The Legionella pneumophila effector DenR hijacks the host NRas proto-oncoprotein to downregulate MAPK signaling.

Small GTPases of the Ras subfamily are best known for their role as proto-oncoproteins, while their function during microbial infection has remained elusive. Here, we show that Legionella pneumophila hijacks the small GTPase NRas to the Legionella-containing vacuole (LCV) surface. A CRISPR interference screen identifies a single L. pneumophila effector, DenR (Lpg1909), required for this process. Recruitment is specific for NRas, while its homologs KRas and HRas are excluded from LCVs. The C-terminal hypervariable tail of NRas is sufficient for recruitment, and interference with either NRas farnesylation or S-acylation sites abrogates recruitment. Intriguingly, we detect markers of active NRas signaling on the LCV, suggesting it acts as a signaling platform. Subsequent phosphoproteomics analyses show that DenR rewires the host NRas signaling landscape, including dampening of the canonical mitogen-activated protein kinase pathway. These results provide evidence for L. pneumophila targeting NRas and suggest a link between NRas GTPase signaling and microbial infection.

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

Physiotherapists' Experiences with and Perspectives on Implementing an Evidence-Based, Chronic Pain Self-Management Programme in Primary Health Care: A Qualitative Study.

Purpose: Evidence suggests that a physiotherapist-led chronic pain self-management programme in primary health care (PHC) improves function for people living with chronic pain; however, implementing a new approach to care can be difficult. In this study, we sought to understand the experiences of physiotherapists who had implemented the ChrOnic pain self-ManageMent support with pain science EducatioN and exerCisE (COMMENCE) programme; its perceived barriers, facilitators, benefits, and drawbacks; and how the physiotherapists tailored the programme to their own clinical contexts. Method: This interpretive description qualitative study used semi-structured interviews with physiotherapists who had implemented the COMMENCE programme in PHC. Results: Themes from 11 interviews included experiences of personal and professional growth, increasing confidence with experience, and changing the culture of pain management. Barriers and drawbacks to implementation included resource intensiveness, balancing programme demands with other clinical work, and challenges with patient attendance and participation. Facilitators included training, programme design and materials, supportive teams, and previous knowledge. Benefits included offering group and individualized support, evidence-based content, and sparking interest in learning more about pain management. The participants made small changes to tailor the programme content and delivery to their context. Conclusions: This study provides a rich understanding of the experiences, barriers, facilitators, benefits, drawbacks, and tailoring related to the COMMENCE programme in PHC. The results will facilitate future implementation of this intervention in PHC settings.

Objectif : selon les données probantes, un programme d'autogestion de la douleur chronique dirigé par un physiothérapeute en soins primaires améliore la fonction des personnes qui vivent avec la douleur chronique, mais il peut être difficile de mettre en œuvre une nouvelle approche des soins. La présente étude visait à comprendre les expériences des physiothérapeutes qui avaient créé le programme COMMENCE (acronyme anglais pour soutien pour l'autoprise en charge de la douleur chronique par l'éducation et l'exercice de la science de la douleur), les obstacles perçus, les incitations, les avantages et les inconvénients, de même que l'adaptation du programme aux contextes cliniques. Méthodologie : étude qualitative par description interprétative faisant appel à des entrevues semi-structurées auprès de physiothérapeutes qui avaient mis en œuvre le programme COMMENCE en soins primaires. Résultats : les thèmes des 11 entrevues portaient sur les expériences de croissance personnelle et professionnelle, l'augmentation de la confiance grâce à l'expérience et le changement de la culture de gestion de la douleur. Les obstacles ou les écueils de mise en œuvre incluaient l'intensité de ressources nécessaires, l'équilibre entre les exigences du programme et le reste du travail clinique et les difficultés relatives à l'assiduité et à la participation des patients. Les incitations incluaient la formation, la conception et le matériel du programme, les équipes solidaires et les connaissances antérieures. Les avantages incluaient l'offre d'un soutien collectif et individuel, le contenu fondé sur des données probantes et l'intérêt à en apprendre davantage sur la gestion de la douleur. Les participants ont apporté de petits changements pour adapter le contenu et la prestation du programme à leur contexte personnel. Conclusions : la présente étude fournit de riches données sur les expériences, les obstacles, les incitations, les avantages, les écueils et l'adaptation du programme COMMENCE en soins primaires. Les résultats faciliteront la future mise en œuvre de cette intervention en soins primaires.

A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila.

Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

Effects of Exercise With and Without Energy Replacement on Substrate Utilization in the Fasting State.

Objective: The present study aimed to examine the interactive effect of exercise and energy balance on energy expenditure and substrate utilization.Method: Seven men and 7 women underwent three 2-day experimental protocols in a random order. Each protocol consisted of no exercise (NE), exercise only (EO), or exercise with a matched energy replacement (ER) on day 1 followed by metabolic testing that occurred after a 12-hour overnight fasting on day 2. Both EO and ER involved treadmill running at 60% maximal oxygen uptake (VO2max) that induced an energy expenditure of ∼ 500 kcal. The replacement meal used in ER contained ∼ 500 kcal made up of 45% carbohydrate, 30% fat, and 25% protein. During metabolic testing, oxygen uptake (VO2), heart rate (HR), respiratory exchange ratio (RER), and rates of carbohydrate (COX) and fat oxidation (FOX) were determined in three successive 15-minute periods including rest and exercise at 50% and 70% VO2max.Results: No differences in VO2 and HR were found at rest among NE, EO, and ER. However, RER was lower in EO than NE (0.840 ± 0.014 vs 0.889 ± 0.012, p < 0.05), COX (g·min-1) was lower in ER than NE (0.144 ± 0.016 vs 0.197 ± 0.019, p < 0.05), and FOX (g·min-1) was higher in EO or ER than NE (0.054 ± 0.010 or 0.057 ± 0.009 vs 0.034 ± 0.007, p < 0.05). No treatment effects were observed for all variables at either intensity.Conclusions: This study demonstrates that an exercise of moderate intensity can increase resting fat oxidation even when the exercise-induced energy expenditure is balanced by energy intake. This finding suggests that muscle action is vital in augmenting fat utilization.

Acute Cardiometabolic Responses to Medicine Ball Interval Training in Children.

Medicine ball interval training (MBIT) has been found to be an effective exercise modality in fitness programs, yet the acute physiological responses to this type of this exercise in youth are unknown. The purpose of this study was to examine the acute cardiometabolic responses to MBIT in children. Fourteen children (mean age 10.1 ± 1.3 yr) were tested for peak oxygen uptake (VO2peak) on a treadmill and subsequently (> 48 hours later) performed a progressive 10 min MBIT protocol of 5 exercises (EX): standing marches (EX1), alternating lunges (EX2), squat swings (EX3), chest passes (EX4) and double arm slams (EX5). A 2.3 kg medicine ball was used for all trials and each exercise was performed twice for 30 sec with a 30 sec rest interval between sets and exercises. Participants exercised while connected to a metabolic system and heart rate (HR) monitor. During the MBIT protocol, mean HR significantly (p<0.05, η2 = 0.89) increased from 121.5 ± 12.3 bpm during EX1 to 178.3 ± 9.4 bpm during EX5 and mean VO2 significantly (p<0.05, η2= 0.88) increased from 15.5 ± 2.9 ml × kg-1 × min-1 during EX1 to 34.9 ± 5.1 ml × kg-1 × min-1during EX5. Mean HR and VO2 values during MBIT ranged from 61.1% to 89.6% of HRpeak and from 28.2% to 63.5% of VO2peak. These descriptive data indicate that MBIT can pose a moderate to vigorous cardiometabolic stimulus in children.

Acute hematological and mood perception effects of bitter orange extract (p-synephrine) consumed alone and in combination with caffeine: A placebo-controlled, double-blind study.

The purpose of this study was to examine acute hematological and mood perception responses to supplementation with p-synephrine alone and in combination with caffeine during quiet sitting. Sixteen subjects visited the laboratory on 6 occasions and were given (in randomized double-blind manner) 103-mg p-synephrine (S), 233-mg caffeine + 104-mg p-synephrine, 240-mg caffeine, 337-mg caffeine + 46-mg p-synephrine, 325-mg caffeine, or a placebo (PL). The subjects sat quietly for 3 hr while completing mood state questionnaires every 30 min. Venous blood samples were collected at baseline (pre) and 3 hr (post) to determine immune, lipid, and chemistry panels. Compared with PL, no significant supplement differences were observed during the S trial with the exception of differential time effects seen in hematocrit (decrease in PL, no change in S), triglycerides and very low-density lipoproteins (no changes in PL, significant decreases in S), and iron (no change in PL, significant elevation in S). Supplements containing caffeine showed increased feelings of attention, excitement, energy, and vigor. These data indicate that consumption of 103-mg p-synephrine does not negatively impact acute blood parameters, does not augment the effects of caffeine, or produce stimulant-like perceptual mood effects.

Acute Cardiorespiratory and Metabolic Effects of a Sandbag Resistance Exercise Protocol.

Ratamess, NA, Kang, J, Kuper, JD, O'Grady, EA, Ellis, NL, Vought, IT, Culleton, E, Bush, JA, and Faigenbaum, AD. Acute cardiorespiratory and metabolic effects of a sandbag resistance exercise protocol. J Strength Cond Res 32(6): 1491-1502, 2018-The purpose of this study was to examine the acute cardiorespiratory and metabolic effects of a sandbag (SB) resistance exercise protocol and compare the responses to time-matched treadmill running protocols. Eight healthy, resistance-trained men (21.1 ± 1.0 years; 86.1 ± 7.8 kg) completed 4 protocols of equal duration in random sequence: (a) SB, (b) treadmill running at 60% of V[Combining Dot Above]O2 reserve (60V[Combining Dot Above]O2R), (c) treadmill running at 80% of V[Combining Dot Above]O2 reserve (80V[Combining Dot Above]O2R), and (d) a control protocol. The SB protocol was 16 minutes in duration and consisted of 3 circuits of 8 multiple-joint exercises (with 11-, 20-, or 48-kg SBs) performed for as many repetitions as possible for 20 seconds followed by a 10-second rest interval before beginning the next exercise. Two minutes of rest was allowed between circuits. Breath-by-breath oxygen consumption (V[Combining Dot Above]O2) and heart rate (HR) were recorded throughout each protocol and for 30 minutes postexercise (PE) and blood lactate was determined before and immediately after each protocol. Blood lactate was significantly higher after SB compared with 60V[Combining Dot Above]O2R and 80V[Combining Dot Above]O2R. Mean and peak HR in SB was significantly higher than 60V[Combining Dot Above]O2R but not different from 80V[Combining Dot Above]O2R. Mean V[Combining Dot Above]O2 and energy expenditure (EE) in SB was significantly lower than 60V[Combining Dot Above]O2R and 80V[Combining Dot Above]O2R during each protocol but significantly higher after SB compared with 60V[Combining Dot Above]O2R and 80V[Combining Dot Above]O2R PE. Compared with 60V[Combining Dot Above]O2R and 80V[Combining Dot Above]O2R, respiratory exchange ratio was significantly higher during SB and through 5 minutes PE, but was significantly lower at 25-30 minutes PE after SB. Sandbag, as performed in this study, provides a superior metabolic stimulus to treadmill running during the PE period; however, the SB results demonstrate inferior EE compared with running at 60V[Combining Dot Above]O2R and 80V[Combining Dot Above]O2R.

Acute cardiovascular effects of bitter orange extract (p-synephrine) consumed alone and in combination with caffeine in human subjects: A placebo-controlled, double-blind study.

The purpose was to examine cardiovascular responses to supplementation with p-synephrine alone and in combination with caffeine during quiet sitting. Sixteen subjects were given (in double-blind manner) either 103 mg of p-synephrine (S), 233 mg of caffeine +104 mg of p-synephrine (LC + S), 240 mg of caffeine (LC), 337 mg of caffeine +46 mg of p-synephrine (HC + S), 325 mg of caffeine (HC), or a placebo. The subjects sat quietly for 3 hr while heart rate (HR) and blood pressure were measured. Only HC + S and HC significantly increased mean systolic blood pressure (SBP) during the second hour and tended to increase mean SBP during the third hour. Mean diastolic blood pressure in S was significantly lower than the other trials during the first and second hours, and mean arterial pressure was significantly lower in S compared to the LC, LC + S, HC, and HC + S trials. No differences were observed in HR. Consumption of p-synephrine may acutely reduce diastolic blood pressure and mean arterial pressure and not affect SBP or HR during quiet sitting. The addition of p-synephrine to caffeine did not augment SBP or HR indicating that consumption of up to 104 mg of p-synephrine does not induce cardiovascular stress during quiet sitting.

Does Persistent Inflammatory Catabolic Syndrome Exist in Critically Ill Neonates?

BACKGROUND: Persistent inflammatory catabolic syndrome (PICS) has not been described in the infant population. This study proposes a definition of PICS in critically ill infants. METHODS: A published adult criterion of PICS was modified using anthropometric and biochemical reference ranges for infants. A prospective chart review of admissions to a tertiary surgical neonatal intensive care unit (NICU) was performed over 65 days. Demographic, anthropometric, biochemical, and other clinical variables such as length of stay and medication use were collected daily throughout admission. Infants were categorized as having or not having PICS. RESULTS: Twenty percent of admitted infants (n = 15) developed PICS using the proposed criteria. Infants with PICS were more likely to be classified as failure to thrive (53%), meeting only 75% of their anticipated weight gain. Significantly more infants with PICS had undergone surgery (100%; P = .01), received inotropic medication (40%; P = .05), and had longer NICU and total hospital length of stay ( P < .001 and P < .001). Infants with PICS had higher peak glucose levels (11.8 ± 7.3 mmol/L) and elevated urea concentrations (7.9 ± 4.6 mmol/L). CONCLUSIONS: PICS does exist in a critically ill neonatal population and may be identified using the definition proposed in this study. Infants with PICS displayed metabolic dysregulation, impaired expected growth velocity, and longer length of stay despite no differences in severity scores or diagnosis between the groups. Validation of this work is required, and research into timely identification of infants with PICS is needed to inform whether these infants would benefit from earlier and novel nutrition intervention.

Catalytically active peptidylglycine alpha-amidating monooxygenase in the media of androgen-independent prostate cancer cell lines.

Peptidylglycine alpha-amidating monooxygenase (PAM) converts inactive terminal-glycine prohormones into their activated alpha-amidated forms. PAM is thought to play a role in the development of antiandrogen drug resistance in prostate cancer (CaP) through PAMactivated autocrine growth. On the basis of the previous finding that many lung cancer cell lines excrete PAM into their culture media, this study investigates PAM levels in media collected from human CaP cell line cultures. Androgen-independent DU145 and PC-3 prostate cancer cell lines exhibited readily detectable levels of PAM activity in extracts and media, whereas the androgen-dependent LNCaP cell line showed little or no activity. Because of the much larger volume of media versus cell extracts, more than 90% of the total PAM activity was located in the media for both the PC-3 and DU145 cell lines, providing a readily accessible source of CaP PAM. A simple, scalable method to obtain PAM from the culture media of androgen-independent human prostate cancer cell lines is described in this article. This approach provides a much easier means of collecting CaP-derived PAM than previously described cell fractionation procedures and should facilitate the investigations of the role and targeting of PAM in hormone-independent CaP.