ARTICLE WATCH

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, Hartwell Center, St. Jude Children’s Research Hospital, 332 North Lauderdale St., Memphis TN 38105-2794. Tel: (901) 495-4844; Fax: (901) 495-2945; email: gro.edujts@rethgualS.evilC or to any member of the editorial board. Article summaries reflect the reviewer’s opinions and not necessarily those of the Association.     

ARTICLE WATCH

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive slaughter, hartwell center, St. Jude children’s Research hospital, 332 north Lauderdale St., Memphis TN 38105–2794. Tel: (901) 495–4844; Fax: (901) 495–2945; email: gro.edujts@rethguals.evilc or to any member of the editorial board. Article summaries reflect the reviewer’s opinions and not necessarily those of the Association.     

Article Watch,April 2010

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information about articles they feel are important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 279 William St., Athens, GA 30607-1777, USA. Tel.: (706) 369-5945: Fax: (706) 369-5936; E-mail: ude.gcm.liam@rethgualsc; or to any member of the editorial board. Article summaries reflect the reviewer''s opinions and not necessarily those of the association.     

Article Watch, April 2012

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, Georgia Health Sciences University-University of Georgia Medical Partnership, 279 William St., Athens GA 30607-1777. Phone: 706-369-5945; Fax: 706-369-5936; E-mail: ude.agu@thgualsc; or to any member of the editorial board. Article summaries reflect the reviewer''s opinions and not necessarily those of the association.     

Article Watch,December 2009

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information about articles they feel is important and useful to Clive Slaughter, MCG-UGA Medical Partnership, 279 William St., Athens, GA 30607-1777, USA; Tel.: (706) 369-5945; Fax: (706) 369-5936; E-mail: cslaughter@mail.mcg.edu; or to any member of the editorial board. Article summaries reflect the reviewer’s opinions and not necessarily those of the association.     

Article Watch: September 2015

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, Georgia Regents University-University of Georgia Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Phone: 706-713-2216; Fax: 706-713-2221; E-mail: ude.agu@thgualsc; or to any member of the Editorial Board. Article summaries reflect the reviewer’s opinions and not necessarily those of the association.     

Article Watch: July 2015

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, Georgia Regents University-University of Georgia Medical Partnership, 1425 Prince Ave., Athens, GA 30606, USA. Phone: 706-713-2216; Fax: 706-713-2221; E-mail: ude.agu@thgualsc; or to any member of the Editorial Board. Article summaries reflect the reviewer’s opinions and not necessarily those of the association.     

Risk of vascular events in patients with polymyalgia rheumatica

Background:

Polymyalgia rheumatica is one of the most common inflammatory rheumatologic conditions in older adults. Other inflammatory rheumatologic disorders are associated with an excess risk of vascular disease. We investigated whether polymyalgia rheumatica is associated with an increased risk of vascular events.

Methods:

We used the General Practice Research Database to identify patients with a diagnosis of incident polymyalgia rheumatica between Jan. 1, 1987, and Dec. 31, 1999. Patients were matched by age, sex and practice with up to 5 patients without polymyalgia rheumatica. Patients were followed until their first vascular event (cardiovascular, cerebrovascular, peripheral vascular) or the end of available records (May 2011). All participants were free of vascular disease before the diagnosis of polymyalgia rheumatica (or matched date). We used Cox regression models to compare time to first vascular event in patients with and without polymyalgia rheumatica.

Results:

A total of 3249 patients with polymyalgia rheumatica and 12 735 patients without were included in the final sample. Over a median follow-up period of 7.8 (interquartile range 3.3–12.4) years, the rate of vascular events was higher among patients with polymyalgia rheumatica than among those without (36.1 v. 12.2 per 1000 person-years; adjusted hazard ratio 2.6, 95% confidence interval 2.4–2.9). The increased risk of a vascular event was similar for each vascular disease end point. The magnitude of risk was higher in early disease and in patients younger than 60 years at diagnosis.

Interpretation:

Patients with polymyalgia rheumatica have an increased risk of vascular events. This risk is greatest in the youngest age groups. As with other forms of inflammatory arthritis, patients with polymyalgia rheumatica should have their vascular risk factors identified and actively managed to reduce this excess risk.Inflammatory rheumatologic disorders such as rheumatoid arthritis,^1,2 systemic lupus erythematosus,^2,3 gout,⁴ psoriatic arthritis^2,5 and ankylosing spondylitis^2,6 are associated with an increased risk of vascular disease, especially cardiovascular disease, leading to substantial morbidity and premature death.^2–6 Recognition of this excess vascular risk has led to management guidelines advocating screening for and management of vascular risk factors.^7–9Polymyalgia rheumatica is one of the most common inflammatory rheumatologic conditions in older adults,¹⁰ with a lifetime risk of 2.4% for women and 1.7% for men.¹¹ To date, evidence regarding the risk of vascular disease in patients with polymyalgia rheumatica is unclear. There are a number of biologically plausible mechanisms between polymyalgia rheumatica and vascular disease. These include the inflammatory burden of the disease,^12,13 the association of the disease with giant cell arteritis (causing an inflammatory vasculopathy, which may lead to subclinical arteritis, stenosis or aneurysms),¹⁴ and the adverse effects of long-term corticosteroid treatment (e.g., diabetes, hypertension and dyslipidemia).^15,16 Paradoxically, however, use of corticosteroids in patients with polymyalgia rheumatica may actually decrease vascular risk by controlling inflammation.¹⁷ A recent systematic review concluded that although some evidence exists to support an association between vascular disease and polymyalgia rheumatica,¹⁸ the existing literature presents conflicting results, with some studies reporting an excess risk of vascular disease^19,20 and vascular death,^21,22 and others reporting no association.^23–26 Most current studies are limited by poor methodologic quality and small samples, and are based on secondary care cohorts, who may have more severe disease, yet most patients with polymyalgia rheumatica receive treatment exclusively in primary care.²⁷The General Practice Research Database (GPRD), based in the United Kingdom, is a large electronic system for primary care records. It has been used as a data source for previous studies,²⁸ including studies on the association of inflammatory conditions with vascular disease²⁹ and on the epidemiology of polymyalgia rheumatica in the UK.³⁰ The aim of the current study was to examine the association between polymyalgia rheumatica and vascular disease in a primary care population.     

Dual Phosphorylation of Btk by Akt/Protein Kinase B Provides Docking for 14-3-3ζ, Regulates Shuttling,and Attenuates both Tonic and Induced Signaling in B Cells

Bruton''s tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.     

Consumption of non–cow’s milk beverages and serum vitamin D levels in early childhood

Background:

Vitamin D fortification of non–cow’s milk beverages is voluntary in North America. The effect of consuming non–cow’s milk beverages on serum 25-hydroxyvitamin D levels in children is unclear. We studied the association between non–cow’s milk consumption and 25-hydroxyvitamin D levels in healthy preschool-aged children. We also explored whether cow’s milk consumption modified this association and analyzed the association between daily non–cow’s milk and cow’s milk consumption.

Methods:

In this cross-sectional study, we recruited children 1–6 years of age attending routinely scheduled well-child visits. Survey responses, and anthropometric and laboratory measurements were collected. The association between non–cow’s milk consumption and 25-hydroxyvitamin D levels was tested using multiple linear regression and logistic regression. Cow’s milk consumption was explored as an effect modifier using an interaction term. The association between daily intake of non–cow’s milk and cow’s milk was explored using multiple linear regression.

Results:

A total of 2831 children were included. The interaction between non–cow’s milk and cow’s milk consumption was statistically significant (p = 0.03). Drinking non–cow’s milk beverages was associated with a 4.2-nmol/L decrease in 25-hydroxyvitamin D level per 250-mL cup consumed among children who also drank cow’s milk (p = 0.008). Children who drank only non–cow’s milk were at higher risk of having a 25-hydroxyvitamin D level below 50 nmol/L than children who drank only cow’s milk (odds ratio 2.7, 95% confidence interval 1.6 to 4.7).

Interpretation:

Consumption of non–cow’s milk beverages was associated with decreased serum 25-hydroxyvitamin D levels in early childhood. This association was modified by cow’s milk consumption, which suggests a trade-off between consumption of cow’s milk fortified with higher levels of vitamin D and non–cow’s milk with lower vitamin D content.Goat’s milk and plant-based milk alternatives made from soy, rice, almonds, coconut, hemp, flax or oats (herein called “non–cow’s milk”) are increasingly available on supermarket shelves. Many consumers may be switching from cow’s milk to these beverages.^1–3 Parents may choose non–cow’s milk beverages for their children because of perceived health benefits. However, it is unclear whether they offer health advantages over cow’s milk or, alternatively, whether they increase the risk of nutritional inadequacy.In the United States and Canada, cow’s milk products are required to contain about 40 IU of vitamin D per 100 mL, making it the major dietary source of vitamin D for children.^4–8 The only other food source with mandatory vitamin D fortification in Canada is margarine, which is required to contain 53 IU per 10 mL (10 g).⁵ Fortification of non–cow’s milk beverages with vitamin D is also possible, but it is voluntary in both countries. Furthermore, there is little regulation on the vitamin D content even if such beverages are fortified.^5,6,9We conducted a study to test the association between total daily consumption of non–cow’s milk and serum 25-hydroxyvitamin D levels in a population of healthy urban preschool-aged children attending routinely scheduled well-child visits. We hypothesized that vitamin D stores would be lower in children who consume non–cow’s milk. The secondary objectives were to explore how consumption of cow’s milk might modify this association and to study the association between daily intake of non–cow’s milk and cow’s milk.     

Biophysical reviews: call for nominations for the 2023 Michèle Auger Award

This Editorial for Issue 3 (Vol. 14 2022) of Biophysical Reviews first describes the Issue’s contents (five commentaries/editorials within the front matter and seven review/letter articles appearing within the main body) before going on to discuss a number of matters of potential importance to the journal and its readers. Amongst this second tranche of content is the opening of the call for nominations for the 2023 Michèle Auger Award for Young Scientists’ Independent Research.

Created in 2009, Biophysical Reviews is the flagship journal of IUPAB, the International Union for Pure and Applied Biophysics (IUPAB 2022). Each of the journal’s six issues per year are divided into a front section, dealing with journal process and points of interest to biophysicists and IUPAB society members, and a main section, that presents scientific Letters and Reviews that are written by recognized experts in the field and are selected with an eye to providing an as wide as possible level of international participation. The first duty of each Issue’s editorial is to provide a precis of both the non-scientific and scientific articles appearing within, and we carry this out forthwith.     

Prognostic Value of Cancer Stem Cell Marker Aldehyde Dehydrogenase in Ovarian Cancer: A Meta-Analysis

Objective

Aldehyde dehydrogenase (ALDH) has recently been reported as a marker of cancer stem-like cells in ovarian cancer. However, the prognostic role of ALDH in ovarian cancer still remains controversial. In this study, we aimed to evaluate the association between the expression of ALDH and the outcome of ovarian cancer patients by performing a meta-analysis.

Methods

We systematically searched for studies investigating the relationships between ALDH expression and outcome of ovarian cancer patients. Only articles in which ALDH expression was detected by immunohistochemical staining were included. A meta-analysis was performed to generate combined hazard ratios (HRs) with 95% confidence intervals (CIs) for overall survival (OS) and disease-free survival (DFS).

Results

A total of 1,258 patients from 7 studies (6 articles) were included in the analysis. Our results showed that high ALDH expression in patients with ovarian cancer was associated with poor prognosis in terms of Os (HR, 1.25; 95% CI, 1.07-1.47; P = 0.005) and DFS (HR, 1.58; 95% CI, 1.00-2.49; P = 0.052), though the difference for DFS was not statistically significant. In addition, there was no evidence of publication bias as suggested by Begg’s and Egger’s tests (Begg’s test, P = 0.707; Egger’s test, P = 0.355).

Conclusion

The present meta-analysis indicated that elevated ALDH expression was associated with poor prognosis in patients with ovarian cancer.     

Efficacy and safety of the ��mother��s kiss�� technique: a systematic review of case reports and case series

Background:

Foreign bodies lodged in the nasal cavity are a common problem in children, and their removal can be challenging. The published studies relating to the “mother’s kiss” all take the form of case reports and case series. We sought to assess the efficacy and safety of this technique.

Methods:

We performed a comprehensive search of the Cochrane library, MEDLINE, CINAHL, Embase, AMED Complementary and Allied Medicine and the British Nursing Index for relevant articles. We restricted the results to only those studies involving humans. In addition, we checked the references of relevant studies to identify further possibly relevant studies. We also checked current controlled trials registers and the World Health Organization search portal. Our primary outcome measures were the successful extraction of the foreign object from the nasal cavity and any reported adverse effects. We assessed the included studies for factors that might predict the chance of success of the technique. We assessed the validity of each study using the Newcastle–Ottawa scale.

Results:

Eight relevant published articles met our inclusion criteria. The overall success rate for all of the case series was 59.9% (91/152). No adverse effects were reported.

Interpretation:

Evidence from case reports and case series suggests that the mother’s kiss technique is a useful and safe first-line option for the removal of foreign bodies from the nasal cavities of children.Nasal foreign bodies are a common problem in children, most frequently occurring between the ages of 2 and 5 years, and their removal can be challenging.^1,2 Children in this age group have a natural fear of the unknown, and providing care to them can be difficult, especially if previous attempts to remove the foreign body have been painful.Potential complications, most notably the risk of aspiration of the foreign body, mean that objects should be removed from the nasal cavity in a timely fashion. Various techniques have been described: instrumental extraction (using a hook or nasal forceps), suction, balloon catheters,³ cyanoacrylate glue⁴ and various positive-pressure techniques, the simplest of which is to ask the child to blow his or her nose while occluding the unaffected nostril. However, this technique is only possible for older children.⁵ Alternatively, a bag valve mask can be applied over the child’s face, the bag then squeezed to apply a puff of air into the child’s mouth;⁶ a male–male tube adaptor can be attached to an oxygen or air outlet via oxygen tubing placed in the unaffected nostril;⁷ or the “mother’s kiss” or “parent’s kiss” technique can be used.The mother’s kiss was first described in 1965 by Vladimir Ctibor, a general practitioner from New Jersey.⁸ The mother, or other trusted adult, places her mouth over the child’s open mouth, forming a firm seal as if about to perform mouth-to-mouth resuscitation. While occluding the unaffected nostril with a finger, the adult then blows until they feel the resistance caused by closure of the child’s glottis, at which point the adult gives a sharp exhalation to deliver a short puff of air into the child’s mouth. This puff of air passes through the nasopharynx, out through the unoccluded nostril and, if successful, results in the expulsion of the foreign body. The procedure is fully explained to the adult before starting, and the child is told that the parent will give him or her a “big kiss” so that minimal distress is caused to the child. The procedure can be repeated a number of times if not initially successful. A modified mother’s kiss technique has been described,⁹ which involves the adult blowing into a straw in the child’s mouth. We did not include this technique in our review.Although the mother’s kiss technique has been sporadically mentioned in the literature in case reports and case series, it has yet to gain widespread acceptance. It is not a suitable intervention for evaluation using a randomized controlled trial, because there is no appropriate control group: nontreatment is unacceptable, and there is no gold standard for comparison.Randomized controlled trials are considered to be the best trial design, but some treatments result in a dramatic effect that may not require randomized trials.¹⁰ The mother’s kiss technique falls into this category, because the foreign body will not usually move without intervention. Hence, case reports are sufficient to show that the technique sometimes works. However, a systematic review is needed to clarify how often it works and under what circumstances.We sought to examine the existing evidence for the efficacy and safety of the mother’s kiss technique, to help clinicians understand this evidence and to confirm or refute the appropriateness of current practice.Although systematic reviews of randomized controlled clinical trials are now common, it is rare to see a report of a systematic review of case reports or case series, and the methods for performing such a review are less clearly defined and tested. The principal elements of a systematic review are the location, appraisal and synthesis of individual studies; however, there are pitfalls to traditional systematic reviews of clinical trials that can introduce bias and inaccuracy in the results, which must be avoided. For this systematic review of case reports and case series, we were ever mindful of the rationale behind the stages in systematic reviews of clinical trials and endeavoured to apply the same principles to reduce bias and improve accuracy.     

Systematic Review: Representativeness of Participants in RCTs of Acetylcholinesterase Inhibitors

Objective

To determine whether there are differences in age and sex distribution and presence of comorbidities between participants included in randomized controlled trials of acetylcholinesterase inhibitors and nationwide cohort of persons with Alzheimer’s disease.

Methods

PubMed, Scopus and Cochrane Library databases were searched for original articles from their inception to January 4, 2015. Double-blind randomized controlled trials with donepezil, rivastigmine or galantamine compared to placebo in participants with Alzheimer’s disease were included. Data from a nationwide cohort of persons with clinically verified diagnoses of Alzheimer’s disease was defined as a reference population.

Results

128 full-text articles were assessed for eligibility, 31 of them fulfilled criteria. Mean age of participants in randomized controlled trials (n = 15,032) was 5.8 years lower (95% CI 5.7 to 5.9, P < 0.001), compared to the mean age of 79.7 years in the reference population with Alzheimer’s disease (n = 28,093). Most of the articles did not report age distribution of participants. The proportion of women was 63.2% (9,475/14,991) in randomized controlled trials and 67.8% (19,043/28,093) (P < 0.001) in the reference population. Information on comorbidities and use of concomitant drugs were lacking or poorly reported in most articles.

Conclusions

There is a discrepancy between participants in randomized controlled trials of acetylcholinesterase inhibitors and real-life population with Alzheimer’s disease. Participants in randomized controlled trials were significantly younger. Further, more detailed reporting of age distribution, comorbidities and concomitant drugs would be important information for clinicians when evaluating conclusions from randomized controlled trials to real-life practice. The existing recommendations of inclusion of older people should be followed to ensure safe pharmacotherapy for older people.     

Women’s Awareness and Knowledge of Abortion Laws: A Systematic Review

Background

Incorrect knowledge of laws may affect how women enter the health system or seek services, and it likely contributes to the disconnect between official laws and practical applications of the laws that influence women’s access to safe, legal abortion services.

Objective

To provide a synthesis of evidence of women’s awareness and knowledge of the legal status of abortion in their country, and the accuracy of women’s knowledge on specific legal grounds and restrictions outlined in a country’s abortion law.

Methods

A systematic search was carried for articles published between 1980–2015. Quantitative, mixed-method data collection, and objectives related to women’s awareness or knowledge of the abortion law was included. Full texts were assessed, and data extraction done by a single reviewer. Final inclusion for analysis was assessed by two reviewers. The results were synthesised into tables, using narrative synthesis.

Results

Of the original 3,126 articles, and 16 hand searched citations, 24 studies were included for analysis. Women’s correct general awareness and knowledge of the legal status was less than 50% in nine studies. In six studies, knowledge of legalization/liberalisation ranged between 32.3% - 68.2%. Correct knowledge of abortion on the grounds of rape ranged from 12.8% – 98%, while in the case of incest, ranged from 9.8% - 64.5%. Abortion on the grounds of fetal impairment and gestational limits, varied widely from 7% - 94% and 0% - 89.5% respectively.

Conclusion

This systematic review synthesizes literature on women’s awareness and knowledge of the abortion law in their own context. The findings show that correct general awareness and knowledge of the abortion law and legal grounds and restrictions amongst women was limited, even in countries where the laws were liberal. Thus, interventions to disseminate accurate information on the legal context are necessary.     

Functional Genetics in the Post-Genomics Era: Building a Better Roadmap in Drosophila

In this commentary, Rob Kulathinal describes two articles from the Perrimon lab, each describing a new online resource that can assist geneticists with the design of their RNA interference (RNAi) experiments. Hu et al.’s “UP-TORR: online tool for accurate and up-to-date annotation of RNAi reagents” and “FlyPrimerBank: An online database for Drosophila melanogaster gene expression analysis and knockdown evaluation of RNAi reagents” are published, respectively, in this month’s issues of GENETICS and G3.     

Environmental Occurrence of the Whipple’s Disease Bacterium (Tropheryma whippelii)

Whipple’s disease is a systemic disorder in which a gram-positive rod-shaped bacterium is constantly present in infected tissues. After numerous unsuccessful attempts to culture this bacterium, it was eventually characterized by 16S rRNA gene analysis to be a member of the actinomycetes. The name Tropheryma whippelii was proposed. Until now, the bacterium has only been found in infected human tissues, but there is no evidence for human-to-human transmission. Here we report the detection of DNA specific for the Whipple’s disease bacterium in 25 of 38 wastewater samples from five different sewage treatment plants in the area of Heidelberg, Germany. These findings provide the first evidence that T. whippelii occurs in the environment, within a polymicrobial community. This is in accordance with the phylogenetic relationship of this bacterium as well as with known epidemiological aspects of Whipple’s disease. Our data argue for an environmental source for infection with the Whipple’s disease bacterium.Whipple’s disease, which was first described in 1907 as intestinal lipodystrophy (18), is a multisystem disorder characterized by the presence of gram-positive, rod-shaped bacteria in infected human tissues (2). Numerous attempts to culture the bacterium associated with Whipple’s disease have failed (2), and eventually its phylogenetic position within the actinomycetes was established by 16S rRNA gene analysis (11, 19). The name Tropheryma whippelii was proposed (11).The clinical presentation of Whipple’s disease is heterogeneous. Frequently, patients suffer for years from arthralgias, chronic diarrhea, and weight loss, and less often from dementia or cardiac insufficiency. If untreated, the disease is usually fatal, but with appropriate antibiotic therapy the prognosis is favorable (2). However, the pathogenesis of Whipple’s disease appears to involve more than just an infection with T. whippelii. Immunological abnormalities are presumed to play a necessary role as predisposing factors (2, 7, 8), a view which is strengthened by the detection of T. whippelii in association with AIDS (5).Two outstanding questions in the epidemiology of Whipple’s disease are the bacterium’s natural habitat and the route of infection. Until now, T. whippelii has never been found outside of infected human hosts, and although an oral route of infection has often been suspected (10), it has not been proven. There is no evidence for human-to-human transmission.A reassessment of its phylogeny revealed a close relationship of the Whipple’s disease bacterium to typical environmental bacteria, such as the cellulomonads and the rare group B peptidoglycan organisms (6). This would support the hypothesis that T. whippelii may be a soil or water inhabitant. This may also explain the difficulties involved with its culture, as an estimated 80 to 99% of bacteria occurring in such natural environments are not culturable on artificial media (14, 17) and uncultured members of the actinomycete line of descent have been found in different environments and geographical locations (12). Soil and water bacteria tend to concentrate in sewage to form communities in which a large variety of different species are found. Therefore, the search for the Whipple’s disease bacterium was concentrated at sewage treatment plants.From September 1995 to July 1996, a total of 38 effluent samples from the sedimentation ponds of five different sewage treatment plants were examined by PCR for the presence of the Whipple’s disease bacterium. The sewage treatment plants are located within a distance of 5 to 25 km from Heidelberg, Germany (Fig. ​(Fig.1).1). Effluent was sampled in 1,000-ml single-use plastic flasks and filtered as described previously (4) in portions of 25 ml through cellulose acetate prefilters (25-mm diameter, 5-μm pore size; Sartorius, Göttingen, Germany). It was then passed through polyvinylidene fluoride filters (25-mm diameter, 0.45-μm pore size; Millipore, Bedford, Mass.). The DNA from the 0.45-μm-pore-size filters was extracted with the EnviroAmp Legionella sample preparation kit (Perkin-Elmer, Norwalk, Conn.), with the addition of 0.2% bovine serum albumin to the water in the final extraction step. Distilled water for negative controls was sampled in the same flasks and treated in the same way as the sewage. Open in a separate windowFIG. 1Map of the area around Heidelberg, Germany, displaying the rivers Rhine (Rhein) and Neckar, the relevant townships (shaded areas with names in capital letters), and the five sewage treatment plants (dots) from which samples were taken. The inset in the figure shows the localization of the area within Germany.PCR amplification and detection were performed as described in detail elsewhere (15). Primers whip1 (5′-AGAGATACGCCCCCCGCAA) and whip2 (5′-ATTCGCTCCACCTTGCGA), which amplify a fragment of 267 bp from the 16S rRNA gene of the bacterium, were used. In addition to hybridization with oligonucleotide whip3 (5′-TGGTACAGAGGGTTGCAATA), a second hybridization in a separate reaction was performed with oligonucleotide whip4 (5′-GTAATGGCGGGGACTCACAG). The specificities of primers whip1 and whip2 and of probe whip3 have been tested previously (15), and testing of probe whip4 with the same set of 37 control bacteria gave the same results as those reported for whip3 (15).Of the 38 sewage samples tested, 25 were found to be positive by PCR and hybridization with both oligonucleotides (Table ​(Table1).1). Positive samples were found at each of the five sewage treatment plants. Most of the positive PCR products displayed relatively weak bands at the expected size of 267 bp. They also contained some extraneous bands, which were not of the expected size, probably due to the large amount of DNA from other organisms present in sewage. On hybridization, however, the PCR products displayed distinct bands, which were consistent between the two oligonucleotides (Fig. ​(Fig.2).2).

TABLE 1

Results of PCR for the Whipple’s disease bacterium from sewage samples

Selectivity of ATP-activated GTP-dependent Ca2+-permeable channels in rat macrophage plasma membrane

Outside-out configuration of the patch clamp technique was used to test whether an intracellular application of G protein activator (GTPS) affects ATP-activated Ca²⁺-permeable channels in rat macrophages without any agonist in the bath solution. With 145 mm K⁺ (pCa 8.0) in the pipette solution, activity of channels permeable to a variety of divalent cations and Na⁺ was observed and general channel characteristics were found to be identical to those of ATP-activated ones. Absence of extracellular ATP makes it possible to avoid the influence of ATP receptor desensitization and to study the channel selectivity using a number of divalent cations (105 mm) and Na⁺ (145 mm) as the charge carriers. Permeability sequence estimated by extrapolated reversal potential measurements was: Ca²⁺ Ba²⁺ Mn²⁺ Sr²⁺ Na⁺ K⁺ = 68 30 26 10 3.5 1. Slope conductances (in pS) for permeant ions rank as follows: Ca²⁺ Sr²⁺ Na⁺ Mn²⁺ Ba²⁺ = 19 18 14 12 10. Unitary Ca²⁺ currents display a tendency to saturate with the Ca²⁺ concentration increase with apparent dissociation constant (K _d ) of 10 mm. No block of Na⁺ permeation by extracellular Ca²⁺ in millimolar range was found. The data obtained suggest that (i) activation of some G protein is sufficient to gate the channels without the ATP receptor being occupied, (ii) the ATP receptor activation results in the gating of a special channel with the properties that differ markedly from those of the receptoroperated or voltage-gated Ca²⁺-permeable channels on the other cell types.DeceasedThe authors are grateful to K. Kiselyov and A. Mamin for technical assistance. The work was supported by the Russian Basic Research Foundation, Grant N 93-04-21722 and was made possible in part by Grant N R4A000 from the International Science Foundation.     

Crystal Structure of a Chimeric Receptor Binding Protein Constructed from Two Lactococcal Phages

Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry to manufacture cheeses, is subject to infection by a diverse population of virulent phages. We have previously determined the structures of three receptor binding proteins (RBPs) from lactococcal phages TP901-1, p2, and bIL170, each of them having a distinct host range. Virulent phages p2 and bIL170 are classified within the 936 group, while the temperate phage TP901-1 is a member of the genetically distinct P335 polythetic group. These RBPs comprise three domains: the N-terminal domain, binding to the virion particle; a β-helical linker domain; and the C-terminal domain, bearing the receptor binding site used for host recognition. Here, we have designed, expressed, and determined the structure of an RBP chimera in which the N-terminal and linker RBP domains of phage TP901-1 (P335) are fused to the C-terminal RBP domain of phage p2 (936). This chimera exhibits a stable structure that closely resembles the parental structures, while a slight displacement of the linker made RBP domain adaptation efficient. The receptor binding site is structurally indistinguishable from that of native p2 RBP and binds glycerol with excellent affinity.A broad number of products are manufactured by large-scale bacterial fermentation, including the value-added fermented dairy products. Most bacterial fermentation industries have experienced problems with phage contamination. Phage outbreaks are costly and time-consuming because they can slow or arrest the fermentation process and adversely affect product quality (15). For decades, the dairy industry has relied on an array of strategies to control this natural phenomenon, including rotation of their bacterial cultures (11, 24, 25). However, in spite of these efforts, new virulent lactococcal phages keep emerging. A better understanding of the various mechanisms affecting the genetic diversity of the phage population is necessary for optimal phage control strategies (18).Lactococcal phages are among the most studied bacterial viruses because of the economic importance of their hosts. Hundreds of lactococcal phages have been isolated, and the vast majority of them have a long, contractile tail, thereby belonging to the Siphoviridae family (1). Lactococcus lactis phages are currently classified into 10 genetically distinct groups (10), but only members of 3 of them are highly adapted to multiply in milk, namely, the 936, c2, and P335 groups (11, 24, 25). The first step for such an effective viral infection is host recognition, which necessitates the interaction between the adsorption device located at the distal tail end of the phage and the cell surface receptor (32). Members of the 936 and P335 groups recognize their host through an interaction between their receptor binding protein (RBP) (13) and receptors, probably lipoteichoic acids, at the host cell surface (27, 29-31).We have previously determined the crystal structures of three RBPs, from the virulent lactococcal phages p2 (30, 31) and bIL170 (936 group) (27) and from the temperate phage TP901-1 (P335 group) (29). The RBPs of these phages have a similar architecture of three protomers related by a threefold axis. Each protomer comprises three domains: the N terminus (named shoulders in p2), the interlaced β-prism linker (the “neck” domain), and the jelly-roll domain (2) at the C terminus (the “head” domain). This last domain harbors a saccharide binding site likely involved in host recognition, as it binds with high affinity to phosphoglycerol, a component of teichoic acid (8, 19, 27, 29-31). We have previously shown that the shoulder and neck domains are highly conserved in the RBPs of 936-like phages (8, 19, 27, 29-31). The individuality of the RBP C-terminal domain sequence likely dictates phage specificity for the receptor, which may specifically recognize different substitutions (H, GlcNAc, or d-Ala) of the phosphoglycerol moieties of the L. lactis teichoic acid polymers. Recently, the complete genomic sequence of the reference virulent phage P335 was determined, and comparative analysis revealed that the C terminus of its RBP showed homology to the RBP of the virulent lactococcal phage P475 of the 936 group (17). Such homology between RBP head domains was surprising because the two lactococcal phage groups rarely shared common genes or domains. This observation suggested that modular shuffling of domains can occur between these otherwise genetically distinct phage groups.The overall fold of the N-terminal RBP domain is different in 936- and P335-like phages. In the P335 group, the N-terminal domain comprises a unique helix that fits into the rest of the phage baseplate (28, 29) (Fig. ​(Fig.1A),1A), while in the 936 group, this 140-residue domain is a large β-sandwich with an external α-helix (30) (Fig. ​(Fig.1B).1B). Nonetheless, the N-terminal domains of the two RBPs may still be, related because both appear to be built using a coiled coil, although the 936-like phages have an additional β-sandwich. The β-prism linkers (neck domain) of the two phage groups also differ in sequence and in radius, but they have a similar fold, the latter being also close to that of T4 phage short fiber (33). The linker domain of phage TP901-1 is wider than that of p2 and exhibits a repeated motif (G-X-Y-X-Y, where X is polar and Y nonpolar). Finally, the C-terminal domains of both species share the same fold, a jelly-roll motif (2) also found in adenovirus (5) and reovirus (3, 4, 6).Open in a separate windowFIG. 1.Structures and sequences of RBPs from lactococcal phages. (A) Three-dimensional structure of the RBP from phage TP901-1 (P335 group; blue). (B) Three-dimensional structure of the RBP from phage p2 (936 group; magenta). (C) View of a model associating domains of TP901-1 (N terminus and linker domain, below red line, blue) and p2 (head, above red line, magenta) RBPs. (D) Three-dimensional crystal structure of chimera form 1 (yellow) assembled according to the model in panel C. (E) Sequence alignment of the RBPs of p2 (part) and TP901-1. The secondary structure is described above the alignment. The binding residues are shown with blue dots. The hinge proline (Pro 162/63) is identified by a red arrow. The chimera is composed of the N-terminal domain (residues 17 to 33) and the linker domain residues (residues 34 to 63) from phage TP901-1 RBP and the C-terminal domain (residues 163 to 264) from phage p2 RBP.The question addressed here was whether exchange between the C-terminal domains of two phage groups would lead to a stable protein with conserved binding capacity. To answer this question, we have generated an RBP chimera comprising the N-terminal and linker domains of phage TP901-1 fused to the C-terminal domain of phage p2. We have produced this chimera and determined its crystal structure and its sugar binding capacity. These results indicate that straightforward domain exchange produced a stable chimera with a conserved binding capacity and a structure close to that of each of the parental parts.