How etiological beliefs contribute to the structure of depression symptom networks.

OBJECTIVES: Each person possesses a unique view surrounding depressive symptomology and etiology that is shaped by idiosyncratic experiences. However, the influence that subjective etiological beliefs regarding a person's depressive symptoms have on actual symptom presentation and organization is seldom considered. METHODS: The current study employed network analytic techniques to examine how subjective views surrounding the cause of depressive symptoms altered actual symptom presentation networks. Additionally, the interaction between depressive symptoms and various etiological beliefs was examined. RESULTS: The results revealed that characterological beliefs, representing the idea that depression is caused by an internal sense of self, are strongly connected to a negative view of self, as well as a saddened mood. Additionally, the characterological beliefs node exhibited the greatest node predictability in its respective network, as well as in an omnibus network consisting of all depression symptoms and potential etiological beliefs. Whereas an achievement-based view of depression has a strong connection with concentration difficulties, a physical view of depression tends to form strong connections with physically based depressive symptoms. CONCLUSION: Subjective views regarding the cause of depression have the potential to influence symptom presentation and organization within a network, which may influence a person's willingness to engage in treatment or specific treatment preferences.

Impact of a Purified Microbiome Therapeutic on Abundance of Antimicrobial Resistance Genes in Patients With Recurrent Clostridioides difficile Infection.

BACKGROUND: The gastrointestinal microbiota is an important line of defense against colonization with antimicrobial resistant (AR) bacteria. In this post hoc analysis of the phase 3 ECOSPOR III trial, we assessed impact of a microbiota-based oral therapeutic (fecal microbiota spores, live; VOWST Oral Spores [VOS], formerly SER-109]; Seres Therapeutics) compared with placebo, on AR gene (ARG) abundance in patients with recurrent Clostridioides difficile infection (rCDI). METHODS: Adults with rCDI were randomized to receive VOS or placebo orally for 3 days following standard-of-care antibiotics. ARG and taxonomic profiles were generated using whole metagenomic sequencing of stool at baseline and weeks 1, 2, 8, and 24 posttreatment. RESULTS: Baseline (n = 151) and serial posttreatment stool samples collected through 24 weeks (total N = 472) from 182 patients (59.9% female; mean age: 65.5 years) in ECOSPOR III as well as 68 stool samples obtained at a single time point from a healthy cohort were analyzed. Baseline ARG abundance was similar between arms and significantly elevated versus the healthy cohort. By week 1, there was a greater decline in ARG abundance in VOS versus placebo (P = .003) in association with marked decline of Proteobacteria and repletion of spore-forming Firmicutes, as compared with baseline. We observed abundance of Proteobacteria and non-spore-forming Firmicutes were associated with ARG abundance, while spore-forming Firmicutes abundance was negatively associated. CONCLUSIONS: This proof-of-concept analysis suggests that microbiome remodeling with Firmicutes spores may be a potential novel approach to reduce ARG colonization in the gastrointestinal tract.

Cohesin contributes to transcriptional repression of stage-specific genes in the human malaria parasite.

The complex life cycle of the human malaria parasite, Plasmodium falciparum, is driven by specific transcriptional programs, but it is unclear how most genes are activated or silenced at specific times. There is an association between transcription and spatial organization; however, the molecular mechanisms behind genome organization are unclear. While P. falciparum lacks key genome-organizing proteins found in metazoans, it has all core components of the cohesin complex. To investigate the role of cohesin in P. falciparum, we functionally characterize the cohesin subunit Structural Maintenance of Chromosomes protein 3 (SMC3). SMC3 knockdown during early stages of the intraerythrocytic developmental cycle (IDC) upregulates a subset of genes involved in erythrocyte egress and invasion, which are normally expressed at later stages. ChIP-seq analyses reveal that during the IDC, SMC3 enrichment at the promoter regions of these genes inversely correlates with gene expression and chromatin accessibility. These data suggest that SMC3 binding contributes to the repression of specific genes until their appropriate time of expression, revealing a new mode of stage-specific gene repression in P. falciparum.

Patient-Perceived Outcomes of Recovery After Trigger Digit Release.

PURPOSE: Trigger finger release (TFR) is one of the most commonly performed hand surgeries; nevertheless, the time until patients subjectively feel recovered has not been well documented. The limited literature on patient perceptions of recovery after any type of surgery has described that patients and surgeons may have differing views on the time until full recovery. Our primary study question was to determine how long it takes for patients to subjectively feel fully recovered after TFR. METHODS: In this prospective study, patients who underwent isolated TFR completed questionnaires before surgery and at multiple time points following surgery until they reported full recovery. Patients completed visual analog scale (VAS) pain scores and QuickDASH (Disabilities of the Arm, Shoulder, and Hand) and were asked if they felt fully recovered at 4 weeks, 6 weeks, and 3, 6, 9, and 12 months. RESULTS: The average time to self-reported full recovery was 6.2 months (SD 2.6), and the median time to self-reported full recovery was 6 months (IQR 4 months). At 12 months, four out of 50 patients (8%) did not feel fully recovered. QuickDASH and VAS pain scores improved significantly from preoperative assessment to final follow-up. All patients reported improvement in both VAS pain scores and QuickDASH scores greater than the minimal clinically important difference between 6 weeks and 3 months after surgery. Higher preoperative VAS and QuickDASH scores were associated with failure to fully recover by 12 months after surgery. CONCLUSIONS: The length of time after surgery until patients felt fully recovered after isolated TFR is longer than the senior authors' expectations. This suggests that patients and surgeons may consider distinctly different parameters when discussing recovery. Surgeons should be aware of this discrepancy when discussing recovery after surgery. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognostic II.

Individuals fearing positivity do not perceive positive affect treatments as strong fits: A novel experimental finding and replication.

BACKGROUND AND OBJECTIVES: Positive affect treatments, which hold great promise to connect with those who are otherwise resistant to depression treatments, attempt to upregulate positive emotions. These treatments have potential advantages over standard therapies because they target cross-diagnostic core symptoms (e.g., anhedonia) that may respond better to interventions aimed at increased positivity. However, the extent to which these treatments are a perceived fit by individuals for whom they were developed (i.e., individuals who are afraid of, avoid, or experience less positivity) is unclear. METHODS: We conducted two independent studies utilizing a cross-sectional, experimental design to examine perceived treatment fit. Participants (Study 1: N = 416; Study 2: N = 321) read counterbalanced treatment descriptions of (1) positive affect treatment and (2) psychodynamic psychotherapy and answered questions regarding perceived treatment fit, effectiveness, and preference of the two treatments. RESULTS: Our findings suggest that individuals fearful of happiness perceived a prospective depression treatment specifically targeting positivity as a poorer fit, demonstrating an opposite pattern to the overall samples' treatment preference in both studies. Thus, as predicted by Reward Devaluation Theory, those fearing positivity exhibited avoidance behaviors for treatments that are to an extent designed, and might otherwise be most effective, for them. LIMITATIONS: The current study utilized a college student sample. CONCLUSION: These empirical findings may ultimately inform psychoeducation of why positive affect treatments, which are in direct contrast with clients' preferences, may be the very treatments they need the most.

SER-109: An Oral Investigational Microbiome Therapeutic for Patients with Recurrent Clostridioides difficile Infection (rCDI).

Clostridioides difficile infection (CDI) is classified as an urgent health threat by the Centers for Disease Control and Prevention (CDC), and affects nearly 500,000 Americans annually. Approximately 20−25% of patients with a primary infection experience a recurrence, and the risk of recurrence increases with subsequent episodes to greater than 40%. The leading risk factor for CDI is broad-spectrum antibiotics, which leads to a loss of microbial diversity and impaired colonization resistance. Current FDA-approved CDI treatment strategies target toxin or toxin-producing bacteria, but do not address microbiome disruption, which is key to the pathogenesis of recurrent CDI. Fecal microbiota transplantation (FMT) reduces the risk of recurrent CDI through the restoration of microbial diversity. However, FDA safety alerts describing hospitalizations and deaths related to pathogen transmission have raised safety concerns with the use of unregulated and unstandardized donor-derived products. SER-109 is an investigational oral microbiome therapeutic composed of purified spore-forming Firmicutes. SER-109 was superior to a placebo in reducing CDI recurrence at Week 8 (12% vs. 40%, respectively; p < 0.001) in adults with a history of recurrent CDI with a favorable observed safety profile. Here, we discuss the role of the microbiome in CDI pathogenesis and the clinical development of SER-109, including its rigorous manufacturing process, which mitigates the risk of pathogen transmission. Additionally, we discuss compositional and functional changes in the gastrointestinal microbiome in patients with recurrent CDI following treatment with SER-109 that are critical to a sustained clinical response.

SER-109, an Oral Microbiome Therapy for Recurrent Clostridioides difficile Infection.

BACKGROUND: Current therapies for recurrent Clostridioides difficile infection do not address the disrupted microbiome, which supports C. difficile spore germination into toxin-producing bacteria. SER-109 is an investigational microbiome therapeutic composed of purified Firmicutes spores for the treatment of recurrent C. difficile infection. METHODS: We conducted a phase 3, double-blind, randomized, placebo-controlled trial in which patients who had had three or more episodes of C. difficile infection (inclusive of the qualifying acute episode) received SER-109 or placebo (four capsules daily for 3 days) after standard-of-care antibiotic treatment. The primary efficacy objective was to show superiority of SER-109 as compared with placebo in reducing the risk of C. difficile infection recurrence up to 8 weeks after treatment. Diagnosis by toxin testing was performed at trial entry, and randomization was stratified according to age and antibiotic agent received. Analyses of safety, microbiome engraftment, and metabolites were also performed. RESULTS: Among the 281 patients screened, 182 were enrolled. The percentage of patients with recurrence of C. difficile infection was 12% in the SER-109 group and 40% in the placebo group (relative risk, 0.32; 95% confidence interval [CI], 0.18 to 0.58; P<0.001 for a relative risk of <1.0; P<0.001 for a relative risk of <0.833). SER-109 led to less frequent recurrence than placebo in analyses stratified according to age stratum (relative risk, 0.24 [95% CI, 0.07 to 0.78] for patients <65 years of age and 0.36 [95% CI, 0.18 to 0.72] for those ≥65 years) and antibiotic received (relative risk, 0.41 [95% CI, 0.22 to 0.79] with vancomycin and 0.09 [95% CI, 0.01 to 0.63] with fidaxomicin). Most adverse events were mild to moderate and were gastrointestinal in nature, with similar numbers in the two groups. SER-109 dose species were detected as early as week 1 and were associated with bile-acid profiles that are known to inhibit C. difficile spore germination. CONCLUSIONS: In patients with symptom resolution of C. difficile infection after treatment with standard-of-care antibiotics, oral administration of SER-109 was superior to placebo in reducing the risk of recurrent infection. The observed safety profile of SER-109 was similar to that of placebo. (Funded by Seres Therapeutics; ECOSPOR III ClinicalTrials.gov number, NCT03183128.).

Chromatin structure can introduce systematic biases in genome-wide analyses of Plasmodium falciparum.

Background: The maintenance, regulation, and dynamics of heterochromatin in the human malaria parasite, Plasmodium falciparum, has drawn increasing attention due to its regulatory role in mutually exclusive virulence gene expression and the silencing of key developmental regulators. The advent of genome-wide analyses such as chromatin-immunoprecipitation followed by sequencing (ChIP-seq) has been instrumental in understanding chromatin composition; however, even in model organisms, ChIP-seq experiments are susceptible to intrinsic experimental biases arising from underlying chromatin structure. Methods: We performed a control ChIP-seq experiment, re-analyzed previously published ChIP-seq datasets and compared different analysis approaches to characterize biases of genome-wide analyses in P. falciparum. Results: We found that heterochromatic regions in input control samples used for ChIP-seq normalization are systematically underrepresented in regard to sequencing coverage across the P. falciparum genome. This underrepresentation, in combination with a non-specific or inefficient immunoprecipitation, can lead to the identification of false enrichment and peaks across these regions. We observed that such biases can also be seen at background levels in specific and efficient ChIP-seq experiments. We further report on how different read mapping approaches can also skew sequencing coverage within highly similar subtelomeric regions and virulence gene families. To ameliorate these issues, we discuss orthogonal methods that can be used to characterize bona fide chromatin-associated proteins. Conclusions: Our results highlight the impact of chromatin structure on genome-wide analyses in the parasite and the need for caution when characterizing chromatin-associated proteins and features.

Functional Characterization of the m6A-Dependent Translational Modulator PfYTH.2 in the Human Malaria Parasite.

Posttranscriptional regulation of gene expression is central to the development and replication of the malaria parasite, Plasmodium falciparum, within its human host. The timely coordination of RNA maturation, homeostasis, and protein synthesis relies on the recruitment of specific RNA-binding proteins to their cognate target mRNAs. One possible mediator of such mRNA-protein interactions is the N6-methylation of adenosines (m6A), a prevalent mRNA modification of parasite mRNA transcripts. Here, we used RNA protein pulldowns, RNA modification mass spectrometry, and quantitative proteomics to identify two P. falciparum YTH domain proteins (PfYTH.1 and PfYTH.2) as m6A-binding proteins during parasite blood-stage development. Interaction proteomics revealed that PfYTH.2 associates with the translation machinery, including multiple subunits of the eukaryotic initiation factor 3 (eIF3) and poly(A)-binding proteins. Furthermore, knock sideways of PfYTH.2 coupled with ribosome profiling showed that this m6A reader is essential for parasite survival and is a repressor of mRNA translation. Together, these data reveal an important missing link in the m6A-mediated mechanism controlling mRNA translation in a unicellular eukaryotic pathogen.IMPORTANCE Infection with the unicellular eukaryotic pathogen Plasmodium falciparum causes malaria, a mosquito-borne disease affecting more than 200 million and killing 400,000 people each year. Underlying the asexual replication within human red blood cells is a tight regulatory network of gene expression and protein synthesis. A widespread mechanism of posttranscriptional gene regulation is the chemical modification of adenosines (m6A), through which the fate of individual mRNA transcripts can be changed. Here, we report on the protein machinery that "reads" this modification and "translates" it into a functional outcome. We provide mechanistic insight into one m6A reader protein and show that it interacts with the translational machinery and acts as a repressor of mRNA translation. This m6A-mediated phenotype has not been described in other eukaryotes as yet, and the functional characterization of the m6A interactome will ultimately open new avenues to combat the disease.

Biofilms harbour Clostridioides difficile, serving as a reservoir for recurrent infection.

C. difficile infection (CDI) is a worldwide healthcare problem with ~30% of cases failing primary therapy, placing a burden on healthcare systems and increasing patient morbidity. We have little understanding of why these therapies fail. Here, we use a clinically validated in vitro gut model to assess the contribution of biofilms towards recurrent disease and to investigate biofilm microbiota-C. difficile interactions. Initial experiments show that C. difficile cells became associated with the colonic biofilm microbiota and are not depleted by vancomycin or faecal microbiota transplant therapies. We observe that transferring biofilm encased C. difficile cells into a C. difficile naïve but CDI susceptible model induces CDI. Members of the biofilm community can impact C. difficile biofilm formation by acting either antagonistically or synergistically. We highlight the importance of biofilms as a reservoir for C. difficile, which can be a cause for recurrent infections.

Anhedonia and the Relationship Between Other Depressive Symptoms and Aggressive Behavior.

Depression has been linked to multiple forms of aggressive behavior in college students; however, it is unclear which aspects of depression explain this connection. Anhedonia, defined as the loss of interest and/or pleasure in previously enjoyed activities, may provide unique information about relationships between depression and aggression. Using cross-sectional data from two independent samples of college students (N = 747 and N = 736 for Study 1 and Study 2, respectively), we examined whether anhedonia helped explain the relationship between broader depressive symptoms and different forms of aggressive and antisocial behavior. Anhedonia accounted for variance in both self-directed aggression and antisocial behavior independent of gender, hostility, anger, other depressive symptoms, and cognitive distortions (Study 2). In addition, there were significant indirect effects of depressive symptoms on self-directed aggression (Studies 1 and 2) and antisocial behavior (Study 2) via anhedonia. Hypotheses involving other-directed aggression received mixed support, with anhedonia atemporally associated with other-directed aggression independent of broader depressive symptoms in Study 1, but not in Study 2. The current findings suggest that anhedonia is an important individual difference that helps explain the relationship between depression and aggressive and antisocial acts and that anhedonia may be differentially associated with various types of aggressive and antisocial behavior.

Ecology of domestic dogs (Canis familiaris) as a host for Guinea worm (Dracunculus medinensis) infection in Ethiopia.

The global programme for the eradication of Guinea worm disease, caused by the parasitic nematode Dracunculus medinensis, has been successful in driving down human cases, but infections in non-human animals, particularly domestic dogs (Canis familiaris), now present a major obstacle to further progress. Dog infections have mainly been found in Chad and, to a lesser extent, in Mali and Ethiopia. While humans classically acquire infection by drinking water containing infected copepods, it has been hypothesized that dogs might additionally or alternatively acquire infection via a novel pathway, such as consumption of fish or frogs as possible transport or paratenic hosts. We characterized the ecology of free-ranging dogs living in three villages in Gog woreda, Gambella region, Ethiopia, in April-May 2018. We analysed their exposure to potential sources of Guinea worm infection and investigated risk factors associated with infection histories. The home ranges of 125 dogs and their activity around water sources were described using GPS tracking, and the diets of 119 dogs were described using stable isotope analysis. Unlike in Chad, where Guinea worm infection is most frequent, we found no ecological or behavioural correlates of infection history in dogs in Ethiopia. Unlike in Chad, there was no effect of variation among dogs in their consumption of aquatic vertebrates (fish or frogs) on their infection history, and we found no evidence to support hypotheses for this novel transmission pathway in Ethiopia. Dog owners had apparently increased the frequency of clean water provision to dogs in response to previous infections. Variations in dog ranging behaviour, owner behaviour and the characteristics of natural water bodies all influenced the exposure of dogs to potential sources of infection. This initial study suggests that the classical transmission pathway should be a focus of attention for Guinea worm control in non-human animals in Ethiopia.

Outcomes of Arthroscopic Surgical Treatment of Osteochondral Lesions of the Elbow in Pediatric and Adolescent Athletes.

BACKGROUND: Osteochondral injuries of the elbow are limiting and affect the ability of pediatric and adolescent athletes to participate in sports. PURPOSE: To report short- and midterm outcomes on athletes undergoing microfracture or fragment fixation of osteochondral elbow lesions and evaluate the effects thereof on sporting activity. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: This was a retrospective study analyzing patients who underwent surgical treatment via microfracture or fragment fixation for osteochondral elbow lesions. Patients were treated at a single institution by a single surgeon between 2012 and 2019. Diagnosis was confirmed with magnetic resonance imaging, and patients were indicated for surgery after having persistent symptoms despite trialing rest, immobilization, and/or activity restriction for at least 3 months. Demographic data including sports of choice were collected preoperatively. Imaging and intraoperative findings were documented, and any complications were noted. Range of motion (ROM) was compared pre- to postoperatively. Return-to-sport evaluation included the ability to play the preoperative sport of choice. RESULTS: In total, 23 patients (25 elbows) were included with a mean follow-up of 23.5 months (range, 6-60.3 months) and a mean age of 13.8 years. Of 25 lesions, 20 (80%) were on the athlete's dominant side. There was significant improvement from pre- to postoperative ROM, including extension (mean ± SD, 6.4° ± 5.3° to 0.04° ± 0.2°; P < .00001), flexion (129.2° ± 10.6° to 138.6° ± 4.4°; P = .00013), and arc of ROM (122.6° ± 13.2° to 138.6° ± 4.4°; P < .00001). Mean lesion size was 81.9 ± 59.3 mm2 (range, 15-225 mm2). All elbows demonstrated radiographic healing postoperatively. Mean time to release to sport was 4.48 ± 1.38 months (range, 2.5-8 months). Six (26.1%) patients changed or stopped their preoperative sporting activity, including 2 of 4 gymnasts and 4 of 11 baseball players. CONCLUSION: Arthroscopic technique with lesion debridement and microfracture or fixation appears safe and results in radiographic healing; however, with these techniques, there remains a high rate of inability to return to sport in patients involved in higher-demand upper extremity activity, such as baseball and gymnastics. Further treatment strategies, including cartilage restoration procedures, may be warranted in this population.

Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite.

Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR-based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 ("dCas9") was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl-CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene-associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin-associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.

Fecal microbiota transplantation for the improvement of metabolism in obesity: The FMT-TRIM double-blind placebo-controlled pilot trial.

BACKGROUND: There is intense interest about whether modulating gut microbiota can impact systemic metabolism. We investigated the safety of weekly oral fecal microbiota transplantation (FMT) capsules from healthy lean donors and their ability to alter gut microbiota and improve metabolic outcomes in patients with obesity. METHODS AND FINDINGS: FMT-TRIM was a 12-week double-blind randomized placebo-controlled pilot trial of oral FMT capsules performed at a single US academic medical center. Between August 2016 and April 2018, we randomized 24 adults with obesity and mild-moderate insulin resistance (homeostatic model assessment of insulin resistance [HOMA-IR] between 2.0 and 8.0) to weekly healthy lean donor FMT versus placebo capsules for 6 weeks. The primary outcome, assessed by intention to treat, was change in insulin sensitivity between 0 and 6 weeks as measured by hyperinsulinemic euglycemic clamps. Additional metabolic parameters were evaluated at 0, 6, and 12 weeks, including HbA1c, body weight, body composition by dual-energy X-ray absorptiometry, and resting energy expenditure by indirect calorimetry. Fecal samples were serially collected and evaluated via 16S V4 rRNA sequencing. Our study population was 71% female, with an average baseline BMI of 38.8 ± 6.7 kg/m2 and 41.3 ± 5.1 kg/m2 in the FMT and placebo groups, respectively. There were no statistically significant improvements in insulin sensitivity in the FMT group compared to the placebo group (+5% ± 12% in FMT group versus -3% ± 32% in placebo group, mean difference 9%, 95% CI -5% to 28%, p = 0.16). There were no statistically significant differences between groups for most of the other secondary metabolic outcomes, including HOMA-IR (mean difference 0.2, 95% CI -0.9 to 0.9, p = 0.96) and body composition (lean mass mean difference -0.1 kg, 95% CI -1.9 to 1.6 kg, p = 0.87; fat mass mean difference 1.2 kg, 95% CI -0.6 to 3.0 kg, p = 0.18), over the 12-week study. We observed variable engraftment of donor bacterial groups among FMT recipients, which persisted throughout the 12-week study. There were no significant differences in adverse events (AEs) (10 versus 5, p = 0.09), and no serious AEs related to FMT. Limitations of this pilot study are the small sample size, inclusion of participants with relatively mild insulin resistance, and lack of concurrent dietary intervention. CONCLUSIONS: Weekly administration of FMT capsules in adults with obesity results in gut microbiota engraftment in most recipients for at least 12 weeks. Despite engraftment, we did not observe clinically significant metabolic effects during the study. TRIAL REGISTRATION: ClinicalTrials.gov NCT02530385.

CRISPR Interference of a Clonally Variant GC-Rich Noncoding RNA Family Leads to General Repression of var Genes in Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum uses mutually exclusive expression of the PfEMP1-encoding var gene family to evade the host immune system. Despite progress in the molecular understanding of the default silencing mechanism, the activation mechanism of the uniquely expressed var member remains elusive. A GC-rich noncoding RNA (ncRNA) gene family has coevolved with Plasmodium species that express var genes. Here, we show that this ncRNA family is transcribed in a clonally variant manner, with predominant transcription of a single member occurring when the ncRNA is located adjacent to and upstream of an active var gene. We developed a specific CRISPR interference (CRISPRi) strategy that allowed for the transcriptional repression of all GC-rich members. A lack of GC-rich ncRNA transcription led to the downregulation of the entire var gene family in ring-stage parasites. Strikingly, in mature blood-stage parasites, the GC-rich ncRNA CRISPRi affected the transcription patterns of other clonally variant gene families, including the downregulation of all Pfmc-2TM members. We provide evidence for the key role of GC-rich ncRNA transcription in var gene activation and discovered a molecular link between the transcriptional control of various clonally variant multigene families involved in parasite virulence. This work opens new avenues for elucidating the molecular processes that control immune evasion and pathogenesis in P. falciparumIMPORTANCEPlasmodium falciparum is the deadliest malaria parasite species, accounting for the vast majority of disease cases and deaths. The virulence of this parasite is reliant upon the mutually exclusive expression of cytoadherence proteins encoded by the 60-member var gene family. Antigenic variation of this multigene family serves as an immune evasion mechanism, ultimately leading to chronic infection and pathogenesis. Understanding the regulation mechanism of antigenic variation is key to developing new therapeutic and control strategies. Our study uncovers a novel layer in the epigenetic regulation of transcription of this family of virulence genes by means of a multigene-targeting CRISPR interference approach.

Transcriptome-wide dynamics of extensive m6A mRNA methylation during Plasmodium falciparum blood-stage development.

Malaria pathogenesis results from the asexual replication of Plasmodium falciparum within human red blood cells, which relies on a precisely timed cascade of gene expression over a 48-h life cycle. Although substantial post-transcriptional regulation of this hardwired program has been observed, it remains unclear how these processes are mediated on a transcriptome-wide level. To this end, we identified mRNA modifications in the P. falciparum transcriptome and performed a comprehensive characterization of N6-methyladenosine (m6A) over the course of blood-stage development. Using mass spectrometry and m6A RNA sequencing, we demonstrate that m6A is highly developmentally regulated, exceeding m6A levels known in any other eukaryote. We characterize a distinct m6A writer complex and show that knockdown of the putative m6A methyltransferase, PfMT-A70, by CRISPR interference leads to increased levels of transcripts that normally contain m6A. In accordance, we find an inverse correlation between m6A methylation and mRNA stability or translational efficiency. We further identify two putative m6A-binding YTH proteins that are likely to be involved in the regulation of these processes across the parasite's life cycle. Our data demonstrate unique features of an extensive m6A mRNA methylation programme in malaria parasites and reveal its crucial role in dynamically fine-tuning the transcriptional cascade of a unicellular eukaryote.

CRISPR in Parasitology: Not Exactly Cut and Dried!

CRISPR/Cas9 technology has been developing rapidly in the field of parasitology, allowing for the dissection of molecular processes with unprecedented efficiency. Optimization and implementation of a new technology like CRISPR, especially in nonmodel organisms, requires communication and collaboration throughout the field. Recently, a 'CRISPR in Parasitology' symposium was held at the Institut Pasteur Paris, bringing together scientists studying Leishmania, Plasmodium, Trypanosoma, and Anopheles. Here we share technological advances and challenges in using CRISPR/Cas9 in the parasite and vector systems that were discussed. As CRISPR/Cas9 continues to be applied to diverse parasite systems, the community should now focus on improvement and standardization of the technique as well as expanding the CRISPR toolkit to include Cas9 alternatives/derivatives for more advanced applications like genome-wide functional screens.

Concurrent and Overlapping Surgery: Perspectives From Parents of Adolescents Undergoing Spinal Posterior Instrumented Fusion for Idiopathic Scoliosis.

STUDY DESIGN: Prospective cross-sectional survey. OBJECTIVE: To determine the perspectives of parents of patients undergoing posterior instrumented fusion for adolescent idiopathic scoliosis (AIS) regarding simultaneous surgery and trainee participation. SUMMARY OF BACKGROUND DATA: Simultaneous ("at the same time") surgery is under scrutiny by the public, government, payers, and the medical community. The objective of this study is to determine the perspectives of parents of patients undergoing posterior instrumented fusion for adolescent idiopathic scoliosis. Our goal is to inform the national conversation on this subject with real patient and family voices. METHODS: A survey was prospectively administered to 31 consecutive parents of patients undergoing posterior instrumented fusion for adolescent idiopathic scoliosis at a large academic medical center. "Overlapping" was defined as simultaneity during "noncritical" parts of an operation. "Concurrent" was defined as simultaneity that includes "critical" part(s) of an operation. Participants were asked to provide levels of agreement with overlapping and concurrent surgery and anesthesia, as well as with trainee involvement. RESULTS: On average, respondents "strongly agree" with the need to be informed about overlapping or concurrent surgery. They "disagree" with both overlapping and concurrent scheduling, and "disagree" with trainees operating without direct supervision, even for "noncritical" parts. Informing parents about the presence of a back-up surgeon or research demonstrating safety of simultaneous surgery did not make them agreeable to simultaneous scheduling. CONCLUSION: Parents have a strong desire to be informed of simultaneous spinal surgery and anesthesia as part of consent on behalf of their children. Their disagreement with simultaneous surgery, as well as with trainees operating without direct supervision, suggests discordance with current guidelines and practice and should inform the national conversation moving forward. LEVEL OF EVIDENCE: N/A.

Differential loss of components of traditional ecological knowledge following a primate extinction event.

Traditional ecological knowledge (TEK), an important component of the modern conservation toolkit, is being eroded in indigenous communities around the world. However, the dynamics of TEK loss in response to ecosystem change and disruption to social-ecological systems, and patterns of variation in vulnerability and resilience of different components of TEK, remain poorly understood. The Hainan gibbon (Nomascus hainanus), a culturally significant primate, was formerly distributed across Hainan Island, China, but became extinct across most of this range within living memory and is now restricted to a single landscape, Bawangling National Nature Reserve. Gibbon-specific TEK (including folktales, natural history information and methods of gibbon exploitation) is still present in indigenous communities across seven Hainanese landscapes, but statistically significant differences in TEK content exist between landscapes with different histories of gibbon persistence: respondents from Bawangling and most landscapes that have recently lost gibbons report more gibbon-related folktales compared with landscapes from which gibbons have been absent for several decades. Species-specific folktales might have been lost more rapidly compared with other components of TEK because older community members are typically the 'cultural repositories' of stories, whereas knowledge about practical interactions with biodiversity might be shared more widely with younger community members.