Orally Bioavailable Macrocyclic Peptide That Inhibits Binding of PCSK9 to the Low Density Lipoprotein Receptor.

BACKGROUND: Inhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design. METHODS: Novel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol). RESULTS: MK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616. CONCLUSIONS: This work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

Chronically activated microglia in ALS gradually lose their immune functions and develop unconventional proteome.

Neuroinflammation and chronic activation of microglial cells are the prominent features of amyotrophic lateral sclerosis (ALS) pathology. While alterations in the mRNA profile of diseased microglia have been well documented, the actual microglia proteome remains poorly characterized. Here we performed a functional characterization together with proteome analyses of microglial cells at different stages of disease in the SOD1-G93A model of ALS. Functional analyses of microglia derived from the lumbar spinal cord of symptomatic mice revealed: (i) remarkably high mitotic index (close to 100% cells are Ki67+) (ii) significant decrease in phagocytic capacity when compared to age-matched control microglia, and (iii) diminished response to innate immune challenges in vitro and in vivo. Proteome analysis revealed a development of two distinct molecular signatures at early and advanced stages of disease. While at early stages of disease, we identified several proteins implicated in microglia immune functions such as GPNMB, HMBOX1, at advanced stages of disease microglia signature at protein level was characterized with a robust upregulation of several unconventional proteins including rootletin, major vaults proteins and STK38. Upregulation of GPNMB and rootletin has been also found in the spinal cord samples of sporadic ALS. Remarkably, the top biological functions of microglia, in particular in the advanced disease, were not related to immunity/immune response, but were highly enriched in terms linked to RNA metabolism. Together, our results suggest that, over the course of disease, chronically activated microglia develop unconventional protein signatures and gradually lose their immune identity ultimately turning into functionally inefficient immune cells.

Outcomes of Integrated Home Dialysis Care: Results From the Canadian Organ Replacement Register.

RATIONALE & OBJECTIVE: The integrated home dialysis model proposes the initiation of kidney replacement therapy (KRT) with peritoneal dialysis (PD) and a timely transition to home hemodialysis (HHD) after PD ends. We compared the outcomes of patients transitioning from PD to HHD with those initiating KRT with HHD. STUDY DESIGN: Observational analysis of the Canadian Organ Replacement Register (CORR). SETTINGS & PARTICIPANTS: All patients who initiated PD or HHD within the first 90 days of KRT between 2005 and 2018. EXPOSURE: Patients transitioning from PD to HHD (PD+HHD group) versus patients initiating KRT with HHD (HHD group). OUTCOME: (1) A composite of all-cause mortality and modality transfer (to in-center hemodialysis or PD for 90 days) and (2) all hospitalizations (considered as recurrent events). ANALYTICAL APPROACH: A propensity score analysis for which PD+HHD patients were matched 1:1 to (1) incident HHD patients ("incident-match" analysis) or (2) HHD patients with a KRT vintage at least equivalent to the vintage of PD+HHD patients at the transition time ("vintage-matched" analysis). Cause-specific hazards models (composite outcome) and shared frailty models (hospitalization) were used to compare groups. RESULTS: Among 63,327 individuals in the CORR, 163 PD+HHD patients (median of 1.9 years in PD) and 711 HHD patients were identified. In the incident-match analysis, compared to the HHD patients, the PD+HHD group had a similar risk of the composite outcome (HR, 0.88 [95% CI, 0.58-1.32]) and hospitalizations (HR, 1.04 [95% CI, 0.76-1.41]). In the vintage-match analysis, PD+HHD patients had a lower hazard for the composite outcome (HR, 0.61 [95% CI, 0.40-0.94]) but a similar hospitalization risk (HR, 0.85 [95% CI, 0.59-1.24]). LIMITATIONS: Risk of survivor bias in the PD+HHD cohort and residual confounding. CONCLUSIONS: Controlling for KRT vintage, the patients transitioning from PD to HHD had better clinical outcomes than the incident HHD patients. These data support the use of integrated home dialysis for patients initiating home-based KRT. PLAIN-LANGUAGE SUMMARY: The integrated home dialysis model proposes the initiation of dialysis with peritoneal dialysis (PD) and subsequent transition to home hemodialysis (HHD) once PD is no longer feasible. It allows patients to benefit from initial lifestyle advantages of PD and to continue home-based treatments after its termination. However, some patients may prefer to initiate dialysis with HHD from the outset. In this study, we compared the long-term clinical outcomes of both approaches using a large Canadian dialysis register. We found that both options led to a similar risk of hospitalization. In contrast, the PD-to-HHD model led to improved survival when controlling for the duration of kidney failure.

Nickel-Catalyzed Reductive Arylation of Redox Active Esters for the Synthesis of α-Aryl Nitriles: Investigation of a Chlorosilane Additive.

A nickel-catalyzed reductive cross-coupling of redox active N-hydroxyphthalimide (NHP) esters and iodoarenes for the synthesis of α-aryl nitriles is described. The NHP ester substrate is derived from cyanoacetic acid, which allows for a modular synthesis of substituted α-aryl nitriles, an important scaffold in the pharmaceutical sciences. The reaction exhibits a broad scope, and many functional groups are compatible under the reaction conditions, including complex highly functionalized medicinal agents. Mechanistic studies reveal that reduction and decarboxylation of the NHP ester to the reactive radical intermediate are accomplished by a combination of a chlorosilane additive and Zn dust. We demonstrate that stoichiometric chlorosilane is essential for product formation and that chlorosilane plays a role beyond activation of the metal reductant.

RNA-based regulation in bacteria-phage interactions.

Interactions of bacteria with their viruses named bacteriophages or phages shape the bacterial genome evolution and contribute to the diversity of phages. RNAs have emerged as key components of several anti-phage defense systems in bacteria including CRISPR-Cas, toxin-antitoxin and abortive infection. Frequent association with mobile genetic elements and interplay between different anti-phage defense systems are largely discussed. Newly discovered defense systems such as retrons and CBASS include RNA components. RNAs also perform their well-recognized regulatory roles in crossroad of phage-bacteria regulatory networks. Both regulatory and defensive function can be sometimes attributed to the same RNA molecules including CRISPR RNAs. This review presents the recent advances on the role of RNAs in the bacteria-phage interactions with a particular focus on clostridial species including an important human pathogen, Clostridioides difficile.

Chronic Diarrhea Caused by a Klebsiella oxytoca Toxin Producer Strain Following Antibiotic-Associated Hemorrhagic Colitis: Successful Treatment by Fecal Microbiota Transplant.

Klebsiella oxytoca is a gram-negative bacterium found in fecal microbiota and known to cause several infections in humans, including antibiotic-associated hemorrhagic colitis. We present here a case of colitis caused by K. oxytoca toxin-producing strains that evolved in chronic diarrhea successfully treated by fecal microbiota transplant.

Novel high-oleic oil consumption for cardiometabolic health: a narrative review.

Several cardiometabolic disorders are risk factors for cardiovascular diseases (CVDs), and prevention is imperative in reducing the burden of these diseases on the healthcare system. Although novel high-oleic acid oils (HOOs) are now commonly used for high-temperature frying in both foodservice and the manufacture of processed foods, there are still limited data regarding their effects on CVD risk. This narrative review aims to clarify these effects by comparing HOOs with saturated fatty acid (SFA)-rich and polyunsaturated fatty acid (PUFA)-rich oils, first regarding their physicochemical properties and then concerning their effects on CVD risk factors using recent randomized controlled trials (RCTs). Overall, although HOOs are more stable than PUFA-rich oils, they do not have the same high-temperature stability as SFA-rich oils. RCTs demonstrate that HOO consumption improves the plasma lipid profile compared with SFA-rich oils while showing similar effects to those of PUFA-rich oils on CVD risk factors. Finally, the current literature lacks information on the actual consumption of HOOs, their long-term effects on cardiometabolic health, and the impact of prolonged heating of these oils on CVD risk factors. In sum, the short-term intake of HOOs may be beneficial for cardiometabolic health; however, more research is needed.

Towards real-time active imaging of greenhouse gases using tunable mid-infrared all-fiber lasers.

We report a tunable all-fiber laser emitting a maximum output power of 2.55 W around 3240 nm. The fiber laser cavity based on a fluoride fiber doped with dysprosium ions yields an efficiency of 42% according to the in-band launched pump power at 2825 nm. Due to a custom piezoelectric fiber Bragg grating (FBG) package, mechanical strains applied to the narrowband FBG used as the input cavity coupler allowed for fast tuning of the emission wavelength over a spectral range of 1.5 nm. This laser was deployed in the field in northern Québec (Canada) to assess its performances for remote sensing of methane in the presence of a significant amount of water vapor, i.e., over a hydroelectric reservoir. The preliminary results acquired during this field campaign confirm the great potential of the proposed approach for the development of a real-time active imaging system of greenhouse gases.

Total Synthesis of Darobactin A.

The collaborative total synthesis of darobactin A, a recently isolated antibiotic that selectively targets Gram-negative bacteria, has been accomplished in a convergent fashion with a longest linear sequence of 16 steps from d-Garner's aldehyde and l-serine. Scalable routes toward three non-canonical amino acids were developed to enable the synthesis. The closure of the bismacrocycle was realized through sequential, halogen-selective Larock indole syntheses, where the proper order of cyclizations proved crucial for the formation of the desired atropisomer of the natural product.

Recent developments in lanthanide-doped mid-infrared fluoride fiber lasers [Invited].

Mid-infrared fiber sources, emitting between 2.5 µm and 5.0 µm, are interesting for their great potential in several application fields such as material processing, biomedicine, remote sensing and infrared countermeasures due to their high-power, their diffraction-limited beam quality as well as their robust monolithic architecture. In this review, we will focus on the recent progress in continuous wave and pulsed mid-infrared fiber lasers and the components that bring these laser sources closer to a field deployment as well as in industrial systems. Accordingly, we will briefly illustrate the potential of such mid-infrared fiber lasers through a few selected applications.

Microglia-derived galectin-3 in neuroinflammation; a bittersweet ligand?

Galectins are soluble ß-galactoside-binding proteins found in all multicellular organisms. Galectins may act as danger-associated molecular patterns in innate immunity and/or as pattern-recognition receptors that bind to pathogen-associated molecular patterns. Among different galectin family members, galectin-3 has been the focus of studies in neurodegenerative diseases in recent years. This lectin modulates brain innate immune responses, microglia activation patterns in physiological and pathophysiological settings in a context-dependent manner. Galectin-3 is considered as a pivotal tuner of macrophage and microglial activity. Indeed galectin-3 acts as a double edged sword in neuroinflammatory context and this multimodal lectin has diverse roles in physiological and pathophysiological conditions. Better understanding of galectin-3 physiology (its extracellular and intracellular actions) and structure (its C terminus vs. N terminus) is instrumental to design molecules that selectively modulate galectin-3 function toward neuroprotective phenotypes. Several experimental studies using different approaches and methods have demonstrated both protective and deleterious effects of galectin-3 in neuroinflammatory diseases. According to the crucial role of galectin-3 in modulation of innate immune response in brain, it is an attractive target in drug discovery of neurodegenerative diseases. The current insight attempts to provide an updated and balanced discussion on the role of galectin-3 as a complex endogenous immune modulator. This helps to have a better insight into the development of galectin-3 modulators with translational value in different neurological disorders including stroke and neurodegenerative diseases, such as Alzheimer's disease, Huntington's disease and Parkinson's disease.

100-W-level single-mode ytterbium-free erbium fiber laser.

We report on an ytterbium-free, erbium-doped single-mode all-fiber laser reaching a record output power of 107 W at 1598 nm, with a slope efficiency of 38.6% according to the absorbed pump power at 981 nm. The erbium-doped gain fiber, co-doped with cerium, aluminum, and phosphorus, was fabricated in-house with adjusted doping concentrations to reduce erbium ions clustering, thereby increasing efficiency while keeping the numerical aperture low to ensure a single-mode laser operation. The addition of cerium co-dopant in the core glass of an erbium system is used for the first time, to the best of our knowledge, in order to adjust the fiber's numerical aperture without increasing the erbium concentration. Numerical modeling, validated by the experimental results, demonstrates that adding aluminum and phosphorus at high concentration mitigates erbium ions clustering, with an estimated erbium paired ions of only 5.0% in the reported gain fiber.

Ultrasound-guided transthoracic needle biopsy of the lung: sensitivity and safety variables.

OBJECTIVES: Variables affecting the performance of ultrasound-guided transthoracic needle biopsy (US-TTNB) are not well established. We examined clinical and imaging variables affecting the sensitivity and the complication rates of US-TTNB. METHODS: We retrospectively reviewed a consecutive series of 528 US-TTNBs performed from 2008 to 2017. Univariate analyses were used to assess the influence of clinical and imaging variables on sensitivity and complication rates. Multivariate logistic regression was used to account for possible confounding variables. RESULTS: In 397 malignant lesions, the sensitivity of US-TTNB was 72% (95% CI 68-77%; 285/397). The overall pneumothorax rate was 15% (95% CI 12-18%; 77/528), leading to a chest tube in 2% (95% CI 1-3%; 9/528). Multivariate analysis showed that increasing pleural contact length (up to 30 mm) was associated with increased sensitivity (OR 1.08 per mm; 95% CI 1.04-1.12; p < 0.001), and pleural contact length (OR 0.98 per mm; 95% CI 0.97-0.99; p = 0.013), lesion size (OR 0.98 per mm; 95% CI 0.96-0.99; p = 0.006), and core needle diameter of 18G (OR 0.47 as compared with 20G; 95% CI 0.26-0.83; p = 0.010) were associated with a decreased pneumothorax rate. Graphical inspection of cubic splines showed that the probability of a positive biopsy rose sharply with increasing pleural contact length up to 30 mm and was stable thereafter. A similar, but inverse, relationship was observed for the probability of a pneumothorax. CONCLUSION: Pleural contact length is a key variable predicting the sensitivity of US-TTNB and pneumothorax rate after US-TTNB. Lesion size also predicts pneumothorax rates. KEY POINTS: • US-TTNB has a high sensitivity and a low complication rate for pleural and pulmonary lesions with pleural contact. • Pleural contact length is a key variable predicting the sensitivity of US-TTNB and pneumothorax rate after US-TTNB. • This study suggests that relying on US-TTNB may not be optimal for lesions < 10 mm for which the risk of pneumothorax is as high as the chance of obtaining diagnosis.

Inactivation of the riboswitch-controlled GMP synthase GuaA in Clostridioides difficile is associated with severe growth defects and poor infectivity in a mouse model of infection.

Clostridioides difficile is the main cause of nosocomial antibiotic-associated diarrhoea. There is a need for new antimicrobials to tackle this pathogen. Guanine riboswitches have been proposed as promising new antimicrobial targets, but experimental evidence of their importance in C. difficile is missing. The genome of C. difficile encodes four distinct guanine riboswitches, each controlling a single gene involved in purine metabolism and transport. One of them controls the expression of guaA, encoding a guanosine monophosphate (GMP) synthase. Here, using in-line probing and GusA reporter assays, we show that these riboswitches are functional in C. difficile and cause premature transcription termination upon binding of guanine. All riboswitches exhibit a high affinity for guanine characterized by Kd values in the low nanomolar range. Xanthine and guanosine also bind the guanine riboswitches, although with less affinity. Inactivating the GMP synthase (guaA) in C. difficile strain 630 led to cell death in minimal growth conditions, but not in rich medium. Importantly, the capacity of a guaA mutant to colonize the mouse gut was significantly reduced. Together, these results demonstrate the importance of de novo GMP biosynthesis in C. difficile during infection, suggesting that targeting guanine riboswitches with analogues could be a viable therapeutic strategy.

Pathophysiology of Vaginal Erosions in Women Using Pessary: A Pilot Study Examining Vaginal Microbiota.

OBJECTIVE: Many women with pelvic organ prolapse opt for a pessary, and some of these women develop erosions of the vaginal mucosa. Ongoing erosions might lead to the discontinuation of this otherwise effective, non-invasive, and inexpensive treatment. The objectives of this study were to investigate the differences in vaginal pH and variations of the vaginal microbiota among pessary and non-pessary users. METHODS: For this descriptive observational study, 30 women, followed in our urogynecology clinic, were recruited to form 3 equal groups: 2 groups of women using a pessary (with and without erosions) and 1 control group of women not using a pessary. Vaginal pH was measured distally and next to the erosion. Vaginal swabs were used to investigate the vaginal microbiota by sequencing the V4 region of the 16S ribosomal RNA gene and analyzing the data with Qiime2. Descriptive statistics were reported using the median values. Vaginal pH comparisons between groups were made using a Kruskal-Wallis test with Dunn's correction for multiple comparisons. RESULTS: The pH of the vagina was more alkaline in women with erosions compared with women in the other 2 groups (P < 0.01). Also, the pH of the distal vagina was not different from the pH next to the erosion (P = 0.25). Patients with erosions displayed significant differences in their vaginal microbiota, which contained a much greater bacterial diversity with an increase in gram-negative bacteria (e.g., Bacteroidetes, Actinobacteria) and a decrease in lactobacilli. CONCLUSION: In our study, women with vaginal erosions had significantly higher vaginal pH and more complex vaginal microbiota than women in the control groups. Treatments focusing on lowering the vaginal pH and/or re-establishing the vaginal microbiota should be considered.

Incidence of fractures in middle-aged individuals with early chronic kidney disease: a population-based analysis of CARTaGENE.

BACKGROUND: Previous studies evaluating fractures in chronic kidney disease (CKD) have mostly focused on hip or major fractures in aged populations with moderate to advanced CKD. We aimed at evaluating the association between early CKD and fracture incidence at all sites across age and sex in middle-aged individuals. METHODS: We analyzed CARTaGENE, a prospective population-based survey of 40- to 69-year-old individuals from Quebec (Canada). Estimated glomerular filtration rate (eGFR) at baseline was evaluated categorically or continuously using restricted cubic splines. Fractures at any site (except toes, hand and craniofacial) for up to 7 years of follow-up were identified through administrative databases using a validated algorithm. Adjusted Cox models were used to evaluate the association of CKD with fracture. Interaction terms for age and sex were also added. RESULTS: A total of 19 391 individuals (756 CKD Stage 3; 9114 Stage 2; 9521 non-CKD) were included and 829 fractures occurred during a median follow-up of 70 months. Compared with the median eGFR of 90 mL/min/1.73 m2, eGFRs of ≤60 mL/min/1.73 m2 were associated with increased fracture incidence in unadjusted and adjusted models [adjusted hazard ratio (HR) = 1.25 (95% confidence interval 1.05-1.49) for 60 mL/min/1.73 m2; 1.65 (1.14-2.37) for 45 mL/min/1.73 m2]. The eGFR was linearly associated with fracture incidence <75 mL/min/1.73 m2 [HR = 1.18 (1.04-1.34) per 10 mL/min/1.73 m2 decrease] but not above [HR = 0.98 (0.91-1.06) per 10 mL/min/1.73 m2 decrease). The effect of decreased eGFR on fracture incidence was more pronounced in younger individuals [HR = 2.45 (1.28-4.67) at 45 years; 1.11 (0.73-1.67) at 65 years] and in men. CONCLUSIONS: Even early CKD increases fracture incidence, especially in younger individuals and in men.

Multilocus Variable-Number Tandem-Repeat Analysis of Clostridioides difficile Clusters in Ribotype 027 Isolates and Lack of Association with Clinical Outcomes.

The epidemiology of Clostridioides difficile infection (CDI) has drastically changed since the emergence of the epidemic strain BI/NAP1/027, also known as ribotype 027 (R027). However, the relationship between the infecting C. difficile strain and clinical outcomes is still debated. We hypothesized that certain subpopulations of R027 isolates could be associated with unfavorable outcomes. We applied high-resolution multilocus variable-number tandem-repeat analysis (MLVA) to characterize C. difficile R027 isolates collected from confirmed CDI patients recruited across 10 Canadian hospitals from 2005 to 2008. PCR ribotyping was performed first to select R027 isolates that were then analyzed by MLVA (n = 450). Complicated CDI (cCDI) was defined by the occurrence of any of admission to an intensive care unit, colonic perforation, toxic megacolon, colectomy, and if CDI was the cause or contributed to death within 30 days after enrollment. Three major MLVA clusters were identified, MC-1, MC-3, and MC-10. MC-1 and MC-3 were exclusive to Quebec centers, while MC-10 was found only in Ontario. Fewer cases infected with MC-1 developed cCDI (4%) than those infected with MC-3 and MC-10 (15% and 16%, respectively), but a statistically significant difference was not reached. Our data did not identify a clear association between subpopulations of R027 and different clinical outcomes; however, the data confirmed the utility of MLVA's higher discrimination potential to better characterize CDI populations in an epidemiological analysis. For a patient with CDI, the progression toward an unfavorable outcome is a complex process that probably includes several interrelated strain and host characteristics.

Pelvic Floor Dysfunction in the Female Athlete.

Pelvic floor dysfunction (PFD) is a term used to describe a variety of disorders involving moderate to severe impairment of the pelvic floor muscles. It can be divided into two broad categories. Relaxing PFD is more common, presenting with urinary incontinence, fecal incontinence, or pelvic organ prolapse. Nonrelaxing PFD symptoms include impaired ability to evacuate urine or stool, sexual dysfunction, and pelvic pain. Unfamiliarity with this diagnosis among medical providers along with nonspecific symptoms often lead to a delayed or missed diagnosis for patients. Here, we present the case of a collegiate soccer player with PFD to illustrate the severity of presentation and progression of this disorder. We review the current literature regarding nonrelaxing PFD and all types of PFD in female athletes to raise awareness and recognition of this condition.

High Prevalence and Genetic Diversity of Large phiCD211 (phiCDIF1296T)-Like Prophages in Clostridioides difficile.

Clostridioides difficile (formerly Clostridium difficile) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified in C. difficile so far, with a genome of 131 kbp. It shares morphological and genomic similarity with other large siphophages, like phage 949, infecting Lactococcus lactis, and phage c-st, infecting Clostridium botulinum A PhageTerm analysis indicated the presence of 378-bp direct terminal repeats at the phiCD211 genome termini. Among striking features of phiCD211, the presence of several transposase and integrase genes suggests past recombination events with other mobile genetic elements. Several gene products potentially influence the bacterial lifestyle and fitness, including a putative AcrB/AcrD/AcrF multidrug resistance protein, an EzrA septation ring formation regulator, and a spore protease. We also identified a CRISPR locus and a cas3 gene. We screened 2,584 C. difficile genomes available and detected 149 prophages sharing ≥80% nucleotide identity with phiCD211 (5% prevalence). Overall, phiCD211-like phages were detected in C. difficile strains corresponding to 21 different multilocus sequence type groups, showing their high prevalence. Comparative genomic analyses revealed the existence of several clusters of highly similar phiCD211-like phages. Of note, large chromosome inversions were observed in some members, as well as multiple gene insertions and module exchanges. This highlights the great plasticity and gene coding potential of the phiCD211/phiCDIF1296T genome. Our analyses also suggest active evolution involving recombination with other mobile genetic elements.IMPORTANCEClostridioides difficile is a clinically important pathogen representing a serious threat to human health. Our hypothesis is that genetic differences between strains caused by the presence of integrated prophages could explain the apparent differences observed in the virulence of different C. difficile strains. In this study, we provide a full characterization of phiCD211, also known as phiCDIF1296T, the largest phage known to infect C. difficile so far. Screening 2,584 C. difficile genomes revealed the presence of highly similar phiCD211-like phages in 5% of the strains analyzed, showing their high prevalence. Multiple-genome comparisons suggest that evolution of the phiCD211-like phage community is dynamic, and some members have acquired genes that could influence bacterial biology and fitness. Our study further supports the relevance of studying phages in C. difficile to better understand the epidemiology of this clinically important human pathogen.

IL-10 Controls Early Microglial Phenotypes and Disease Onset in ALS Caused by Misfolded Superoxide Dismutase 1.

While reactive microgliosis is a hallmark of advanced stages of amyotrophic lateral sclerosis (ALS), the role of microglial cells in events initiating and/or precipitating disease onset is largely unknown. Here we provide novel in vivo evidence of a distinct adaptive shift in functional microglial phenotypes in preclinical stages of superoxide dismutase 1 (SOD1)-mutant-mediated disease. Using a mouse model for live imaging of microglial activation crossed with SOD1(G93A) and SOD1(G37R) mouse models, we discovered that the preonset phase of SOD1-mediated disease is characterized by development of distinct anti-inflammatory profile and attenuated innate immune/TLR2 responses to lipopolysaccharide (LPS) challenge. This microglial phenotype was associated with a 16-fold overexpression of anti-inflammatory cytokine IL-10 in baseline conditions followed by a 4.5-fold increase following LPS challenge. While infusion of IL-10R blocking antibody, initiated at day 60, caused a significant increase in markers of microglial activation and precipitated clinical onset of disease, a targeted overexpression of IL-10 in microglial cells, delivered via viral vectors expressed under CD11b promoter, significantly delayed disease onset and increased survival of SOD1(G93A) mice. We propose that the high IL-10 levels in resident microglia in early ALS represent a homeostatic and compensatory "adaptive immune escape" mechanism acting as a nonneuronal determinant of clinical onset of disease. Significance statement: We report here for the first time that changing the immune profile of brain microglia may significantly affect clinical onset and duration of disease in ALS models. We discovered that in presymptomatic disease microglial cells overexpress anti-inflammatory cytokine IL-10. Given that IL-10 is major homeostatic cytokine and its production becomes deregulated with aging, this may suggest that the capacity of microglia to adequately produce IL-10 may be compromised in ALS. We show that blocking IL-10 increased inflammation and precipitated clinical disease onset, whereas overexpression of IL-10 in microglia using a gene therapy approach significantly delayed disease onset and increased survival of ALS mice. Based on our results, we propose that targeted overexpression of IL-10 in microglia may have therapeutic potential in ALS.