IRG and GBP Host Resistance Factors Target Aberrant, “Non-self” Vacuoles Characterized by the Missing of “Self” IRGM Proteins

Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with “non-self” PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on “self” organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of “self” IRGM proteins from these structures.     

Subcellular localization of proteases in wheat and corn mesophyll protoplasts

Mesophyll protoplasts were isolated from the leaves of wheat and corn seedlings. After purification the protoplasts were judged to be free of contaminating proteases in the isolation enzymes based on specific activity of the proteases in comparison to leaf tissue and their response to inhibitors that “differentiated” between leaf and isolation enzyme proteases. Wheat protoplasts showed rates of photosynthesis of 95 to 100 micromoles O₂ per milligram chlorophyll per hour, while corn exhibited rates of 35 to 85 micromoles O₂ per milligram chlorophyll per hour, indicating the intactness of the chloroplasts within the protoplasts. These chloroplasts were isolated from the protoplasts using the procedure of Robinson and Walker (1979 Arch Biochem Biophys 196: 319-323). Yields of 91 and 82% intact chloroplasts were obtained from wheat and corn, respectively, based on the distribution of ribulose bisphosphate carboxylase in wheat and NADP-malate dehydrogenase in corn. Vacuoles were obtained from the protoplasts using a modification of the techniques of Wagner and Siegelman (1975 Science 190: 1298-1299) and Saunders (1979 Plant Physiol 64: 74-78). The vacuoles were at least 98% free of protoplast contamination as determined by assaying for “marker” enzymes of chloroplasts, mitochondria, and endoplasmic reticulum. Assuming one vacuole per protoplast, the vacuoles contained 4% of the soluble protein of the protoplasts in wheat and 8% in corn. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuoles. Although the isolated chloroplasts always retained about 13% of the proteolytic activity of the protoplasts, this could be accounted for by that which became associated with the chloroplasts during their isolation.     

Placebo Trends across the Border: US versus Canada

Background

Physicians around the world report to using placebos in a variety of situations and with varying degrees of frequency. Inconsistent methodologies, however, complicate interpretation and prevent direct comparisons across studies. While US- and Canada-based physicians share similar professional standards, Canada harbours a less-litigious universal healthcare model with no formal placebo-related policy—factors that may impact how physicians view and use placebos.

Methods

To compare American and Canadian data, we circulated an online survey to academic physicians practicing in Canada, collected anonymous responses, and extracted those of internists and rheumatologists for comparison to US data obtained through parallel methodologies.

Results

Whereas our data show overall concordance across the border—from definitions to ethical limitations and therapeutic potential—differences between American- and Canadian-based placebo practices merit acknowledgement. For example, compared to 45%-80% among US-based respondents, only 23±7% of Canada-based respondents reported using placebos in clinical practice. However, 79±7% of Canada-respondents—a figure comparable to US data—professed to prescribing at least one form of treatment without proven or expected efficacy. Placebo interventions including unwarranted vitamins and herbal supplements (impure placebos) as well as sugar pills and saline injections (pure placebos) appear more common in Canada, where more doctors described placebos as “placebos” (rather than “medications”) and used them as a “diagnostic” tool (rather than a means of placating patient demands for treatment).

Interpretation

Cross-border variation in the use of clinical placebos appears minor despite substantial differences in health care delivery system, malpractice climate, and placebo-related policy. The prevalence of impure placebos in both Canadian and US clinics raises ethical and practical questions currently unaddressed by policy and warranting investigation.     

The Effectiveness of a ‘Train the Trainer’ Model of Resuscitation Education for Rural Peripheral Hospital Doctors in Sri Lanka

Background

Sri Lankan rural doctors based in isolated peripheral hospitals routinely resuscitate critically ill patients but have difficulty accessing training. We tested a train-the-trainer model that could be utilised in isolated rural hospitals.

Methods

Eight selected rural hospital non-specialist doctors attended a 2-day instructor course. These “trained trainers” educated their colleagues in advanced cardiac life support at peripheral hospital workshops and we tested their students in resuscitation knowledge and skills pre and post training, and at 6- and 12-weeks. Knowledge was assessed through 30 multiple choice questions (MCQ), and resuscitation skills were assessed by performance in a video recorded simulated scenario of a cardiac arrest using a Resuci Anne Skill Trainer mannequin.

Results/Discussion/Conclusion

Fifty seven doctors were trained. Pre and post training assessment was possible in 51 participants, and 6-week and 12-week follow up was possible for 43, and 38 participants respectively. Mean MCQ scores significantly improved over time (p<0.001), and a significant improvement was noted in “average ventilation volume”, “compression count”, and “compressions with no error”, “adequate depth”, “average depth”, and “compression rate” (p<0.01). The proportion of participants with compression depth ≥40mm increased post intervention (p<0.05) and at 12-week follow up (p<0.05), and proportion of ventilation volumes between 400-1000mls increased post intervention (p<0.001). A significant increase in the proportion of participants who “checked for responsiveness”, “opened the airway”, “performed a breathing check”, who used the “correct compression ratio”, and who used an “appropriate facemask technique” was also noted (p<0.001). A train-the-trainer model of resuscitation education was effective in improving resuscitation knowledge and skills in Sri Lankan rural peripheral hospital doctors. Improvement was sustained to 12 weeks for most components of resuscitation knowledge and skills. Further research is needed to identify which components of training are most effective in leading to sustained improvement in resuscitation.     

Cell vacuolation induced by Haemophilus influenzae supernatants in HEp-2 cells

Haemophilus influenzae belongs to respiratory tract microbiota. We observed vacuoles formation in previous studies with H. influenzae culture supernatants, so in this work we characterised that cytotoxic effect. We observed an abundant production of acidic cytoplasmic vacuoles due to the presence of a “vacuolating factor” in H. influenzae supernatants which was characterised as thermolabile. Greatest vacuolating activity was observed when utilizing the fraction > 50 kDa. The presence of a large number of vacuoles in HEp-2 cells was verified by transmission electron microscopy and some vacuoles were identified with a double membrane and/or being surrounded by ribosomes. These results suggest similar behaviour to that of vacuolating effects described by autotransporter proteins an undescribed cytotoxic effect induced by H. influenzae .     

Blood Pressure: A Consideration of Terminology

Concepts of hypertension have changed and changes in terminology to reflect this state of affairs are suggested. Statistically, the best mortality experience is associated with blood pressure commonly regarded as subnormal, and increments of blood pressure above this level are associated with progressive increases in mortality. The terms “normal”, “benign” and “essential” in relation to blood pressure should be abandoned. “Optimal”, “acceptable” and “hypertensive” ranges of blood pressure are suggested. Hypertension is regarded as a symptom of disease, rather than as a disease in itself, and “hypertension”, when used as a diagnostic label, should be qualified always by the primary disease, if known, or by the modifying phrase, “of unknown cause”, if not known.     

Isolation and identification of lymphocytic and myelogenous leukemia-specific sequences in genomes of gibbon oncornaviruses

Five gibbon ape leukemia virus substrains (two from gibbons with lymphocytic leukemia and three from gibbons with myelogenous leukemia) were examined for unique genomic sequences specific for each form of leukemia. By using sequential adsorption procedures, the genome from each gibbon ape leukemia virus was fractionated into four sets of distinct nucleotide sequences. Based on their hybridization specificities toward DNAs of leukemic tissues, these sequences were designated as follows: (i) “COM,” (ii) “LYM” or “MYE,” (iii) “UNI,” and (iv) “UND.” The COM fraction represented sequences common to all of the viral genomes. The LYM fraction, which was isolated only from gibbon ape leukemia viruses associated with lymphocytic leukemia, represented genomic sequences associated with lymphocytic leukemia since the RNA hybridized at a 4- to 15-fold-higher rate to infected tissue DNA from lymphocytic leukemic gibbons than to infected tissue DNA from myelogenous leukemic gibbons. The MYE fraction, which was isolated only from gibbon ape leukemia viruses associated with myelogenous leukemia, represented genomic sequences associated with myelogenous leukemia since the RNA hybridized at a 5- to 15-fold-higher rate to infected tissue DNA from myelogenous leukemic gibbons than to infected tissue DNA from lymphocytic leukemic gibbons. The UNI fraction contained sequences unique to one virus substrain. The UND fraction contained sequences which varied depending upon the substrains involved in the adsorption procedures. These findings suggest that each gibbon ape leukemia virus examined in this study contains subgenomic sequences that are specifically identifiable only with the form of leukemia from which the virus was isolated.     

Understanding Neighborhood Environment Related to Hong Kong Children’s Physical Activity: A Qualitative Study Using Nominal Group Technique

Background

Relationships between the neighborhood environment and children’s physical activity have been well documented in Western countries but are less investigated in ultra-dense Asian cities. The aim of this study was to identify the environmental facilitators and barriers of physical activity behaviors among Hong Kong Chinese children using nominal group technique.

Methods

Five nominal groups were conducted among 34 children aged 10–11 years from four types of neighborhoods varying in socio-economic status and walkability in Hong Kong. Environmental factors were generated by children in response to the question “What neighborhood environments do you think would increase or decrease your willingness to do physical activity?” Factors were prioritized in order of their importance to children’s physical activity.

Results

Sixteen unique environmental factors, which were perceived as the most important to children’s physical activity, were identified. Factors perceived as physical activity-facilitators included “Sufficient lighting”, “Bridge or tunnel”, “Few cars on roads”, “Convenient transportation”, “Subway station”, “Recreation grounds”, “Shopping malls with air conditioning”, “Fresh air”, “Interesting animals”, and “Perfume shop”. Factors perceived as physical activity-barriers included “People who make me feel unsafe”, “Crimes nearby”, “Afraid of being taken or hurt at night”, “Hard to find toilet in shopping mall”, “Too much noise”, and “Too many people in recreation grounds”.

Conclusions

Specific physical activity-related environmental facilitators and barriers, which are unique in an ultra-dense city, were identified by Hong Kong children. These initial findings can inform future examinations of the physical activity-environment relationship among children in Hong Kong and similar Asian cities.     

Pathological States and Criminal Responsibility

The doctor is embarrassed in court when asked to testify to the effects of illness on a defendant''s capability “of appreciating the nature and quality of an act or omission or of knowing that an act or omission is wrong”. The source of his difficulty is traced to the legal concepts of “guilt”, “crime” and “punishment” which imply a legal view of man at variance with our modern biological view. To abolish this discrepancy we need not accept a medical model for criminal law where “crime” is analogous to “disease”, and “punishment” to “treatment”. A pragmatic approach to the handling of the criminal could exclude the notions of “guilt” and “punishment” and yet fulfil the rational goals of protecting society from the criminal and of compensating his victims.     

Fueling the Flames: Mammalian Programmed Necrosis in Inflammatory Diseases

Programmed necrosis or necroptosis is an inflammatory form of cell death driven by TNF-like death cytokines, toll-like receptors, and antigen receptors. Unlike necrosis induced by physical trauma, a dedicated pathway is involved in programmed necrosis. In particular, a kinase complex composed of the receptor interacting protein kinase 1 (RIPK1) and RIPK3 is a central step in necrotic cell death. Assembly and activation of this RIPK1–RIPK3 “necrosome” is critically controlled by protein ubiquitination, phosphorylation, and caspase-mediated cleavage events. The molecular signals cumulate in formation of intracellular vacuoles, organelle swelling, internal membrane leakage, and eventually plasma membrane rupture. These morphological changes can result in spillage of intracellular adjuvants to promote inflammation and further exacerbate tissue injury. Because of the inflammatory nature of necrosis, it is an attractive pathway for therapeutic intervention in acute inflammatory diseases.Necrosis and tissue inflammation are two tightly linked phenomena. Cell injury induced by excessive trauma such as heat shock and osmotic shock can result in cell death with “necrotic morphology.” These relatively nonspecific means to trigger necrosis have contributed to the notion that necrosis is caused by excessive insults and does not involve elaborate intracellular signaling pathways. In contrast to the notion that necrosis is associated with harmful pathologies, recent work indicates that necrosis can have beneficial roles in certain biological responses. Proteomic approaches and RNA interference screens have identified several crucial regulators of necrosis induced by TNF-like death cytokines. Because a dedicated molecular circuitry is involved, the terms “programmed necrosis” and “necroptosis” have been used to distinguish these types of necrotic cell death from necrosis induced by physical trauma or insults. Here I will discuss the molecular pathway that regulates programmed necrosis/necroptosis. For the sake of simplicity, we will use the term necrosis to refer to programmed necrosis induced by defined death cytokines.     

Effect of Energy Under-Reporting on Secular Trends of Dietary Patterns in a Mediterranean Population

BackgroundDiet is an important factor in the prevention of chronic diseases. Analysis of secular trends of dietary patterns can be biased by energy under-reporting. Therefore, the objective of the present study was to analyse the impact of energy under-reporting on dietary patterns and secular trends in dietary patterns defined by cluster analysis.ResultsThree clusters, “healthy”, “mixed” and “western”, were identified for both surveys. The “mixed” cluster was the predominant cluster in both surveys. Excluding EUR reduced the proportion of the “mixed” cluster up to 6.40% in the 2000 survey; this caused secular trend increase in the prevalence of the “mixed” pattern. Cross-classification analysis of all participants and PER’ data showed substantial agreement in cluster assignments: 68.7% in 2000 and 84.4% in 2005. Excluding EUR did not cause meaningful (≥15%) changes in the “healthy” pattern. It provoked changes in consumption of some food groups in the “mixed” and “western” patterns: mainly decreases of unhealthy foods within the 2000 and increases of unhealthy foods within the 2005 surveys. Secular trend effects of EUR were similar to those within the 2005 survey. Excluding EUR reversed the direction of secular trends in consumption of several food groups in PER in the “mixed” and “western” patterns.ConclusionsEUR affected distribution of participants between dietary patterns within and between surveys, secular trends in food group consumption and amount of food consumed in all, but not in the “healthy” pattern. Our findings emphasize threats from energy under-reporting in dietary data analysis.     

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the “sick sinus syndrome” is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. “Biological pacemakers” generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines “forward programming” of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of >80% physiologically functional pacemaker cells. These “induced-sinoatrial-bodies” (“iSABs”) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.     

Certifying achievement in the control of Chagas disease native vectors: what is a viable scenario?

As an evaluation scheme, we propose certifying for “control”, as alternative to “interruption”, of Chagas disease transmission by native vectors, to project a more achievable and measurable goal and sharing good practices through an “open online platform” rather than “formal certification” to make the key knowledge more accumulable and accessible.     

Hospital Infections Caused by a Group of Recently Recognized Strains of Staphylococcus aureus

A survey was made of the phage-types of staphylococci responsible for cross-infection in a large veterans'' hospital between 1961 and 1964. An earlier survey had shown that in 1959 most of the infections were caused by staphylocci of the “80/81/82” group. In 1961 a new group of staphylococci were first recognized and provisionally designated as “Atypical Group III” strains; these were non-typable by the usual typing phages but showed inhibition patterns with some of the Group III phages. The “Atypical Group III” staphylococci all showed one or other of four patterns of multiple antibiotic resistance. By 1963 these resistant “Atypical Group III” staphylococci had become more frequent than “80/81/82” strains as causative agents of cross-infection, although both groups have continued to cause infections in the hospital. “Atypical Group III” strains mainly infected surgical wounds and skin ulcers, whereas “80/81/82” strains commonly produced primary skin sepsis, such as boils.     

Characterization of the Denitrification-Associated Phosphorus Uptake Properties of “Candidatus Accumulibacter phosphatis” Clades in Sludge Subjected to Enhanced Biological Phosphorus Removal

To characterize the denitrifying phosphorus (P) uptake properties of “Candidatus Accumulibacter phosphatis,” a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of “Ca. Accumulibacter” and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescence in situ hybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized “Ca. Accumulibacter” subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria (Dechloromonas [from 1.2% to 19.2%] and “Candidatus Competibacter phosphatis” [from 16.4% to 20.0%]), while the overall “Ca. Accumulibacter” abundance decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses was performed to characterize the denitrifying P uptake properties of the “Ca. Accumulibacter” clades. In FISH/MAR experiments using slightly diluted sludge (∼0.5 g/liter), all “Ca. Accumulibacter” clades successfully took up phosphorus in the presence of nitrate. However, the “Ca. Accumulibacter” clades showed no P uptake in the presence of nitrate when the sludge was highly diluted (∼0.005 g/liter); under these conditions, reduction of nitrate to nitrite did not occur, whereas P uptake by “Ca. Accumulibacter” clades occurred when nitrite was added. These results suggest that the “Ca. Accumulibacter” cells lack nitrate reduction capabilities and that P uptake by “Ca. Accumulibacter” is dependent upon nitrite generated by associated nitrate-reducing bacteria such as Dechloromonas and “Ca. Competibacter.”     

The Histological Components of the Phoniatrical Body-Cover Model in Minipigs of Different Ages

Pigs are models in human phoniatry. However, features of maturation and ageing have not been considered with regard to the so-called body-cover model in this species. Therefore, the glottis of “young” (2–3 months; n = 6) and “old” (4–7 years; n = 6) minipigs was investigated. Their cranial (CraF) and caudal (CauF) vocal folds were histomorphometrically and stratigraphically analysed with emphasis on their amounts of collagen structures and elastic fibres. A dense subepithelial layer (SEL) was a distinct feature of CraF and CauF of both age groups; it was spread upon the underlying loose, flexible “cover” like a fibro-elastic membrane. The “cover” was characterised by the so-called superficial layer (SL), which was distinctly loose in the “young” minipigs, but had a much denser texture in the “old” minipigs. Here, the SL was dominated by elastic fibres in the CraF, but was of mixed qualities (collagenous and elastic) in the CauF. The structural requirements for the SL’s function as a loose “cover” were thus met only in the “young” animals. A clearly demarcated intermediate layer (IL)—characterised by high amounts of elastic fibres (as in humans)—was only found in the CraF of the “young” animals. In the “old” animals, it had lost its demarcation. In the depth of the CraF of the “old” animals, many thick collagen fibre bundles were detected in a location equivalent to that of the vocal muscle in the CauF. The development of their large diameters was interpreted as part of the maturation process, thereby supporting the hypothesis of their functional importance as a component of the “body.” In the CauF, the amounts of collagen structures increased throughout the entire lamina propria, resulting in a loss of demarcated stratigraphical subdivisions in the “old” minipigs. This situation resembled that described in the vocal fold of geriatric humans.     

END RESULTS IN PAROTID TUMORS

Management of parotid tumors can be based on a clinical classification of these lesions as being either “encapsulated” or infiltrating.The Warthin tumor (papillary cystadenolymphomatosum) is a benign encapsulated tumor, often occurring multicentrically or bilaterally especially in the lower pole area of the parotid. It is characterized clinically by its softness and fluctuation in size and a high incidence in elderly men.The so-called “capsule” of well demarcated mixed and mucoepidermoid tumors is represented by a condensation of host fibrous stroma, in the interstices of which tumor cells may be present.The “encapsulated” tumors should be excised with a “shell” of uninvolved parotid tissue. To do this safely, the facial nerve should first be isolated.Total parotidectomy is necessary only if the size of the tumor, the multiplicity of recurrences, or the infiltrating nature of the tumor are such that complete eradication of the primary site must be done.Radical neck dissection is never performed electively except in the small group of nonencapsulated infiltrating primary lesions.In a series of cases of previously untreated parotid tumors treated by the method outlined, the local parotid recurrence rate was less than 1 per cent.     

Time-Lapse Photography of Bacillus subtilis L-Forms Replicating in Liquid Medium

Phase-contrast observations indicate that an L-form of Bacillus subtilis divides in liquid medium by a “budding-like” mechanism without participation of “large bodies” or “elementary bodies.”     

Genome Wide Association Study Identifies New Loci Associated with Undesired Coat Color Phenotypes in Saanen Goats

This paper reports a quantitative genetics and genomic analysis of undesirable coat color patterns in goats. Two undesirable coat colors have routinely been recorded for the past 15 years in French Saanen goats. One fifth of Saanen females have been phenotyped “pink” (8.0%) or “pink neck” (11.5%) and consequently have not been included in the breeding program as elite animals. Heritability of the binary “pink” and “pink neck” phenotype, estimated from 103,443 females was 0.26 for “pink” and 0.21 for “pink neck”. Genome wide association studies (using haplotypes or single SNPs) were implemented using a daughter design of 810 Saanen goats sired by 9 Artificial Insemination bucks genotyped with the goatSNP50 chip. A highly significant signal (-log10pvalue = 10.2) was associated with the “pink neck” phenotype on chromosome 11, suggesting the presence of a major gene. Highly significant signals for the “pink” phenotype were found on chromosomes 5 and 13 (-log10p values of 7.2 and, 7.7 respectively). The most significant SNP on chromosome 13 was in the ASIP gene region, well known for its association with coat color phenotypes. Nine significant signals were also found for both traits. The highest signal for each trait was detected by both single SNP and haplotype approaches, whereas the smaller signals were not consistently detected by the two methods. Altogether these results demonstrated a strong genetic control of the “pink” and “pink neck” phenotypes in French Saanen goats suggesting that SNP information could be used to identify and remove undesired colored animals from the breeding program.