover, on 13 occasions where wastewater SARS-CoV-2 concentrations were less than 1.8 gene copies per mL, one or more passive samplers were positive. Finally, there was a statistically significant (p < 0.001) positive relationship between the concentrations of SARS-CoV-2 in wastewater and the levels found on the passive samplers, indicating that with further evaluation, these devices could yield semi-quantitative results in the future. Passive samplers have the potential for wide use in WBE with attractive feasibility attributes of cost, ease of deployment at small-scale locations, and continuous sampling of the wastewater. Further research will focus on the optimization of laboratory methods including elution and extraction and continued parallel deployment and evaluations in a variety of settings to inform optimal use in wastewater surveillance.

First report of anatoxin-a producing cyanobacteria in Australia illustrates need to regularly up-date monitoring strategies in a shifting global distribution.

Routine monitoring of toxic cyanobacteria depends on up-to-date epidemiological information about their distribution. In Australia, anatoxin producing cyanobacteria are not regularly tested for and thought to be rare if not absent from the continent. Our study investigated the presence of anatoxin-a (ATX-a) producing cyanobacteria in surface water samples (n = 226 from 67 sampling locations) collected from 2010 to 2017 across the state of Victoria, Australia. We (1) detected the presence and distribution of anaC (anatoxin-a synthetase C) gene sequences previously associated with various cyanobacteria, including Cuspidothrix issatschenkoi, Aphanizomenon sp., D. circinale, Anabaena sp., and Oscillatoria sp., from 31 sampling locations, and (2) determined the concentration of ATX-a in samples tested using ELISA, in two instances detected at >4 µg · L-1. These data present the first confirmation of ATX-a producers in Australia. Our study indicates that ATX-a should be included in regular testing of cyanobacterial blooms in Australia and highlights the importance of regular investigation of the distributions of toxic cyanobacteria worldwide, particularly amid the known expanding distribution of many cyanobacterial taxa in a period of increased eutrophication and rising surface water temperatures.

The last 2 years of life for children with severe physical disability: Observations from a tertiary paediatric centre.

AIM: To describe the clinical course for children with severe physical disability (SPD) in the 2 years prior to their death and to identify whether these children had palliative care involvement and advance care planning prior to death. To investigate whether there is a difference between children with progressive (PSPD) and non-progressive (NPSPD) aetiologies of SPD. METHODS: A retrospective cohort analysis of 48 children with SPD who died between 1 January 2013 and 1 January 2015 at The Royal Children's Hospital, Melbourne. Clinical charts were reviewed to collect data about the type of SPD, frequency and duration of hospital admissions, duration of palliative care involvement (if any) and presence of an advance care plan. RESULTS: The majority of children were admitted in the 6 months before their death, and over a third were admitted to the intensive care unit. There was a significant increase in the frequency of hospital admissions as the study cohort approached death (P = 0.003). The majority of children with SPD were offered a referral to a palliative care service, with referrals more likely in children with PSPD (90%) compared to children with NPSPD (57%). While approximately 60% of children in each cohort had an advance care plan, there was a trend towards this being formalised earlier in children with PSPD (P = 0.09). CONCLUSION: The increase in hospital admissions prior to death in children with SPD suggests an opportunity for greater consistency in offering advanced care planning and palliative care, especially to those with NPSPD.

Trihexyphenidyl for dystonia in cerebral palsy.

BACKGROUND: Cerebral palsy occurs in up to 2.1 of every 1000 live births and encompasses a range of motor problems and movement disorders. One commonly occurring movement disorder amongst those with cerebral palsy is dystonia: sustained or intermittent involuntary muscle spasms and contractions that cause twisting, repetitive movements and abnormal postures. The involuntary contractions are often very painful and distressing and cause significant limitations to activity and participation.Oral medications are often the first line of medical treatment for dystonia. Trihexyphenidyl is one such medication that clinicians often use to treat dystonia in people with cerebral palsy. OBJECTIVES: To assess the effects of trihexyphenidyl in people with dystonic cerebral palsy, according to the World Health Organization's (WHO) International Classification of Functioning, Disability and Health (ICF) domains of impairment, activity and participation. We also assessed the type and incidence of adverse effects in people taking the drug. SEARCH METHODS: We searched CENTRAL, MEDLINE, Embase, eight other databases and two trials registers in May 2017, and we checked reference lists and citations to identify additional studies. SELECTION CRITERIA: We included randomised controlled trials comparing oral trihexyphenidyl versus placebo for dystonia in cerebral palsy. We included studies in children and adults of any age with dystonic cerebral palsy, either in isolation or with the associated movement disorders of spasticity, ataxia, chorea, athetosis and/or hypotonia. We included studies regardless of whether or not the study authors specified the method used to diagnose dystonia in their study population. Primary outcomes were change in dystonia and adverse effects. Secondary outcomes were: activity, including mobility and upper limb function; participation in activities of daily living; pain; and quality of life. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. MAIN RESULTS: We identified one study, which was set in Australia, that met the inclusion criteria. This was a randomised, double-blind, placebo-controlled, cross-over trial in 16 children (10 boys and 6 girls) with predominant dystonic cerebral palsy and a mean age of 9 years (standard deviation 4.3 years, range 2 to 17 years). We considered the trial to be at low risk of selection, performance, detection, attrition, reporting and other sources of bias. We rated the GRADE quality of the evidence as low.We found no difference in mean follow-up scores for change in dystonia as measured by the Barry Albright Dystonia Scale (BADS), which assesses eight body regions for dystonia on a 5-point scale (0 = none to 4 = severe), resulting in a total score of 0 to 32. The BADS score was 2.67 points higher (95% confidence interval (CI) -2.55 to 7.90; low-quality evidence), that is, worse dystonia, in the treated group. Trihexyphenidyl may be associated with an increased risk of adverse effects (risk ratio 2.54, 95% CI 1.38 to 4.67; low-quality evidence).There was no difference in mean follow-up scores for upper limb function as measured by the Quality of Upper Extremity Skills Test, which has four domains that collectively assess 36 items (each scored 1 or 2) and produces a total score of 0 to 100. The score in the treated group was 4.62 points lower (95% CI -10.98 to 20.22; low-quality evidence), corresponding to worse function, than in the control group. We found low-quality evidence for improved participation (as represented by higher scores) in the treated group in activities of daily living, as measured by three tools: 18.86 points higher (95% CI 5.68 to 32.03) for the Goal Attainment Scale (up to five functional goals scored on 5-point scale (-2 = much less than expected to +2 = much more than expected)), 2.91 points higher (95% CI 1.01 to 4.82) for the satisfaction subscale of the Canadian Occupational Performance Measure (COPM; satisfaction with performance in up to five problem areas scored on a 10-point scale (1 = not satisfied at all to 10 = extremely satisfied)), and 2.24 points higher (95% CI 0.64 to 3.84) for performance subscale of the COPM (performance in up to five problem areas scored on a 10-point scale (1 = not able to do to; 10 = able to do extremely well)).The study did not report on pain or quality of life. AUTHORS' CONCLUSIONS: At present, there is insufficient evidence regarding the effectiveness of trihexyphenidyl for people with cerebral palsy for the outcomes of: change in dystonia, adverse effects, increased upper limb function and improved participation in activities of daily living. The study did not measure pain or quality of life. There is a need for larger randomised, controlled, multicentre trials that also examine the effect on pain and quality of life in order to determine the effectiveness of trihexyphenidyl for people with cerebral palsy.

Differential protein expression and post-translational modifications in metronidazole-resistant Giardia duodenalis.

Background: Metronidazole (Mtz) is the frontline drug treatment for multiple anaerobic pathogens, including the gastrointestinal protist, Giardia duodenalis. However, treatment failure is common and linked to in vivo drug resistance. In Giardia, in vitro drug-resistant lines allow controlled experimental interrogation of resistance mechanisms in isogenic cultures. However, resistance-associated changes are inconsistent between lines, phenotypic data are incomplete, and resistance is rarely genetically fixed, highlighted by reversion to sensitivity after drug selection ceases or via passage through the life cycle. Comprehensive quantitative approaches are required to resolve isolate variability, fully define Mtz resistance phenotypes, and explore the role of post-translational modifications therein. Findings: We performed quantitative proteomics to describe differentially expressed proteins in 3 seminal Mtz-resistant lines compared to their isogenic, Mtz-susceptible, parental line. We also probed changes in post-translational modifications including protein acetylation, methylation, ubiquitination, and phosphorylation via immunoblotting. We quantified more than 1,000 proteins in each genotype, recording substantial genotypic variation in differentially expressed proteins between isotypes. Our data confirm substantial changes in the antioxidant network, glycolysis, and electron transport and indicate links between protein acetylation and Mtz resistance, including cross-resistance to deacetylase inhibitor trichostatin A in Mtz-resistant lines. Finally, we performed the first controlled, longitudinal study of Mtz resistance stability, monitoring lines after cessation of drug selection, revealing isolate-dependent phenotypic plasticity. Conclusions: Our data demonstrate understanding that Mtz resistance must be broadened to post-transcriptional and post-translational responses and that Mtz resistance is polygenic, driven by isolate-dependent variation, and is correlated with changes in protein acetylation networks.

An improved method for PCR-based detection and routine monitoring of geosmin-producing cyanobacterial blooms.

Production of taste and odour (T/O) compounds, principally geosmin, by complex cyanobacterial blooms is a major water quality issue globally. Control of these cyanobacteria imposes a significant cost on water producing and dependent industries, and requires routine monitoring and management. Classic monitoring methods, including microscopy and direct chemical analysis, lack sensitivity, are laborious, expensive or cannot reliably identify the source of geosmin production. Polymerase Chain Reaction (PCR) based tools targeting the geosmin synthase gene (geoA) provide a novel tool for routine monitoring. However, geoA is variable at the nucleotide level and potential geosmin producers represent a broad taxonomic distribution, such that multiple PCR primers with distinct amplification protocols are needed to target all potential sources of this important T/O compound. Development of novel primers is hindered by a lack of sequence data and limited field and laboratory data on geosmin producers prevents prioritizing taxa for PCR testing. Here we performed a genetic screen of 253 bloom samples from Victoria, Australia using each existing PCR protocol targeting geoA. We detected Dolichospermum ucrainicum as the major geosmin producer (87% of sequenced samples) along with 3 unknown geoA sequence types. Using these data, we designed a novel, short amplicon, PCR protocol utilising a single standardised primer pair, capable of amplifying all geoA positive samples in our study, as well as a Nostoc punctiforme positive control. This single protocol geoA PCR can further be tested on other geosmin producers and will simplify routine monitoring of T/O producing cyanobacteria.

Management of elderly patients with glioblastoma-multiforme-a systematic review.

The management of elderly patients with glioblastoma-multiforme (GBM) remains poorly defined with many experts in the past advocating best supportive care, in view of limited evidence on efficacy of more aggressive treatment protocols. There is randomised evidence (NORDIC and NA-O8 studies) to support the use of surgery followed by adjuvant monotherapy with either radiotherapy (RT) using hypofractionated regimes (e.g. 36 Gy in 6 fractions OR 40 Gy in 15 fractions) or chemotherapy with temozolomide (TMZ) in patients expressing methylation of promoter for O6-methylguanine-DNA methyltransferase enzyme. However, the role of combined-modality therapy involving the use of combined RT and TMZ protocols has remained controversial with data from the EORTC (European Organisation for Research and Treatment of Cancer)-NCIC (National Cancer Institute of Canada) studies indicating that patients more than 65 years of age may not benefit significantly from combining standard RT fractionation using 60 Gy in 30 fractions with concurrent and adjuvant TMZ. More recently, randomised data has emerged on combining hypofractionated RT with concurrent and adjuvant TMZ. We provide a comprehensive review of literature with the aim of defining an evidence-based algorithm for management of elderly glioblastoma-multiforme population.

Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines.

Giardia duodenalis is an intestinal parasite that causes 200-300 million episodes of diarrhoea annually. Metronidazole (Mtz) is a front-line anti-giardial, but treatment failure is common and clinical resistance has been demonstrated. Mtz is thought to be activated within the parasite by oxidoreductase enzymes, and to kill by causing oxidative damage. In G. duodenalis, Mtz resistance involves active and passive mechanisms. Relatively low activity of iron-sulfur binding proteins, namely pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxins, and nitroreductase-1, enable resistant cells to passively avoid Mtz activation. Additionally, low expression of oxygen-detoxification enzymes can allow passive (non-enzymatic) Mtz detoxification via futile redox cycling. In contrast, active resistance mechanisms include complete enzymatic detoxification of the pro-drug by nitroreductase-2 and enhanced repair of oxidized biomolecules via thioredoxin-dependent antioxidant enzymes. Molecular resistance mechanisms may be largely founded on reversible transcriptional changes, as some resistant lines revert to drug sensitivity during drug-free culture in vitro, or passage through the life cycle. To comprehensively characterize these changes, we undertook strand-specific RNA sequencing of three laboratory-derived Mtz-resistant lines, 106-2ID10, 713-M3, and WB-M3, and compared transcription relative to their susceptible parents. Common up-regulated genes encoded variant-specific surface proteins (VSPs), a high cysteine membrane protein, calcium and zinc channels, a Mad-2 cell cycle regulator and a putative fatty acid α-oxidase. Down-regulated genes included nitroreductase-1, putative chromate and quinone reductases, and numerous genes that act proximal to PFOR. Transcriptional changes in 106-2ID10 diverged from those in 713-r and WB-r (r ≤ 0.2), which were more similar to each other (r = 0.47). In 106-2ID10, a nonsense mutation in nitroreductase-1 transcripts could enhance passive resistance whereas increased transcription of nitroreductase-2, and a MATE transmembrane pump system, suggest active drug detoxification and efflux, respectively. By contrast, transcriptional changes in 713-M3 and WB-M3 indicated a higher oxidative stress load, attributed to Mtz- and oxygen-derived radicals, respectively. Quantitative comparisons of orthologous gene transcription between Mtz-resistant G. duodenalis and Trichomonas vaginalis, a closely related parasite, revealed changes in transcripts encoding peroxidases, heat shock proteins, and FMN-binding oxidoreductases, as prominent correlates of resistance. This work provides deep insight into Mtz-resistant G. duodenalis, and illuminates resistance-associated features across parasitic species.

Outcomes after scoliosis surgery for children with cerebral palsy: a systematic review.

AIM: This study aims (1) to evaluate and synthesize the evidence for the postoperative outcomes after scoliosis surgery for children with cerebral palsy (CP), and (2) to identify preoperative risk factors for adverse outcomes after surgery. METHOD: Medline, EMBASE, CINAHL, and PubMed were searched for relevant literature. Included studies were assessed for risk of bias using the Cochrane Effective Practice and Organisation of Care tool. Quality of evidence for overall function, quality of life (QoL), gross motor function, caregiver outcomes, deformity correction, and postoperative complications were assessed using GRADE (Grades of Recommendation, Assessment, Development and Evaluation). RESULTS: Fifty-one studies met inclusion criteria, including 35 case series designs. Risk of bias was high across all studies. On average good deformity correction was achieved, the trend appears positive for caregiver and QoL outcomes, but there was minimal to no change for gross motor or overall function. Inconsistent measurement limited synthesis. A mean overall complication rate of 38.1% (95% confidence interval 27.3-53.3) was found. The quality of evidence was very low across all functional outcomes. INTERPRETATION: Limited high-quality evidence exists for outcomes after scoliosis surgery in children with CP, a procedure associated with a moderately high complication rate. The intervention appears indicated for deformity correction, but currently there is insufficient evidence to make recommendations for this surgery as a way to also improve functional outcomes, caregiver outcomes, and quality of life.

Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis.

Understanding how parasites respond to stress can help to identify essential biological processes. Giardia duodenalis is a parasitic protist that infects the human gastrointestinal tract and causes 200 to 300 million cases of diarrhea annually. Metronidazole, a major antigiardial drug, is thought to cause oxidative damage within the infective trophozoite form. However, treatment efficacy is suboptimal, due partly to metronidazole-resistant infections. To elucidate conserved and stress-specific responses, we calibrated sublethal metronidazole, hydrogen peroxide, and thermal stresses to exert approximately equal pressure on trophozoite growth and compared transcriptional responses after 24 h of exposure. We identified 252 genes that were differentially transcribed in response to all three stressors, including glycolytic and DNA repair enzymes, a mitogen-activated protein (MAP) kinase, high-cysteine membrane proteins, flavin adenine dinucleotide (FAD) synthetase, and histone modification enzymes. Transcriptional responses appeared to diverge according to physiological or xenobiotic stress. Downregulation of the antioxidant system and α-giardins was observed only under metronidazole-induced stress, whereas upregulation of GARP-like transcription factors and their subordinate genes was observed in response to hydrogen peroxide and thermal stressors. Limited evidence was found in support of stress-specific response elements upstream of differentially transcribed genes; however, antisense derepression and differential regulation of RNA interference machinery suggest multiple epigenetic mechanisms of transcriptional control.

Time-Dependent Transcriptional Changes in Axenic Giardia duodenalis Trophozoites.

Giardia duodenalis is the most common gastrointestinal protozoan parasite of humans and a significant contributor to the global burden of both diarrheal disease and post-infectious chronic disorders. Although G. duodenalis can be cultured axenically, significant gaps exist in our understanding of the molecular biology and metabolism of this pathogen. The present study employed RNA sequencing to characterize the mRNA transcriptome of G. duodenalis trophozoites in axenic culture, at log (48 h of growth), stationary (60 h), and declining (96 h) growth phases. Using ~400-times coverage of the transcriptome, we identified 754 differentially transcribed genes (DTGs), mainly representing two large DTG groups: 438 that were down-regulated in the declining phase relative to log and stationary phases, and 281 that were up-regulated. Differential transcription of prominent antioxidant and glycolytic enzymes implicated oxygen tension as a key factor influencing the transcriptional program of axenic trophozoites. Systematic bioinformatic characterization of numerous DTGs encoding hypothetical proteins of unknown function was achieved using structural homology searching. This powerful approach greatly informed the differential transcription analysis and revealed putative novel antioxidant-coding genes, and the presence of a near-complete two-component-like signaling system that may link cytosolic redox or metabolite sensing to the observed transcriptional changes. Motif searching applied to promoter regions of the two large DTG groups identified different putative transcription factor-binding motifs that may underpin global transcriptional regulation. This study provides new insights into the drivers and potential mediators of transcriptional variation in axenic G. duodenalis and provides context for static transcriptional studies.

Daytime intrusive thoughts and subjective insomnia symptoms.

Insomnia is increasingly recognised as a 24h complaint that is associated with an increased risk of mood and anxiety disorders. However, the effects of insomnia symptoms on maladaptive daytime patterns of thinking are poorly understood. We examined the relationship between subjective insomnia symptoms, attentional control and negative thought intrusions during daytime in a large sample of undergraduates experiencing poor sleep. A total of 109 participants completed self-report measures of sleep quality, current sleepiness, anxiety and attentional control. A behavioural measure of intrusive thought required participants to control their attention during two focus periods separated by a 5min period of self-referential worry. Thought intrusions were sampled throughout the pre- and post-worry periods. Perceived insomnia severity was associated with the reduced ability to focus attention and uniquely associated with increased negative thought intrusions in the pre-worry period. These results support suggestions that acute episodes of poor sleep can dysregulate key networks involved in attentional control and emotion regulation, and that promote negative cognitive activity.